METHOD FOR PREPARING FERMENTABLE SUGARS FROM LIGNOCELLULOSIC BIOMASS

Abstract

A method is disclosed for providing a c5 / c6 product from a lignocellulosic material, said method comprising the steps of: (i) pretreatment of the lignocellulosic material; (ii) solid / liquid separation of the pretreated lignocellulosic material from step (a) into a first solid fraction and a first liquid fraction; (iii) enzymatic hydrolysis of the fiber from said first solid fraction from step (b) using an enzyme composition capable of degrading lignocellulosic material, thereby providing a c5 / c6 fiber paste comprising c5 and / or c6 sugars; (iv) solid / liquid separation of the c5 / c6 fiber paste from step (c) into a second solid fraction and a second liquid fraction; and optionally (v) combining said first liquid fraction and said second liquid fraction for enzymatic hydrolysis of mixed sugar (msh), thereby providing a c5 / c6 msh product.

Description

1 / 116 “METHOD FOR PREPARING FERMENTABLE SUGARS FROM LIGNOCELLULOSIC BIOMASS” FIELD

[0001] The present invention relates to a method for processing lignocellulosic biomass into fermentable sugars, to a method for producing a fermentation product, such as by using a two-step fermentation method, and to a method for producing lignin. BACKGROUND

[0002] The historical dependence on oil and other fossil fuels has been associated with dramatic and alarming increases in atmospheric levels of greenhouse gases. International efforts are underway to mitigate the accumulation of greenhouse gases, supported by formal policy guidelines in many countries. A central focus of these mitigation efforts has been the development of processes and technologies for the use of renewable plant biomass to replace oil as a source of fuel precursors and other chemicals.

[0003] The industrial manufacture of fuel ethanol from sugar and starch-based plant materials, such as sugarcane, root crops, and grains, is already widely used globally. However, environmental, economic, and moral objections have been raised to these first-generation bioethanol processes, for example, because they place the demand for crops for human consumption in direct competition with the demand for fuel for personal automobiles.

[0004] Thus, there has been great interest in Petition 870190096087, dated 09 / 26 / 2019, page 8 / 149 2 / 116 Development of biomass conversion systems that do not consume food crops – the so-called second-generation biorefining, in which bioethanol and other products can be produced from lignocellulosic biomass, such as agricultural residues (stems, ears, kernels, stalks, husks, peels, etc.), grasses, straw, wood chips, paper waste and the like. In second-generation technology, fermentable 6-carbon (C6) sugars derived mainly from cellulose and fermentable 5-carbon (C5) sugars derived from hemicellulose are released from biomass polysaccharide polymer chains by enzymatic hydrolysis or, in some cases, by pure chemical hydrolysis.Fermentable sugars obtained from the conversion of biomass in a second-generation biorefinery can be used to produce, for example, ethanol, acetone, butanol, lactic acid and / or other useful compounds such as, for example, fuel or precursors of chemical products, for example, various polymers, etc.

[0005] The total yield of both C5 and C6 products is a key factor in the economic viability of commercializing lignocellulosic biomass processing. Due to the limitations of its physical structure, lignocellulosic biomass cannot be effectively converted into fermentable sugars by enzymatic hydrolysis without some pretreatment process. A wide variety of different pretreatment schemes have been reported, each scheme offering different advantages and disadvantages.

[0006] Document WO2014 / 019589, incorporated herein by reference in its entirety, discloses a method Petition 870190096087, dated 09 / 26 / 2019, page 9 / 149 3 / 116 for processing lignocellulosic biomass comprising a pretreatment and enzymatic processing of a solid fraction in order to produce a C5 / C6 product.

[0007] Document WO2015 / 014364, incorporated herein by reference in its entirety, discloses a method for processing lignocellulosic biomass using a single-step autohydrolysis pretreatment and enzymatic hydrolysis. BRIEF DESCRIPTION OF THE FIGURES

[0008] Figure 1: Schematic sketch of the process steps relating to methods according to the present invention.

[0009] Figure 2: Process scheme (1) describes a process scheme of a relatively simple process configuration, such as a “full folder” process as described in WO2015 / 014364.

[0010] Figure 3: Process scheme (2) describes a more complex process scheme comprising a derivation of C5, also referred to in this document as V2, as are the processes described in document WO 2014 / 019589.

[0011] Figure 4: Process scheme (3) describes a process embodiment / system according to the present invention (also referred to herein as V2.X or “two-step hydrolysis and mixed sugar hydrolysis”).

[0012] Figure 5: Experimental design for comparing total carbohydrate conversion in the V2 and V2.X methods.

[0013] Figure 6: Glucan conversion as a function of enzyme dose for the V2.X, V2 and C5 derivation methods Petition 870190096087, dated 09 / 26 / 2019, p. 10 / 149 4 / 116 with lines to guide the eye.

[0014] Figure 7: Xylan conversion as a function of enzyme dose for the V2.X method and C5 derivation with lines to guide the eye.

[0015] Figure 8: Arabin conversion as a function of enzyme dose for the V2.X method and for the C5 derivation method with lines to guide the eye.

[0016] Figure 9: Fiber glucan conversion for the V2.X method and C5 derivation for five different pretreatment dates.

[0017] Figure 10: Conversion of xylan fibers to the Version V2.X and C5 derivation method for five different pretreatment dates.

[0018] Figure 11: Total Glucan Conversion: Gray circle - One-step hydrolysis with 75 g of Cellic® CTec3 / kg of glucan added during fiber hydrolysis (FH) and 22% by weight of SS, Dark gray triangle - One-step hydrolysis with 75 g of Cellic® CTec3 / kg of glucan added during fiber hydrolysis (FH) and 18% by weight of SS, Light gray cross - Two-step hydrolysis with 75 g of Cellic® CTec3 / kg of glucan added during fiber hydrolysis (FH) and 22% by weight of SS during fiber hydrolysis and 22% by weight of SS during fiber cake hydrolysis, and Unfilled black circle - Two-step hydrolysis with 50 g of Cellic® CTec3 / kg of glucan added during fiber hydrolysis (FH) and 22% by weight of SS during fiber cake hydrolysis. fiber and 25 g of Cellic® CTec3 / kg FH of glucan (giving a total of 75 g of Cellic® CTec3 / kg FH of glucan) were added to the fiber cake hydrolysis (FCH), and 22% by weight of SS was added to the fiber cake hydrolysis.

[0019] Figure 12: Conversion of xylan to MSH. Code of Petition 870190096087, dated 09 / 26 / 2019, page 11 / 149 5 / 116 dark gray: MSH including fibers; light gray: MSH where fibers are removed before the addition of the C5 derivation.

[0020] Figure 13: Total conversion of xylan to MSH as a function of enzyme dose at 48 hours reaction time (0 hours for heat treatment without enzymes) at pH 5, 250 rpm and 50 °C.

[0021] Figure 14: Modality of a V2 configuration with improved fermentation.

[0022] Figure 15: Schematic sketch of a two-step fermentation.

[0023] Figure 16: Changes in xylan conversion in increasing proportions of liquid C5 in post-hydrolysis. Filtrate may refer to the liquid fraction after a hydrolysis step, such as hydrolysis step (c) in Figure 14. SUMMARY OF THE INVENTION

[0024] In a first aspect, the present invention relates to a method for providing a C5 / C6 product from a lignocellulosic material comprising the steps of: a) Pre-treatment of the lignocellulosic material; b) Solid / liquid separation of the pretreated lignocellulosic material from step (a) into a first solid fraction and a first liquid fraction; c) Enzymatic hydrolysis of the fiber from the aforementioned first solid fraction of step (b) through the use of an enzyme composition capable of degrading lignocellulosic material, thereby providing a C5 / C6 fiber paste comprising C5 and / or C6 sugars; Petition 870190096087, dated 09 / 26 / 2019, page 12 / 149 6 / 116 d) Solid / liquid separation of the C5 / C6 fiber paste from step (c) into a second solid fraction and a second liquid fraction; and optionally (e) Combination of the aforementioned first liquid fraction and the aforementioned second liquid fraction for enzymatic hydrolysis of mixed sugar (MSH), through which a C5 / C6 MSH product is provided.

[0025] In a second aspect, the present invention relates to a method for providing a fermentation product wherein said method comprises the steps of: m) Supply of at least one C5 / C6 product in accordance with the method of any of the preceding embodiments; and n) Supply of the fermentation product by means of fermentation of said C5 / C6 product with a microorganism. [0 026] In a third aspect, the present invention relates to a two-step fermentation method comprising the steps of: aa) Pre-treatment of lignocellulosic material; bb) Solid / liquid separation of the pre-treated lignocellulosic material from step (aa) into a first solid fraction and a first liquid fraction; cc) Enzymatic hydrolysis of the fiber from the aforementioned first solid fraction of step (bb) through the use of an enzyme composition capable of degrading lignocellulosic material, thereby providing a C5 / C6 fiber paste; dd) Solid / liquid separation of the C5 / C6 fiber paste from step (cc) into a second solid fraction and a second liquid fraction; Petition 870190096087, dated 09 / 26 / 2019, page 13 / 149 7 / 116 ee) Enzymatic hydrolysis of mixed sugar (MSH) from a mixture of the first liquid fraction of step (bb) and the C5 / C6 fiber paste of step (cc), or the first liquid fraction of step (bb) and the second liquid fraction of step (dd), thereby providing an MSH C5 / C6 product; ff) Provision of a first fermentation substrate comprising at least a portion of the “C5 / C6 fiber hydrolysis paste” and / or the second liquid fraction; gg) Provision of a second fermentation substrate comprising at least a portion of the MSH C5 / C6 product; hh) Fermentation of the first fermentation substrate in a first fermentation with a microorganism; and ii) Fermentation of the second fermentation substrate in a subsequent second fermentation; where the step (dd) is optional.

[0027] In a fourth aspect, the present invention relates to a method for preparing ethanol and lignin from a lignocellulosic material comprising the steps of: - Supply of at least one C5 / C6 product according to the method in accordance with any of the preceding aspects; - Fermentation of said, at least one, C5 / C6 product in order to convert sugars into ethanol in the fermentation broth with a yeast; - Isolation of an ethanol-rich fraction from the fermentation broth; and optionally - Lignin isolation.

[0028] In a fifth aspect, the present invention relates to lignin provided from lignocellulosic biomass in accordance with any of the preceding aspects.

[0029] In a sixth aspect, the present invention relates Petition 870190096087, dated 09 / 26 / 2019, p. 14 / 149 8 / 116 a product C5 / C6 provided in accordance with any of the preceding aspects.

[0030] In a seventh aspect, the present invention relates to a fermentation substrate comprising a C5 / C6 product provided by a method in accordance with any of the preceding aspects.

[0031] In an eighth aspect, the present invention relates to a first or a second fermentation substrate provided by a method in accordance with any of the preceding aspects.

[0032] In a ninth aspect, the present invention relates to compositions comprising lignin obtained or obtainable by a method in accordance with any of the preceding aspects, including uses other than said compositions comprising lignin. DETAILED DESCRIPTION

[0033] Methods for the preparation of hydrolyzed lignocellulosic biomass that can be fermented by microorganisms to produce small organic molecules are only suitable for large-scale industrial use if such methods are economically competitive. To be economically competitive, such methods must have a high yield of C6 and / or C5 sugars, the lowest possible consumption of energy, enzymes and other cost-effective prerequisites, and preferably provide by-products of significant value.

[0034] The present inventors have surprisingly discovered a method for preparing a C5 / C6 and / or C6+C5 product from a lignocellulosic material that exhibits relatively low energy input, rapid hydrolysis. Petition 870190096087, dated 09 / 26 / 2019, p. 15 / 149 9 / 116 and efficient of lignocellulosic material for C6 and / or C5 sugars, while at the same time providing a substantial amount of high-value lignin as a byproduct.

[0035] The particular advantage of the present invention is that the separate hydrolysis of liquid and solid fractions of pretreated lignocellulosic biomass produces at least two C5 / C6 and / or C6+C5 product fractions, one, for example, having mainly C6 sugars and a low concentration of fermentation-inhibiting substances and one, for example, having mainly C5 sugars and a higher quantity of fermentation-inhibiting substances. Thus, the fermentation of the C5 / C6 and / or C6+C5 products can be advantageously carried out by first fermenting the low-inhibiting fraction and then subsequently adding and fermenting the high-inhibiting fraction.

[0036] In some embodiments, the present invention relates to a method for preparing at least one C5 / C6 product, such as a C6 and / or C5 sugar, from a lignocellulosic material comprising the steps of: I. Pre-treatment of the lignocellulosic material, II. Solid / liquid separation into a first solid fraction and a first liquid fraction; III. Enzymatic hydrolysis of the aforementioned first solid fraction from step II) using an enzyme composition comprising at least one cellulase and / or hemicellulase (such as a xylanase), IV. Solid / liquid separation of the reaction mixture from step III) into a second solid fraction and a second liquid fraction, and optionally Petition 870190096087, dated 09 / 26 / 2019, page 16 / 149 10 / 116 V. Mixture of the aforementioned first liquid fraction and the aforementioned second liquid fraction for enzymatic hydrolysis in order to obtain a C5 / C6 product, and optionally VI. Recycling of enzymes present after enzymatic hydrolysis in step III). [003 7] Figure 1 shows different embodiments of the invention, in particular a method for providing a C5 / C6 product from a lignocellulosic material comprising the steps of: a) Pre-treatment of lignocellulosic material; b) Solid / liquid separation of the pretreated lignocellulosic material from step (a) into a first solid fraction and a first liquid fraction; c) Enzymatic hydrolysis of the fiber from the aforementioned first solid fraction of step (b) through the use of an enzyme composition capable of degrading lignocellulosic material, thereby providing a C5 / C6 fiber hydrolysis paste comprising C5 and / or C6 sugars; d) Solid / liquid separation of the C5 / C6 fiber paste from step (c) into a second solid fraction and a second liquid fraction; and optionally (e) Combination of the aforementioned first liquid fraction and the aforementioned second liquid fraction for the enzymatic hydrolysis of mixed sugar (MSH), through which an MSH C5 / C6 product is provided; and optionally f) Enzymatic hydrolysis of the fiber cake from the aforementioned second solid fraction of step (d) in order to obtain a C5 / C6 paste product; and optionally Petition 870190096087, dated 09 / 26 / 2019, page 17 / 149 11 / 116 g) Solid / liquid separation of the C5 / C6 paste product from step (f) into a third solid fraction and a liquid C5 / C6 product; and optionally h) Combination of at least one portion of the MSH C5 / C6 product with at least one portion of one or more of: the C5 / C6 paste product from step (f), the liquid C5 / C6 product from step (g), and / or the second liquid fraction from step (d) in order to obtain a combined C5 / C6 product; and optionally i) Ultrafiltration step for recycling enzymes present after MSH in step (e).

[0038] The liquid fraction provided by the solid / liquid separations of steps b) and d) can be kept separate from the solid fractions during enzymatic hydrolysis, as shown in Figure 1. Enzymatic hydrolysis separate from the solid fractions can take place in the hydrolysis of fibers in step c) and the hydrolysis of fiber cake in step f), thus providing advantages of the present invention compared to the prior art, such as a higher yield of C6 and C5 sugars from the solid fraction and leaving the C6+C5 product paste rich in high-value lignin in the solid part. Typically, the liquid fractions obtained in steps b) and d) of solid / liquid separation can be combined and subjected to mixed sugar hydrolysis (MSH), as disclosed in step e), as shown in Figure 1.

[0039] As used in this document, the following terms have the following meaning:

[0040] The terms product C5 / C6 and / or product C6 / C5 may be used interchangeably, and are intended to encompass a composition comprising at least one Petition 870190096087, dated 09 / 26 / 2019, page 18 / 149 12 / 116 C6 sugar and / or at least one C5 sugar, wherein the C6 sugar and the C5 sugar may be any carbohydrate possessing six or five carbon atoms, respectively.

[0041] The terms C5+C6 product and / or C6+C5 product may be used interchangeably, and are intended to encompass a composition comprising at least one C6 sugar and at least one C5 sugar, wherein the C6 sugar and the C5 sugar may be any carbohydrate having six or five carbon atoms, respectively.

[0042] The C5 / C6 and / or C6+C5 product may be a liquid, a suspension or paste, or a solid composition and may contain additional compounds in addition to the C6 sugar and / or the C5 sugar, such as compounds resulting from a degradation process to release the C6 and C5 sugars from macromolecules. Such additional compounds may be, for example, poly-, oligo- or disaccharides, furfural, salts, etc., but also lignin and / or compounds and / or compositions derived from lignin.

[0043] Non-limiting examples of C6 sugars are, for example, glucose, galactose, mannose, rhamnose, and the like. Non-limiting examples of C5 sugars are xylose, arabinose, etc. When a C6+C5 product is obtained by hydrolysis of lignocellulosic material, the C6 sugar glucose is mainly obtained from the cellulose part, while the C5 sugars, mannose, galactose, and rhamnose, are mainly obtained from the hemicellulose part of the lignocellulosic material. The aforementioned C5 and / or C6 sugar(s) may be modified, such as esterified or similar.

[0044] In some embodiments, C6 sugar is a fermentable C6 sugar, for example, carbohydrates having six atoms of Petition 870190096087, dated 09 / 26 / 2019, p. 19 / 149 13 / 116 carbon and which can be fermented by well-known microorganisms, such as naturally occurring microorganisms or genetically modified microorganisms.

[0045] In some embodiments, C5 sugar is a fermentable C5 sugar, for example, carbohydrates having five carbon atoms and which can be fermented by well-known microorganisms such as naturally occurring microorganisms or genetically modified microorganisms.

[0046] The term C1-C4 product, as used in this document, means a low molecular weight organic compound having one to four carbon atoms. Non-limiting examples of C1-C4 products are methanol, ethanol, butanol, acetone, formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, lactic acid, malic acid, and / or any combination thereof.

[0047] The term fermentation product may include a C1-C4 product, as in the context of the present invention, the term fermentation product(s) is intended to include any product that can be provided through fermentation with one or more microorganisms. Fermentations according to the invention may include aerobic or anaerobic fermentations, e.g. fermentations where pyruvate is reduced to fermentation products such as ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propanediol, ethylene, glycerol, butanol, a Plactam antibiotic and a cephalosporin. A “fermentation product” may also include value-added products including, but not limited to, one or more of: Petition 870190096087, dated 09 / 26 / 2019, page 20 / 149 14 / 116 biofuels (including methanol, ethanol, propanol and butanol); alcohol, aldehyde, ketone, lactic acid; 3-hydroxypropionic acid; acrylic acid; acetic acid; 1,3-propanediol; ethylene; glycerol; a plastic; a specialty chemical; an organic acid, including citric acid, succinic acid and maleic acid; a solvent; an animal feed supplement; a drug such as a β-lactam antibiotic or a cephalosporin; a vitamin; an amino acid, such as lysine, methionine, tryptophan, threonine and aspartic acid; a peptide, a protein, an enzyme, such as a protease, a cellulase, a hemicellulase, a xylanase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductase, an esterase or a transferase; a chemical feedstock; or a food supplement for animals.

[0048] About, as used in this document, generally with reference to a number or quantitative range, can refer to + / - 1, 2, 5 or even 10% in relative terms to the number or range to which it refers.

[0049] Autohydrolysis refers to a lignocellulosic biomass pretreatment process in which acetic acid is released from hemicellulose during said process, which is believed to further catalyze and / or enhance the hydrolysis of hemicellulose. Autohydrolysis of lignocellulosic biomass is thus carried out without or essentially without the addition of any additional chemicals, such as acid(s) or base(s), and is commonly performed at a pH between 3.5 and 9.0.

[0050] Preparation of commercially available cellulase optimized for lignocellulosic biomass conversion if Petition 870190096087, dated 09 / 26 / 2019, p. 21 / 149 15 / 116 refers to a commercially available mixture of enzymatic activities that is sufficient to provide enzymatic hydrolysis of pretreated lignocellulosic biomass and that typically comprises endocellulase (endoglucanase), exocellulase (exoglucanase), endoxylanase, acetyl xylanesterase, xylosidase, and β-glucosidase activities. The term optimized for lignocellulosic biomass conversion refers to a product development process in which enzyme mixtures have been selected and / or modified for the specific purpose of improving hydrolysis yields and / or reducing enzyme consumption in the hydrolysis of pretreated lignocellulosic biomass to fermentable sugars.

[0051] The term Cellulase(s) refers to one or more enzymes capable of degrading cellulose and / or related compounds. Cellulase is one of several enzymes commonly produced by fungi, bacteria, and protozoa that catalyze cellulolysis, the breakdown of cellulose and / or related polysaccharides. Cellulase can also be used for any mixture or complex of several of these enzymes, which act in series or synergistically to decompose cellulosic material. Cellulases break down the cellulose molecule into monosaccharides (simple sugars), such as beta-glucose, and / or shorter polysaccharides and oligosaccharides. Specific reactions may involve hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, hemicellulose, lichenine, and beta-D-glucans from cereals. Several different types of cellulases are known, which differ structurally and mechanistically. Synonyms, derivatives and / or specific enzymes associated with the name cellulase. Petition 870190096087, dated 09 / 26 / 2019, page 22 / 149 16 / 116 comprise endo-1,4-beta-D-glucanase (beta-1,4-glucanase, beta-1,4-endoglucan hydrolase, endoglucanase D, 1,4-(1,3,1,4)-beta-D-glucan 4-glucan hydrolase), carboxymethylcellulase (CMCase), avicelase, celludextrinase, cellulase A, cellulosin AP, alkaline cellulase, cellulase A 3, 9,5 cellulase and pancelase SS.

[0052] Cellulases can also be classified based on the type of reaction catalyzed, where endocellulases (EC 3.2.1.4) randomly cleave internal linkages at amorphous sites that create new chain ends, exocellulases or cellobiohydrolases (EC 3.2.1.91) cleave two to four units from the exposed chain ends produced by endocellulase, resulting in tetra-, tri-, or disaccharides, such as cellobiose. Exocellulases are further classified into type I – which processively function from the reducing end of the cellulose chain, and type II – which processively function from the non-reducing end. Cellobioses (EC 3.2.1.21) or beta-glucosidases hydrolyze the exocellulase product into individual monosaccharides. Oxidative cellulases depolymerize cellulose through radical reactions, such as cellobiose dehydrogenase (the acceptor).Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.

[0053] The term Hemicellulase(s) refers to one or more enzymes capable of and / or contributing to the breakdown of hemicellulose, one of the main components of plant cell walls. Some of the main polysaccharides that constitute hemicellulose are believed to be xylan, arabinoxylan, xyloglucan, glucuronoxylan, and glucomannan. Petition 870190096087, dated 09 / 26 / 2019, page 23 / 149 17 / 116 In the context of the present invention, the term hemicellulase(s) is intended to include: xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s) and / or esterase(s), including any combination thereof.

[0054] The term Xylanase(s) is intended to encompass one or more enzymes capable of degrading xylan and / or related compounds. Xylanase is any of several enzymes produced, for example, by microorganisms such as yeasts, that catalyze the decomposition of xylan and / or related polysaccharides. Xylanase may also be used for any mixture or complex of several of these enzymes, which act in series or synergistically to decompose xylanosic material. Synonyms, derivatives and specific enzymes associated with the name xylanase may include EC 3.2.1.8, 4-xylan endo-(1->4)-beta-xylanase, endo-1,4-xylanase, endo-1,4-beta-xylanase, beta-1,4-xylanase, endo-1,4-beta-D-xylanase, 1,4beta-xylan xylane hydrolase, beta-xylanase, beta-1,4-xylan xylane hydrolase, beta-D-xylanase and / or xylosidase capable of degrading xylan, such as beta-1,4-xylan into xylose, thus contributing to the breakdown of hemicellulose, one of the main components of the plant cell wall.

[0055] Xylosidase, as used in this document, is intended to encompass the enzyme xylan 1,4-beta-xylosidase (EC 3.2.1.37), also known as xylobiase, beta-xylosidase, exo-1,4-beta-D-xylosidase, or 4-beta-D-xylan xylohydrolase. This enzyme catalyzes the hydrolysis of (1-4)-beta-D-xylans by removing successive D-xylose residues from the non-reducing terminals of Petition 870190096087, dated 09 / 26 / 2019, page 24 / 149 18 / 116 substrate, for example, hemicellulose and the disaccharide xylobiose. This enzyme is believed to be commercially available either as an essentially pure xylosidase enzyme or, for example, as part of cellulase preparations.

[0056] The term Arabinoxylanase(s) is intended to encompass one or more enzymes capable of degrading arabinoxylan and / or related compounds, including, for example, glucuronoarabinoxylan endo-1,4beta-xylanase (EC 3.2.1.136), feraxane endoxylanase, feraxanase, endoarabinoxylanase, glucuronoxylan xylohydrolase, glucuronooxylanase, glucuronoxylan xylane hydrolase, glucuronoarabinoxylan 1,4-beta-D-xylane hydrolase) and glucuronoarabinoxylan 4-beta-D-xylane hydrolase. It is believed that glucuronoarabinoxylan 4beta-D-xylan hydrolase hydrolyzes the (1->4)-beta-D-xylosyl linkages in some glucuronoarabinoxylans. This enzyme is also believed to have high activity towards feruloylated arabinoxylans. (Nishitani, K.; Nevins, DJ (1988). Enzymatic analysis of ferulated arabinoxylans (Feraxan) derived from Zea mays cell walls. I. Purification of novel enzymes capable of dissociating Feraxan fragments from Zea mays coleoptile cell wall. Plant Physiol. 87: 883—890.)

[0057] The term “Xyloglucanase(s)” is intended to encompass one or more enzymes capable of degrading xyloglucan and / or related compounds, including, for example, xyloglucan-specific endobeta-1,4-glucanase (EC 3.2.1.151), which is an enzyme believed to catalyze the chemical reaction:

[0058] xyloglucan + H2O ^ xyloglucan oligosaccharides. This enzyme belongs to the hydrolase family, specifically those glycosidases that hydrolyze the. Petition 870190096087, dated 09 / 26 / 2019, p. 25 / 149 19 / 116 O- and S-glycosyl compounds. The systematic name for this class of enzymes is [(1->6)-alpha-D-xylo]-(1->4)-beta-D-glucanhydrolase. Other commonly used names may include XEG, xyloglucan endobeta-1,4-glucanase, xyloglucanase, xyloglucaneendohydrolase, XH, and 1,4-beta-D-glucanhydrolase.

[0059] The term Glucuronoxylanase(s) is intended to encompass one or more enzymes capable of degrading glucuronoxylan and / or related compounds.

[0060] The term Glucomannanase(s) is intended to encompass one or more enzymes capable of degrading glucomannanase and / or related compounds.

[0061] The term Esterase(s) refers to one or more enzymes capable of separating an ester into an acid and an alcohol. Examples of esterases include acetylsterases and feroyl esterase.

[0062] The term Acetylesterase(s) refers to an enzyme capable of cleaving acetyl groups. An acetylesterase (EC 3.1.1.6) is an enzyme that catalyzes the chemical reaction:

[0063] Acetic ester + H2O ^ alcohol + acetate. This enzyme belongs to the hydrolase family, specifically those that act on carboxylic ester bonds. The systematic name for this class of enzymes is acetyl ester hydrolase. Other commonly used names include C-esterase (in animal tissues), acetic ester hydrolase, chloroesterase, p-nitrophenyl acetate esterase, and Citrus acetylesterase.

[0100] The term(s) “feroyl esterase(s)” and “feruloyl esterase(s)” can be used interchangeably. Petition 870190096087, dated 09 / 26 / 2019, page 26 / 149 20 / 116 interchangeable, and aims to understand an enzyme that catalyzes the chemical reaction feruloyl-(poly-, oligo- or mono-)polysaccharide + H2O ^ ferulic acid + (poly-, oligo- or mono-)saccharide. Feruloyl esterase belongs to the family of hydrolases, specifically those that act on carboxylic ester bonds. The systematic name of this class of enzymes is feruloyl esterase (EC 3.1.1.73); other names may include ferulic acid esterase (FAE), hydroxycinnamoyl esterase, hemicellulase accessory enzyme, and cinnamoyl ester hydrolase (cinnAE).

[0101] Suitable microbial enzymes, such as cellulases, hemicellulase(s) including xylanases, and / or esterases, can be expressed in suitable hosts using methods known in the art. Such enzymes are also commercially available, either in pure form or in enzyme cocktails. Particular enzymatic activities can be purified from commercially available enzyme cocktails, again using methods known in the art – see, for example, Sorensen et al. (2005) “Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial fermentation residue” (Enzyme and Microbial Technology 36 (2005) 773-784), where a beta-xylosidase from Trichoderma reesei is purified from Celluclast (Finizima) and additional commercial enzyme preparations are disclosed.

[0102] Conducting a treatment / process, such as a pretreatment at a dry matter level, refers to the dry matter content of the raw material at the beginning of said treatment. Similarly, conducting a treatment / process, Petition 870190096087, dated 09 / 26 / 2019, p. 27 / 149 21 / 116, such as a pretreatment at a pH, refers to the pH of the aqueous content of the biomass at the beginning of said treatment.

[0103] In the context of the present invention, the term “adjusted to pH and temperature” is intended to encompass adjustments of pH and / or temperature in order to allow hydrolysis and / or enzymatic fermentation to occur under suitable pH and / or temperature conditions.

[0104] Dry matter, also appearing as DM, refers to total solids, both soluble and insoluble, and effectively means non-water content. Dry matter content is measured by drying at 105 °C until a constant weight is achieved. Fiber structure is maintained insofar as the average size of fiber fragments after pretreatment is > 750 μm.

[0105] Hydrothermal pretreatment or sometimes just pretreatment generally refers to the use of water, either as liquid, steam or hot pressurized steam comprising high temperature liquid or steam or both, to cook biomass at temperatures of 120 °C or higher, with or without the addition of acids or other chemicals. In the context of the present invention, “hydrothermal pretreatment” is intended to comprise methods, unit operations and / or processes related to the softening of lignocellulosic biomass through the use of temperature and water, and usually also pressure, aiming to provide a pretreated biomass suitable for enzymatic digestion.

[0106] Single-stage pressurized hydrothermal pretreatment refers to a pretreatment in which biomass is subjected to hydrothermal pretreatment. Petition 870190096087, dated 09 / 26 / 2019, page 28 / 149 22 / 116 pressurized in a single reactor configured to heat biomass in a single pass and in which no additional pressurized hydrothermal pretreatment is applied after a solid / liquid separation step to remove the liquid fraction of the feedstock subjected to pressurized hydrothermal pretreatment.

[0107] Process water refers to water of a quality suitable for its intended use in an industrial process. Process water is commonly of lower quality than, for example, potable water. Process water may comprise water that is recycled from an industrial process, such as a process according to the present invention. Process water may be adjusted in terms of mineral / salt content, pH, and the like.

[0108] Solid / liquid separation refers to an active mechanical process and / or unit operation(s) in which liquid is separated from solid by applying force through, for example, pressing, centrifugation, sedimentation, decantation or similar. Commonly, a solid / liquid (s / l) separation yields a liquid and a solid fraction.

[0109] Solid fraction and liquid fraction refer to the fractionation of pretreated and / or hydrolyzed biomass in solid / liquid separation. The separated liquid is collectively referred to as the liquid fraction. The residual fraction comprising a considerable amount of insoluble solids is referred to as the solid fraction. A solid fraction will have a substantial dry matter content and will typically also comprise a considerable residue of liquid fraction, thus having the form of a solid or a paste. Petition 870190096087, dated 09 / 26 / 2019, page 29 / 149 23 / 116

[0110] Lignocellulosic biomass refers to plant biomass comprising cellulose and lignin, and usually also hemicellulose.

[0111] Soft lignocellulosic biomass refers to plant biomass other than wood, comprising cellulose and lignin, and usually also hemicellulose.

[0112] The term lignin is intended to encompass a complex phenolic polymer that forms an integral part of the secondary cell walls of various plants. Lignin is believed to be one of the most abundant organic polymers on earth, exceeded only by cellulose, and constituting 25 to 33% of the dry mass of wood and 20 to 25% of annual crops. “Lignin” is also used for a lignin component obtained in the process of refining biomass, generally comprising pretreatment. Thus, the term “lignin” in the present description and appended claims refers to the polymer denoted as such and being present in unprocessed lignocellulosic plant material, as well as “lignin” that has been subjected to various physical and / or chemical treatments, usually imposing only minor alterations to the structure of the lignin polymer, such as maintaining its polymeric character.Examples of such physical and / or chemical treatments include processes and methods for providing a C5 / C6 product as disclosed herein. "Lignin" may comprise significant amounts of hemicellulose and cellulose and / or other sugars. Thus, "lignin," as used in this description and the appended claims, may refer to lignin that has undergone minor structural modifications and / or comprises some... Petition 870190096087, dated 09 / 26 / 2019, page 30 / 149 24 / 116 quantity of chemical residues resulting from its manufacturing process, or resulting from native compounds in the lignocellulosic material from which it is isolated.

[0113] In the context of the present invention, the term inhibitor is intended to encompass one or more components or chemicals reducing (i) the effectiveness of the process, such as a chemical reaction, e.g. catalyzed by a catalyst such as an enzyme; (ii) growth of a microorganism; and / or (iii) reduction of metabolism, in particular of product yield, such as reduction in the product yield of a fermentation product. “Fermentation inhibitors are inhibitors of type (ii) and / or (iii).At least three categories of fermentation inhibitors are typically formed during autohydrolysis pretreatment: (1) furans, mainly 2-furfural and 5-hydroxymethylfurfural (5-HMF), which are degradation products from mono- or oligosaccharides; (2) monomeric phenols, which are degradation products of the lignin structure; and (3) small organic acids, mainly acetic acid, which originate from acetyl groups in hemicellulose and lignin. Further details related to the inhibitors found in pretreated biomass, and methods for their determination and analysis, can, for example, be found in Rasmussen (2016) “Carbohydrate degradation mechanisms and compounds from pretreated biomass” PhD thesis, Technical University of Denmark.

[0114] Theoretical yield refers to the equivalent molar mass of pure monomeric sugars obtained from polymeric cellulose, or from structures of Petition 870190096087, dated 09 / 26 / 2019, p. 31 / 149 25 / 116 polymeric hemicellulose, in which the constituent monomeric sugars may also be esterified or otherwise substituted. C5 monomer yields as a percentage of the theoretical yield are determined as follows: Before pretreatment, the biomass feedstock is analyzed for carbohydrates using strong acid hydrolysis and an HPLC system in which galactose and mannose coelute with xylose. Examples of such systems are REZEX™, Phenomenex H+ monosaccharide column, and a Biorad AMINEX HPX 87C™ column. During strong acid hydrolysis, esters and acid-labile substitutions are removed. Unless otherwise indicated, the total amount of Xylose + Arabinose determined in the unpretreated biomass is taken as a theoretical 100% C5 monomer recovery, which may be referred to as C5 monomer recovery.Monomeric sugar determinations are made using HPLC characterization based on standard curves with purified external standards. The actual recovery of C5 monomer is determined by HPLC characterization of samples for direct measurement of C5 monomers, which are then expressed as a percentage of the theoretical yield. The xylan number refers to a characterization of pretreated biomass determined as follows: The pretreated biomass is subjected to solid / liquid separation to provide a solid fraction of approximately 30% total solids and a liquid fraction. This solid fraction is then partially washed by mixing with water at 70 °C in a total solids (DM) to water ratio of 1:3 wt. The solid fraction washed in this manner is then pressed to approximately 30% total solids. Alternatively, the pretreated biomass... Petition 870190096087, dated 09 / 26 / 2019, page 32 / 149 26 / 116 can be subjected to solid / liquid separation to provide a solid fraction of approximately 50% total solids and a liquid fraction. With both methods, approximately 25% of the dissolved solids remain in the solid fraction with the suspended solids. The xylan content of the washed solid fraction in this manner can be determined using, for example, the method of A. Sluiter, et al., Determination of structural carbohydrates and lignin in biomass, Laboratory Analytical Procedure (LAP) of the US National Renewable Energy Laboratory (NREL) submitted on April 25, 2008, as described in Technical Report NREL / TP-51042618, revised in April 2008, which is expressly incorporated by reference herein in its entirety. This measurement of the xylan content as described will include some contribution from the soluble material from the residual liquid fraction that is not washed from the solid fraction under these conditions.Consequently, in the context of the present invention, the term xylan number(s) refers to (pre-)treatment gravities and refers to a composite measurement and / or values ​​that reflect a weighted combination of both the residual xylan content remaining within insoluble solids and also the concentration of xylose and soluble xylo-oligomers within the liquid fraction. The lower the Ro gravity, the higher the xylan numbers. Thus, the higher the xylan number, the lower the pre-treatment gravity. Xylan numbers provide a negative linear correlation with the log R0 of conventional gravity measurement, even at low gravity levels where the residual xylan content within insoluble solids is above 10%. Generally, pre-treatment gravities... Petition 870190096087, dated 09 / 26 / 2019, page 33 / 149 27 / 116 low, medium and high treatments provide xylan numbers > 10%, 6-10% and <6%, respectively.

[0115] In the context of the present invention, unless otherwise indicated, % indicates % by weight / weight (w / w).

[0116] In the context of the present invention, the terms about, around, approximately, or the symbol ~ may be used interchangeably, and should encompass variations generally accepted in the art, for example, including analytical errors and the like. Thus, about may also indicate the measurement uncertainty commonly experienced in the art, which may be on the order of magnitude of, for example, + / - 1, 2, 5, 10, 20, or even 50 percent.

[0117] The term comprising should be interpreted as specifying the presence of the stated or similar parts, steps, features, components, but does not exclude the presence of one or more additional parts, steps, features, components, etc. For example, a composition comprising a chemical compound may thereby comprise additional chemical compounds.

[0118] A derivative is a compound that is derived from a similar compound by a chemical reaction.

[0119] An isomer is a molecule with the same molecular formula as another molecule, but with a different chemical structure. That is, isomers contain the same number of atoms of each element, but have different arrangements of their atoms. Isomers do not necessarily share similar properties unless they also have the same functional groups. There are two main forms of isomerism: structural isomerism (or isomerism). Petition 870190096087, dated 09 / 26 / 2019, page 34 / 149 28 / 116 constitutional) and stereoisomerism (or spatial isomerism).

[0120] A “structural analogue, also known as a chemical analogue or simply an analogue, is a compound having a structure similar to that of another, but which differs from it with respect to a particular component.

[0121] This may differ in one or more atoms, functional groups, or substructures, which are replaced by other atoms, groups, or substructures. A structural analogue can be imagined as being formed, at least theoretically, from the other compound.

[0122] In the context of the present invention, the terms related to “recover,” “isolate,” “purify,” and “concentrate” may be used interchangeably, and are intended to encompass processes and / or unit operations aimed at providing a desired product, compound, and the like, such as a fermentation product or lignin in a more concentrated, less contaminated, and / or purer form. Suitable processes, operations, and / or processes are considered to be well known in the art.

[0123] Lignocellulosic biomass comprises crystalline cellulose fibrils interspersed within a weakly organized hemicellulose matrix and sealed within a hydrophobic lignin-rich environment. While cellulose itself comprises long, linear chain polymers of D-glucose, hemicellulose is a heterogeneous mixture of short, branched-chain carbohydrates, including monomers of all 5-carbon aldopentoses (C5 sugars), as well as some 6-carbon (C6) sugars including glucose and mannose. Lignin is a highly polymeric Petition 870190096087, dated 09 / 26 / 2019, page 35 / 149 29 / 116 heterogeneous, lacking any particular primary structure and comprising hydrophobic phenylpropanoid monomers. Suitable lignocellulosic biomass typically comprises cellulose in amounts between 20 and 50% of the dry mass before pretreatment, lignin in amounts between 10 and 40% of the dry mass before pretreatment, and hemicellulose in amounts between 15 and 40%.

[0124] In some embodiments, biomass raw materials may be subjected to particle size reduction and / or other mechanical treatment, such as granulation, shearing, grinding, crushing, cutting or other processes prior to hydrothermal pretreatment. Other mechanical treatments may include cleaning / purification means, such as means to remove non-biomass components or objects, such as stones, debris, sand, dust and / or foreign objects, such as metal or plastic objects and the like.

[0125] In some embodiments, biomass feedstocks may be washed and / or leached of valuable salts prior to pressurized pretreatment. In some embodiments, feedstocks may be soaked prior to pressurized pretreatment at temperatures up to 99°C. Said washing and / or leaching is generally carried out around ambient pressure.

[0126] In some embodiments, the feedstock is first soaked in an aqueous solution before hydrothermal pretreatment. In some embodiments, the feedstock is soaked in a liquid containing acetic acid obtained from a subsequent step in the pretreatments, as described in US patent 8,123,864, which is incorporated in Petition 870190096087, dated 09 / 26 / 2019, p. 36 / 149 30 / 116 of this document by reference in its entirety. It may be advantageous to conduct the treatment with the highest possible dry matter content, as described in US document 12 / 935,587, which is incorporated herein by reference in its entirety. Conducting the pretreatment with high dry matter avoids the process energy expenditure on unnecessary water heating. However, some water content is necessary to achieve optimum sugar yields from enzymatic hydrolysis. Typically, it is advantageous to pretreat biomass feedstocks to or near their inherent water-holding capacity. This is the water content level that a given feedstock will reach after immersion in excess water, followed by pressing to the mechanical limits of a common screw press (typically between 30 and 45% DM). In some embodiments, hydrothermal pretreatment is carried out at a DM content of at least 35%.It will be readily understood by a person skilled in the art that the DM content may decrease during hydrothermal pretreatment when some water content is added during heating. In some embodiments, the feedstocks are pretreated with a DM content of at least 20%, or at least 25%, or at least 30%, or at least 40%, or less than 40%, or less than 35%, or less than 30%. Suitable additional DM contents may be described elsewhere in this document.

[0127] In some embodiments, immersion / humidification with an aqueous solution can serve to adjust the pH before pretreatment to the range between 3.5 and 9.0, which is typically advantageous for autohydrolysis. It will be quickly understood that the pH can change during pretreatment. Petition 870190096087, dated 09 / 26 / 2019, p. 37 / 149 31 / 116 treatment, typically to more acidic levels, as acetic acid is released from the solubilized hemicellulose. Additional suitable pH values ​​may be disclosed elsewhere in this document.

[0128] The xylan number is particularly useful as a measure of the severity of the pretreatment where different pretreated biomass feedstocks having an equivalent xylan number exhibit equivalent C5 monomer recovery. In contrast, conventional Ro gravity is simply an empirical description of pretreatment conditions, which does not provide a rational basis for comparisons between different biomass feedstocks. For example, a single-stage autohydrolysis for log Ro gravity = 3.75 yields pretreated sugarcane bagasse and corn straw having a xylan number between 6-7%, while with typical varieties of wheat straw, the resulting xylan number of pretreated feedstock is around 10%.

[0129] It may be advantageous for biomass feedstocks to be pretreated to low gravity, where the xylan number of the pretreated feedstock is greater than 10%. This low gravity level corresponds to a process in which the total hemicellulose content of the feedstock before pretreatment is minimized, as it is either solubilized or irretrievably lost during pretreatment. At xylan numbers of 10% or more, with typical strains of wheat straw, sugarcane bagasse, sweet sorghum bagasse, corn straw, and empty fruit bunches (from palm oil), at least 60% of the original C5 content of the feedstock can be recovered after pretreatment. Petition 870190096087, dated 09 / 26 / 2019, page 38 / 149 32 / 116 of single-stage autohydrolysis, where both the xylan in the solid fraction and the xylose and soluble xylo-oligomers in the liquid fraction are accounted for. High final C5 monomer yields of at least 55% theoretical, at least 60%, or at least 65% can be obtained without appreciable loss of C6 monomer yields after enzymatic hydrolysis of the pretreated feedstocks to very low gravity by single-stage autohydrolysis. At very low gravity levels, a large fraction of the hemicellulose content of the feedstock remains within the solid fraction after pretreatment, where it can subsequently be hydrolyzed to C5 monomers with high recovery using enzymatic hydrolysis.

[0130] It should be noted that reports relating to xylose recovery are often expressed in terms that may not be directly comparable with the xylose recoveries reported in this document. For example, reported xylose recoveries often refer only to xylose recovery from pretreated biomass, not expressed as a percentage of the original hemicellulose content of the feed before pretreatment.

[0131] Another surprising characteristic of biomass that has been pretreated by single-stage autohydrolysis at very low gravity levels is that the concentrations of pretreatment byproducts that serve as inhibitors of fermentative organisms are maintained at very low levels. Consequently, it is often possible to use hydrolyzed biomass obtained by methods of the invention directly in fermentations, without the need for any washing or other detoxification step. As is well known Petition 870190096087, dated 09 / 26 / 2019, pp. 39 / 149 33 / 116 known in the art, the hydrothermal autohydrolysis pretreatment typically produces a variety of soluble byproducts that act as fermentation inhibitors, insofar as they inhibit the growth and / or metabolism of fermentative organisms. Different fermentation inhibitors are produced in different quantities, depending on the properties of the lignocellulosic feedstock and the severity of the pretreatment. At least three categories of fermentation inhibitors are typically formed during autohydrolysis pretreatment: (1) furans, mainly 2-furfural and 5-hydroxymethylfurfural (5-HMF), which are degradation products from mono- or oligosaccharides; (2) monomeric phenols, which are degradation products of the lignin structure; and (3) small organic acids, mainly acetic acid, which originate from acetyl groups in hemicellulose and lignin.The mixture of different inhibitors is believed to act synergistically to inhibit microorganisms such as yeasts and E. coli.

[0132] In some embodiments, the pretreated biomass is subjected to rapid evaporation using methods well known in the art in order to reduce the levels of volatile inhibitors, most notably furfural. When autohydrolysis is used with typical biomass feedstock strains, such as wheat straw, sweet sorghum bagasse, sugarcane bagasse, corn straw and empty fruit bunches, pretreated with xylan at 10% or more, the levels of acetic acid and furfural are believed to be potentially inhibitory to fermentative organisms. Where biomass feedstocks are pretreated in DM at 35% Petition 870190096087, dated 09 / 26 / 2019, p. 40 / 149 34 / 116 or more for xylan number at 10% or more, and where the solid fraction is subsequently enzymatically hydrolyzed to 25% or less DM, with the addition of water to adjust the DM but without washing steps, furfural levels in the hydrolysate can typically be maintained below 3 g / kg and acetic acid levels below 9 g / kg. These levels are typically acceptable for yeast fermentations using specialized strains. During enzymatic hydrolysis, some additional acetic acid may be released from the degradation of hemicellulose in the solid fraction. In some embodiments, it may be advantageous to remove some acetic acid content from the liquid fraction and / or the hydrolyzed solid fraction using electrodialysis and / or other methods known in the art.

[0133] Lignocellulosic biomass, such as soft lignocellulosic biomass feedstocks, such as agricultural residues such as cereal straw, e.g. wheat, barley, rye or sorghum, grass, leaves, sugarcane bagasse, sweet sorghum bagasse, corn straw and empty fruit bunches etc. are pretreated, usually preceded by a cleaning step, where for example sand, stones, foreign objects and the like are removed, and / or after a single-stage autohydrolysis until the xylan number is 10% or higher, typically comprise a small component of C6 monomers (1x), mainly glucose with some other sugars; a larger component of soluble C6 oligomers (about 2x - 7x); a larger component of C5 monomers (about 4x - 8x), mainly xylose with some arabinose and other sugars; and a much larger component of soluble xylo-oligomers (approximately 18x - 30x) where nx refers to the number of sugar units, i.e., 1x = Petition 870190096087, dated 09 / 26 / 2019, page 41 / 149 35 / 116 monomer, 2x = dimer, and so on. Soluble xylo-oligomers typically include xylohexose, xylopentose, xylotetraose, xylotriose, and xylobiose, with some higher-chain oligomers. Xylo-oligomers can also be modified, such as esterified.

[0134] Different feedstocks can be pretreated using single-stage autohydrolysis to, for example, xylan counts of 10% or higher by a variety of different combinations of residence times and reactor temperatures. An expert in the art will quickly determine, through routine experimentation, an appropriate pretreatment routine to apply with any feedstock, using any reactor, and with any biomass reactor loading and reactor discharge system.Where raw materials are pre-treated using a continuous reactor, loaded either by a sluice system or by a screw feeder, and discharged either by a particle pump sluice system or a hydrocyclone system, a very low xylan number of 10% or higher can, for example, be obtained using typical strains of wheat straw or empty fruit bunches at a temperature of 180 °C and a reactor residence time of 24 minutes. For typical biomass feedstocks, such as soft lignocellulosic biomass from commonly used varieties of corn straw, sugarcane bagasse, and sweet sorghum bagasse, it is believed that low gravities, such as xylan number > 10%, can be achieved at around 180 °C and a reactor residence time of around 12 minutes, or using a temperature of around 175 °C and a reactor residence time of around 12 minutes. Petition 870190096087, dated 09 / 26 / 2019, page 42 / 14936 / 116 of 17 minutes. It will be readily understood by someone skilled in the art that the residence times and temperatures can be adjusted to achieve comparable Ro severity levels. After pretreatment, the pretreated biomass is separated into a solid fraction and a liquid fraction by a solid / liquid separation step. It will be readily understood that the solid and liquid fractions can be further subdivided or processed. In some embodiments, the biomass can be removed from a pretreatment reactor simultaneously with the solid / liquid separation. In some embodiments, the pretreated biomass is subjected to a liquid / solid separation step after being discharged from the reactor, typically using a simple and low-cost screw press system, in order to generate a solid fraction and a liquid fraction.The activities of the cellulase enzyme are inhibited by the liquid fraction, mainly due to the xylo-oligomer content but possibly also due to the phenol content and / or other as yet unidentified compounds. It may be advantageous to achieve the highest possible levels of dry matter content in the solid fraction or, alternatively, to remove as much dissolved solids as possible from the solid fraction. In some embodiments, solid / liquid separation achieves a solid fraction having a DM content of at least 40%, at least 45%, at least 50%, or at least 55%. Solid / liquid separation using common screw press systems can typically achieve DM levels as high as 50% in the solid fraction, especially when the biomass feedstock has been pre-treated and processed in such a way that the fiber structure is maintained. Petition 870190096087, dated 09 / 26 / 2019, page 43 / 149 37 / 116

[0135] In some embodiments, it may be advantageous to incur higher factory capital expenditures in order to achieve more effective solid / liquid separation, for example, by using a membrane filter press system. In some embodiments, dissolved solids can be removed from a solid fraction by serial washing and pressing or by displacement washing techniques known in the pulp and paper art. In some embodiments, either by direct solid / liquid separation or by some combination of washing and solid / liquid separation, the dissolved solids content of the solid fraction is reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75%.Enzymatic hydrolysis of pretreated raw materials with a xylan number of 10% or higher can typically be carried out using commercially viable enzymes, without the need for specific washing or detoxification steps, where the solid fraction is pressed to at least 40% DM or where the dissolved solids content is reduced by at least 50%.

[0136] In some embodiments, the hydrothermal pretreatment is carried out without supplemental oxygen as required for wet oxidation pretreatments, or without the addition of organic solvent as required for organosolvent pretreatment, or without the use of microwave heating as required for microwave pretreatments. In some embodiments, the hydrothermal pretreatment is carried out at temperatures of 140 °C or higher, or 150 °C or higher, or 160 °C or higher, or between 160 and 200 °C, or between 170 and 190 °C, or 180 °C or lower, Petition 870190096087, dated 09 / 26 / 2019, pp. 44 / 149 38 / 116 or 170 °C or less. In some embodiments, some C5 content may be removed by an immersion step prior to pressurized pretreatment. In some embodiments, the single reactor may be configured to heat biomass to a single target temperature. Alternatively, the single reactor may be configured to effect a temperature gradient within the reactor, so that the biomass is exposed, during a single pass, to more than one temperature region. In some embodiments, it may be advantageous to partially remove some solubilized biomass components from within the pressurized reactor during the course of pretreatment.

[0137] Suitable hydrothermal pretreatment reactors typically include most known pulp reactors in the pulp and paper industry. In some embodiments, hydrothermal pretreatment is administered by steam in a reactor pressurized to 10 bar or less, or 12 bar or less, or 4 bar or more, or 8 bar or more, or between 8 and 18 bar, or between 18 and 20 bar. In some embodiments, the pretreatment reactor is configured for a continuous influx of raw material.

[0138] In some embodiments, wet biomass is transported through the reactor under pressure for a certain duration or residence time. The residence time is advantageously kept short in order to facilitate a higher biomass yield. However, the severity of the pretreatment obtained is determined by both the temperature and the residence time. The temperature during the hydrothermal pretreatment is advantageously kept lower, not only because the methods of the invention Petition 870190096087, dated 09 / 26 / 2019, pp. 45 / 149 39 / 116 seek to achieve a very low pretreatment gravity, but also because lower temperatures can be achieved using lower vapor pressures. Insofar as the pretreatment temperature can be 180 °C or lower and, consequently, the saturated vapor pressures remain at 10 bar or less, a lower tendency for corrosion is experienced and much lower pressure fittings and steel compositions can be used, which reduces plant capital costs. In some embodiments, the reactor is configured to heat the biomass to a single target temperature between 160 and 200 °C, or between 170 and 190 °C.

[0139] The dwell times in some modalities are less than 60, less than 30, less than 20, less than 15, less than 14, less than 13, less than 12, less than 10, less than 8 or less than 5 minutes.Additional options related to appropriate lengths of stay may be disclosed elsewhere.

[0140] Biomass feedstocks, such as lignocellulosic biomass, can be fed at atmospheric pressure into a pressurized reactor by a variety of means. In some embodiments, a sluice-type particle pump system can be used to feed biomass feedstocks, such as the systems described in, for example, WO 2003 / 013714 or WO 2011 / 024145, both of which are incorporated herein by reference in their entirety. In some embodiments, it may be advantageous to feed a pretreatment reactor using a screw feeder.

[0141] Pretreated biomass can be discharged to Petition 870190096087, dated 09 / 26 / 2019, pp. 46 / 149 40 / 116 from a reactor pressurized by a variety of means. In some embodiments, the pretreated biomass is discharged in a manner that preserves the fiber structure of the material. Preserving the fiber structure of the pretreated biomass is advantageous because it allows the solid fraction of the pretreated material to be compressed during solid / liquid separation to comparatively high levels of dry matter using common screw press equipment, thus avoiding the added expense and complexity of membrane filter press systems. The fiber structure can be maintained by removing the feedstock from the pressurized reactor in a non-explosive manner. In some embodiments, non-explosive removal can be achieved and the fiber structure thus maintained using sluice-type systems, such as those described previously.In some embodiments, non-explosive removal can be achieved and the fiber structure thus preserved using a hydrocyclone removal system, such as those described in WO 2009 / 147512, which are incorporated in their entirety herein by reference.

[0142] In some embodiments, pretreated biomass can be removed from a pressurized pretreatment reactor using steam explosion, which involves the explosive release of the pretreated material. Steam-exploded pretreated biomass does not retain its fiber structure and consequently requires more elaborate solid / liquid separation systems in order to achieve dry matter content comparable to dry matter contents that can be obtained using, for example, systems Petition 870190096087, dated 09 / 26 / 2019, pp. 47 / 149 41 / 116 conventional screw presses with pre-treated biomass that retain their fiber structure.

[0143] As will be readily understood by one skilled in the art, the composition of enzyme mixtures suitable for practicing the methods practiced of the invention may vary within comparatively wide limits. Suitable enzyme preparations include commercially available xylanase preparations and cellulase preparations optimized for lignocellulosic biomass conversion. The selection and modification of enzyme mixtures during optimization may include genetic engineering techniques. Commercially available cellulase preparations optimized for lignocellulosic biomass conversion are typically identified by the manufacturer and / or supplier as such. These are typically distinct from commercially available cellulase preparations for general use or optimized for use in the production of animal feed, food, textile detergents, or in the paper industry.In some embodiments, a commercially available cellulase preparation optimized for lignocellulosic biomass conversion is used, such as, for example, one provided by GENENCOR™ (currently DuPont), DSM, or NOVOZYMES™. Typically, such compositions comprise cellulase(s) and / or hemicellulase(s), such as one or more exoglucanases, endoglucanases, endoxylanases, xylosidases, acetylxylan esterases, and beta-glucosidases, including any combination thereof. Such enzymes may, for example, be isolated from fermentations of genetically modified Trichoderma reesei, such as, for example, the commercial cellulase preparation sold under the brand name. Petition 870190096087, dated 09 / 26 / 2019, pp. 48 / 149 42 / 116 registered ACCELLERASE TRIO™.

[0144] In some embodiments, a commercially available cellulase preparation optimized for lignocellulosic biomass conversion is used and provided by NOVOZYMES™ and comprising exoglucanases, endoglucanases, endoxylanases, xylosidases, acetylxylan esterases and beta glucosidases, such as, for example, the commercial cellulase preparations sold under either of the trade names Cellic® CTec2 or Cellic® CTec3.

[0145] It is believed that the specific enzymatic activities present in commercially available cellulase preparations optimized for lignocellulosic biomass conversion can be analyzed in detail using methods known in the art.

[0146] It is believed that three different cellulase preparations, Accellerase® TRIO™ from DuPont (and / or GENENCOR) and Cellic® CTec2 and Cellic® CTec3 from Novozymes™, are effective at enzyme dosage levels within a range suggested by the manufacturers.

[0147] Suitable cellulase preparations optimized for lignocellulosic biomass conversion typically comprise multiple enzymatic activities, including exoglucanase, endoglucanase, hemicellulases (including xylanases), and β-glucosidases. Enzyme preparations may be expressed in different activities / units, such as carboxymethylcellulase units (CMC U), acid birch wood xylanase units (ABXU), and pNP-glucosidase units (pNPG U). For example, ACCELLERASE TRIO™ comprises: endoglucanase activity: 2000 - 2600 CMC U / g, Petition 870190096087, dated 09 / 26 / 2019, pp. 49 / 149 43 / 116 xylanase activity: > 3000 ABX U / g, and beta-glucosidase activity: > 2000 pNPG U / g; wherein one unit of CMC activity releases 1 gmol of reducing sugars (expressed as glucose equivalents) in one minute at 50 °C and pH 4.8; one ABX unit defined as the amount of enzyme required to generate 1 gmol of xylose reducing sugar equivalents per minute at 50 °C and pH 5.3; and one pNPG unit indicates 1 gmol of nitrophenol released from para-nitrophenylBD-glucopyranoside per minute at 50 °C and pH 4.8.

[0148] Based on information available in the public domain, it is believed that a person skilled in the art is able to provide suitable enzymatic preparations for enzymatic hydrolysis according to the present invention, in particular for any of the enzymatic hydrolysis steps disclosed herein, such as fiber hydrolysis, fiber cake hydrolysis and MSH (mixed sugar hydrolysis).

[0149] The present invention appears well suited for industrial applications, including large-scale industrial applications. In some embodiments, the methods of the invention are practiced using at least about 100, 200, 500 kg of biomass feedstock, or at least 1,000 kg, or at least 5,000 kg.

[0150] In a first aspect, the present invention relates to a method for providing a C5 / C6 product from a lignocellulosic material comprising the steps of: a) Pre-treatment of the lignocellulosic material; b) Solid / liquid separation of the pretreated lignocellulosic material from step (a) into a first solid fraction and a first liquid fraction; Petition 870190096087, dated 09 / 26 / 2019, p. 50 / 149 44 / 116 c) Enzymatic hydrolysis of the fiber from the aforementioned first solid fraction of step (b) through the use of an enzyme composition capable of degrading lignocellulosic material, thereby providing a C5 / C6 fiber paste comprising C5 and / or C6 sugars; d) Solid / liquid separation of the C5 / C6 fiber paste from step (c) into a second solid fraction and a second liquid fraction; and optionally (e) Combination of the aforementioned first liquid fraction and the aforementioned second liquid fraction for the enzymatic hydrolysis of mixed sugar (MSH), through which an MSH C5 / C6 product is provided.

[0151] In some embodiments, the said method may also comprise an additional step (f): Enzymatic hydrolysis of the fiber cake of said second solid fraction of step (d) in order to obtain a C5 / C6 paste product.

[0152] According to the present invention, sustainable lignocellulosic biomass may comprise soft lignocellulosic biomass, such as wheat straw, corn straw, corn cobs, empty fruit bunches, rice straw, oat straw, barley straw, canola straw, rye straw, sorghum, sweet sorghum, soybean straw, spelt grass, Bermuda grass and other grasses, bagasse, beet pulp, corn fiber or any combination thereof. Lignocellulosic biomass may comprise other lignocellulosic materials, such as wood, wood chips, but also paper, newsprint, cardboard or other municipal or office waste. Lignocellulosic biomass may be used as a mixture of materials derived from different raw materials, which may be Petition 870190096087, dated 09 / 26 / 2019, p. 51 / 149 45 / 116 fresh, partially dried, fully dried, or any combination thereof. Lignocellulosic biomass is commonly considered a waste product.

[0153] In some embodiments, the lignocellulosic material is lignocellulosic biomass, e.g., agricultural residues such as one or more wheat straw, corn straw, corn cobs, empty fruit bunches, rice straw, oat straw, barley straw, canola straw, rye straw, sorghum, sweet sorghum, soybean straw, swarf grass, Bermuda grass and other grasses, bagasse, beet pulp, corn fiber or any combination thereof. In some embodiments, the lignocellulosic biomass may also be predominantly or entirely ensiled biomass, or comprise ensiled biomass, such as at least 5, 10, 25, 50%, 75%, 90%, 95%, 99% or more of ensiled biomass.

[0154] In some embodiments, the lignocellulosic material is not soft lignocellulosic biomass. Examples of such non-soft lignocellulosic biomass include, for example, wood, wood chips, bark, branches, but also paper, newsprint, cardboard or even municipal waste, such as separated or unseparated municipal waste, or office waste. In some embodiments, the lignocellulosic biomass may also be predominantly or entirely non-soft lignocellulosic biomass, or comprise non-soft lignocellulosic biomass, such as at least 5, 10, 25, 50%, or more than 50% of non-soft lignocellulosic biomass.

[0155] In some embodiments, the pretreatment is carried out at a dry matter (DM) content in the range of 5-80%, such as 10-70%, such as 20-60%, or such as 30-50%, or at Petition 870190096087, dated 09 / 26 / 2019, p. 52 / 149 46 / 116 a DM content of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or with a DM content greater than 80%. In some other modalities, pretreatment is performed at a DM content of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, or even 70-80%. In some additional modalities, pretreatment is performed at a DM content of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or with a DM content greater than 80%.

[0156] In some embodiments, the pretreatment is carried out at low, medium, or high gravity. In some embodiments, the pretreatment is carried out under conditions that provide a xylan number > 10%, 6-10%, or <6%. It is believed that relevant advantages according to the invention can also be obtained at medium or high pretreatment gravities. In some embodiments, the biomass feedstock is pretreated at medium gravity, such that the pretreated biomass is characterized by having a xylan number of 6-10%. In some embodiments, the biomass is pretreated to a xylan number of 6-7%, 7-8%, or 9-10%. In further embodiments, the biomass feedstock is pretreated at medium gravity, such that the pretreated biomass is characterized by having a xylan number of less than 6%. In some embodiments, the biomass is pretreated to a xylan number below 6%, 5% or lower, 4% or lower, 3% or lower, 2% or lower, or 1% or lower.

[0157] In some embodiments, enzymatic fiber hydrolysis, fiber bolus hydrolysis and / or MSH hydrolysis is / are carried out over a period of at least 6h, 12h, 24h, Petition 870190096087, dated 09 / 26 / 2019, p. 53 / 149 47 / 116 48h or 72h, such as 6-120h, 12-100h, or 48-96h, or approximately 12h, 24h, 48h, 72h, 96h, or 120h.

[0158] In some embodiments, the enzymatic hydrolysis of the fiber, the hydrolysis of the fiber cake and / or MSH is / are carried out at a pH in the range of at least pH 3.0, such as in the pH range of 3.0-6.0, such as pH 4.0-5.5 and / or such as pH 4.2-5.4.

[0159] In some embodiments, the enzymatic hydrolysis of fiber, fiber cake hydrolysis and / or MSH is / are carried out at a pH of approximately 4.2, 4.5, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3 or 5.4.

[0160] In some embodiments, the enzymatic hydrolysis of the fiber, the hydrolysis of the fiber cake and / or MSH is / are carried out at a temperature in the range of 30-70 °C, 40-65 °C, 50-62 °C, or 55-60 °C and / or approximately 40 °C, 42 °C, 44 °C, 46 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C, 62 °C, 64 °C, 66 °C, 68 °C, or 70 °C.

[0161] In some embodiments, the enzymatic hydrolysis of the fiber, the hydrolysis of the fiber cake and / or MSH is / are carried out at an appropriate DM content, such as a DM content of at least 10%, such as 15%. In some embodiments, the DM content is about 15-45%, 20-40%, 25-35% and / or at a DM content of about 15%, 20%, 25%, 30%, 35% or 40%. In some embodiments, the DM content is about 40% or higher.

[0162] In some embodiments, the enzyme composition capable of degrading lignocellulosic material comprises a cellulase and / or a hemicellulase.

[0163] In some embodiments, the enzyme composition capable of degrading lignocellulosic material comprises a Petition 870190096087, dated 09 / 26 / 2019, page 54 / 149 48 / 116 mixture of cellulase(s) and / or hemicellulase(s).

[0164] In some embodiments, hemicellulase is or comprises one or more xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), esterase(s) and any combination thereof.

[0165] Sugars, such as C5 and / or C6 mono-, oligo- and / or polymers, can be modified, such as esterified, for example, comprising ferulic acid. Ferulic acid can be efficiently released by esterase(s), such as a ferulic acid esterase, for example FAE-III from Aspergillus niger (see Faulds and Williamson, Appl. Microbiol. Biotechnol. Nov 1995; 43(6): 1082-7), which released ferulic acid from wheat bran. Said release was improved in the presence of a xylanase, such as a xylanase from Trichoderma viride. Thus, in some embodiments, an esterase, such as a ferulic acid esterase, and optionally cellulase and / or at least one xylanase are added in an enzymatic hydrolysis step, such as any fiber hydrolysis, fiber cake hydrolysis, and / or mixed sugar hydrolysis.

[0166] In some embodiments, esterases comprise one or more acetylsterases and / or feroyl esterases.

[0167] In some embodiments, the enzyme composition capable of degrading lignocellulosic material comprises one or more endocellulase(s), endoglucanase(s), exocellulase(s), exoglucanase(s), endoxylanase(s), acetylxylan esterase(s), xylosidase(s), β-glucosidase and any combination thereof.

[0168] In some modalities, the aforementioned method for Petition 870190096087, dated 09 / 26 / 2019, p. 55 / 149 49 / 116 providing a C5 / C6 product from a lignocellulosic material comprises step (e), namely, combining said first liquid fraction and said second liquid fraction for enzymatic hydrolysis of mixed sugar (MSH). By combining said first liquid fraction and said second liquid fraction and enzymatically hydrolyzing the mixed sugar, an MSH C5 / C6 product is provided.

[0169] In some forms, hemicellulase(s) is / are also present in step (e).

[0170] In some embodiments, the hemicellulase(s) present in step (e) comprise(s) xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), esterase(s), acetylesterases, feroyl esterase(s) and any combination thereof.

[0171] In some embodiments, all or at least a fraction of the hemicellulase(s) present in step (e) was / were added in step (c).

[0172] In some embodiments, one or more hemicellulase(s) are added in step (e).

[0173] In some embodiments, one or more additional enzyme(s) is / are added in step (e). In some other additional embodiments, the additional enzyme(s) is / are not essentially present (e.g., less than 1% of the total enzyme activity, present only as a minor secondary activity and / or contamination, etc.) in the enzyme composition capable of degrading lignocellulosic material added in step (c). In other embodiments, the additional enzyme(s) is / are one or Petition 870190096087, dated 09 / 26 / 2019, p. 56 / 149 50 / 116 more than: hemicellulase(s), xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), esterase(s), acetylesterases, feroyl esterase(s) and any combination thereof.

[0174] In some embodiments, step (e) comprises an ultrafiltration step for separating the hydrolyzed sugars present in the permeate from the hemicellulase(s) present in the retentate, in order to recycle at least part of the hemicellulase(s). The cost of the enzymes constitutes a significant proportion of the variable costs of the method, and the ultrafiltration step limits this cost by recycling the hemicellulase(s). Thus, in some embodiments, said step (e) comprises or is followed by an ultrafiltration step (j) to recycle enzymes present after MSH. In further embodiments, the ultrafiltration step (j) is adapted to allow the recycling of at least 30% (w / w), 50% (w / w), 75% (w / w), 80% (w / w) or 90% (w / w) of the enzyme activity.

[0175] In some embodiments, cellulase(s) is / are also present in step (f). In some embodiments, at least one cellulase has been added, such that the said second solid fraction in step (f) comprises at least one cellulase. In some embodiments, the cellulase(s) present in step (f) was / were added in step (c). The cellulases bind to the fibers in the solid fraction, and thus the addition of cellulase to the solid fraction of step (b) can serve to complete the hydrolysis performed in both step (c) (fiber hydrolysis) and step (f) (fiber cake hydrolysis). Therefore, in some embodiments, all, or essentially all, of the cellulase Petition 870190096087, dated 09 / 26 / 2019, page 57 / 149 51 / 116 present in step (f) was added in step (c).

[0176] In some embodiments, at least a fraction of the cellulase used in step (f) was added in step (c).

[0177] In some embodiments, one or more cellulase(s) and optionally hemicellulase(s) is / are added in step (f).

[0178] In some embodiments, cellulase(s) and / or hemicellulase(s) are added in step (c), such as by adding a mixture comprising one or more cellulase and one or more hemicellulase.

[0179] In some embodiments, MSH and / or fiber bolus hydrolysis is performed without the addition of one or more enzyme(s).

[0180] In some embodiments, MSH and / or fiber cake hydrolysis is performed without the addition of one or more cellulose(s) and / or one or more hemicellulose(s).

[0181] In some embodiments, the said method for providing a C5 / C6 product from a lignocellulosic material comprises step (g), namely the solid / liquid separation of the C5 / C6 product in paste form from step (f) into a third solid fraction and a liquid C5 / C6 product.

[0182] In some embodiments, the second liquid fraction has a lower inhibitor concentration than the first liquid fraction.

[0183] In some embodiments, the second liquid fraction has a lower concentration of inhibitor than the MSH C5 / C6 product.

[0184] In some embodiments, the C5 / C6 paste product has a lower inhibitor concentration than the MSH C5 / C6 product. Petition 870190096087, dated 09 / 26 / 2019, page 58 / 149 52 / 116

[0185] In some embodiments, the said method for providing a C5 / C6 product from a lignocellulosic material comprises step (k), that is, combining at least one portion of the MSH C5 / C6 product with at least one portion of one or more of: the C5 / C6 paste product from step (f), the liquid C5 / C6 product from step (g), and / or the second liquid fraction from step (d) in order to obtain a combined C5 / C6 product.

[0186] In some embodiments, the combined C5 / C6 product consists or essentially consists of the MSH C5 / C6 product and the paste C5 / C6 product from step (f); the MSH C5 / C6 product and the liquid C5 / C6 product from step (g); or the MSH C5 / C6 product and the second liquid fraction from step (d).

[0187] In some embodiments, the combined C5 / C6 product has a ratio of MSH C5 / C6 product to liquid C5 / C6 product; C5 / C6 product paste or second liquid fraction of step (d) in the range of 100:0.1-0.1:100 (w / w), such as 10:0.1-0.1:10 (w / w), or such as 10:1-1:10 (w / w), such as 5:1-1:5 (w / w); such as 4:1-1:4 (w / w), such as 3:1-1:3 (w / w), such as 2.5-1:2.5 (w / w), such as 2:1-1:2 (w / w) or such as 1.5-1:1-1.5 (w / w).

[0188] In some embodiments, the combined C5 / C6 product has a ratio of MSH C5 / C6 product to liquid C5 / C6 product; paste of product C5 / C6 or second liquid fraction of step (d) in the range of 50:1 (w / w), 25:1 (w / w), 20:1 (w / w), 15:1 (w / w), 10:1 (w / w), 9:1 (w / w), 8:1 (w / w), 7:1 (w / w), 6:1 (w / w), 5:1 (w / w), 4:1 (w / w), 3:1 (w / w), 2:1 (w / w), 1:1 (w / w), 1:1.5 (w / w), 1:2 (w / w), 1:2.5 (w / w), 1:3 (w / w), 1:4 (w / w), 1:5 (w / w), 1:6 (w / w), 1:7 (w / w), 1:8 (w / w), 1:9 (w / w), 1:10 (p / p), 1:15 (p / p), 1:20 (p / p), 1:25 (p / p), or 1:50 (p / p). In Petition 870190096087, dated 09 / 26 / 2019, p. 59 / 149 53 / 116 some preferred modalities, the aforementioned ratio is, or is approximately 1:1.5 (p / p), 1:2 (p / p), or 1:2.5 (p / p).

[0189] In some embodiments, the aforementioned method for providing a C5 / C6 product from a lignocellulosic material comprises a lignin recovery step. This step may comprise one or more of: water removal, compaction and / or granulation.

[0190] In some embodiments, the aforementioned lignin recovery is carried out on the second or third solid fraction provided in steps (d) or (g).

[0191] In some embodiments, any C5 / C6 product is a C5+C6 product, that is, a product comprising C5 and C6 carbohydrates, such as xylose and glucose, including structural analogues, isomers and / or derivatives thereof.

[0192] In some embodiments, the C5+C6 product comprises glucose and xylose.

[0193] In a second aspect, the present invention relates to a method for providing a fermentation product wherein said method comprises the steps of: (m) Supply of at least one C5 / C6 product according to the method of any of the preceding embodiments according to the first aspect; and n) Supply of the fermentation product by means of fermentation of said C5 / C6 product with a microorganism.

[0194] In some embodiments, the C5 / C6 product comprises one or more of: MSH C5 / C6 product, C5 / C6 paste product, liquid C5 / C6 product, combined C5 / C6 product, first liquid fraction or second liquid fraction, and any combination thereof. Petition 870190096087, dated 09 / 26 / 2019, page 60 / 149 54 / 116

[0195] Normally, the fermentation product is provided in a fermentation broth. Thus, in some embodiments, the said method of providing a fermentation product comprises an additional step (the): recovering said fermentation product from a fermentation broth.

[0196] In some embodiments, the aforementioned method comprises step (p): recovering lignin from spent fermentation broth and / or a proportionate fraction in steps (n) or (o).

[0197] In some embodiments, fermentation is carried out in at least a first and a second fermentation step, in which a first and a second fermentation substrate are fermented.

[0198] Providing two fermentation substrates with different inhibitors and / or concentrations of fermentation inhibitors can be advantageous, particularly useful when the fermentation is carried out by a microorganism sensitive to said inhibitors, which are predominantly present in the C6 + C5 product obtained in step b). An increase in fermentation productivity can thus be achieved, for example, through a shorter fermentation duration and / or higher product yield.

[0199] In particular, when the combined fractions of the MSH C5 / C6 + paste product or liquid C5 / C6 product also comprise an excessively high inhibitor concentration, the present invention provides an alternative that does not require dilution of the fermentation substrate with water, which is undesirable. Such dilution with water can be carried out in a first fermentation, such as a fermentation Petition 870190096087, dated 09 / 26 / 2019, page 61 / 149 55 / 116 discontinuous, before the fermentation of the combined fractions, for example, in a fed-batch fermentation.

[0200] In some embodiments, the present invention relates to a method, as defined in the previous embodiments, in which said fermentation is carried out by first discontinuous fermentation of the liquid C5 / C6 product obtained, for example, in step (g) or of the C5 / C6 paste product obtained in step (f) and, subsequently, discontinuous fermentation of the MSH C5 / C6 product obtained in step (e) or (j), generally in combination with additional quantities of the liquid C5 / C6 product obtained, for example, in step (g) or of the C5 / C6 paste product obtained in step (f).

[0201] Additional embodiments relating to two-step fermentations are also presented below.

[0202] In a third aspect, the present invention relates to a two-step fermentation method comprising the steps of: aa) Pre-treatment of lignocellulosic material; bb) Solid / liquid separation of the pre-treated lignocellulosic material from step (aa) into a first solid fraction and a first liquid fraction; cc) Enzymatic hydrolysis of the fiber from the aforementioned first solid fraction of step (bb) through the use of an enzyme composition capable of degrading lignocellulosic material, thereby providing a C5 / C6 fiber paste; dd) Solid / liquid separation of the C5 / C6 fiber paste from step (cc) into a second solid fraction and a second liquid fraction; ee) Enzymatic hydrolysis of mixed sugar (MSH) from a mixture of the first liquid fraction from step (bb) and the fiber paste Petition 870190096087, dated 09 / 26 / 2019, page 62 / 149 56 / 116 C5 / C6 of step (cc), or the first liquid fraction of step (bb) and the second liquid fraction of step (dd), thus providing an MSH C5 / C6 product; ff) Provision of a first fermentation substrate comprising at least a portion of the C5 / C6 fiber paste and / or the second liquid fraction; gg) Provision of a second fermentation substrate comprising at least a portion of the MSH C5 / C6 product; hh) Fermentation of the first fermentation substrate in a first fermentation with a microorganism; and ii) Fermentation of the second fermentation substrate in a subsequent second fermentation;

[0203] where the (dd) step is optional.

[0204] In some forms, any of the steps aa, bb, cc, dd and / or ee may correspond to steps a, b, c, de and / or e according to any of the previous aspects, respectively.

[0205] In some embodiments, the first fermentation substrate has a significantly lower inhibitor concentration than the second fermentation substrate.

[0206] In some forms, the first fermentation is a batch or fed-batch fermentation.

[0207] In some embodiments, the first fermentation is carried out by providing a first fermentation substrate comprising: (i) the second liquid fraction provided in step (d) or (dd); (ii) the C5 / C6 fiber paste provided in step (c) or (cc); and / or (iii) the C5 / C6 product obtained in step (f), i.e., the liquid C5 / C6 product or the C5 / C6 paste product. Petition 870190096087, dated 09 / 26 / 2019, page 63 / 149 57 / 116

[0208] In some embodiments, the first fermentation is carried out by providing a first fermentation substrate consisting essentially of: (i) the second liquid fraction provided in step (d) or (dd); (ii) the C5 / C6 fiber paste provided in step (c) or (cc); and / or (iii) the C5 / C6 product obtained in step (f), i.e., the liquid C5 / C6 product or the C5 / C6 paste product.

[0209] In some embodiments, the first fermentation substrate comprises or consists essentially of a mixture of the second liquid fraction and the C5 / C6 product obtained in step (f), i.e., the liquid C5 / C6 product or the paste-like C5 / C6 product.

[0210] In some embodiments, the ratio between the second liquid fraction and the C5 / C6 product is in the range of 100:0.1-0.1:100 (w / w), such as 10:0.1-0.1:10 (w / w) or such as 10:1-1:10 (w / w).

[0211] In some embodiments, the ratio of the second liquid fraction and the C5 / C6 product is in the range of approximately 50:1 (w / w), 25:1 (w / w), 20:1 (w / w), 15:1 (w / w), 10:1 (w / w), 9:1 (w / w), 8:1 (w / w), 7:1 (w / w), 6:1 (w / w), 5:1 (w / w), 4:1 (w / w), 3:1 (w / w), 2:1 (w / w), 1:1 (w / w), 1:2 (w / w), 1:3 (w / w), 1:4 (w / w), 1:5 (w / w), 1:6 (w / w), 1:7 (w / w), 1:8 (w / w), 1:9 (w / w), 1:10 (w / w), 1:15 (w / w), 1:20 (w / w), 1:25 (w / w), or 1:50 (w / w).

[0212] In some forms, the first fermentation substrate is provided essentially without dilution with process water.

[0213] In some embodiments, the second fermentation is a fed-batch fermentation or a continuous fermentation, optionally carried out in the same fermenter as Petition 870190096087, dated 09 / 26 / 2019, pp. 64 / 149 58 / 116 first fermentation.

[0214] In some embodiments, the aforementioned fed-batch fermentation is carried out using linear or exponential feeding.

[0215] In some forms, the second fermentation is carried out with the same microorganisms as in the first fermentation.

[0216] In some embodiments, the second fermentation is carried out by providing a second fermentation substrate comprising or consisting essentially of a mixture of the C5 / C6 product obtained in step (f) (i.e., the liquid C5 / C6 product or the paste C5 / C6 product) and the C5 / C6 product obtained from step (e) (i.e., the MSH C5 / C6 product).

[0217] In some embodiments, the ratio between the liquid C5 / C6 product or the paste C5 / C6 product and the C5 / C6 product obtained from step (e) (i.e., MSH C5 / C6 product) is in the range of 100:0.1-0.1:100 (w / w), such as 10:0.1-0.1:10 (w / w), or such as 10:1-1:10 (w / w), such as 5:1-1:5 (w / w); such as 4:1-1:4 (w / w), such as 3:1-1:3 (w / w), such as 2.5-1:2.5 (w / w), such as 2:1-1:2 (w / w) or such as 1.5-1:11.5 (w / w). In some embodiments, the said ratio is 2.5-1:2.5 (w / w).

[0218] In some embodiments, the ratio between the liquid C5 / C6 product or the paste C5 / C6 product and the C5 / C6 product obtained from step (e) (i.e., MSH C5 / C6 product) is in the range of about 50:1 (w / w), 25:1 (w / w), 20:1 (w / w), 15:1 (w / w), 10:1 (w / w), 9:1 (w / w), 8:1 (w / w), 7:1 (w / w), 6:1 (w / w), 5:1 (w / w), 4:1 (w / w), 3:1 (w / w), 2:1 (w / w), 1:1 (w / w), 1:1.5 (w / w), 1:2 (w / w), 1:25 (w / w), 1:3 (w / w), 1:4 (w / w), 1:5 (b / w), 1:6 (b / w), 1:7 (b / w), 1:8 (b / w), 1:9 (b / w), 1:10 Petition 870190096087, dated 09 / 26 / 2019, p. 65 / 149 59 / 116 (p / p), 1:15 (p / p), 1:20 (p / p), 1:25 (p / p), or 1:50 (p / p). In some preferred embodiments, the said ratio is, or is approximately, 1:1.5 (p / p), 1:2 (p / p), or 1:2.5 (p / p).

[0219] In some embodiments, the second fermentation is carried out by providing a second fermentation substrate comprising or consisting essentially of the MSH C5 / C6 product provided in step (ee).

[0220] In some forms, the second fermentation is provided essentially without dilution with process water.

[0221] In some forms, the volume of the first fermentation is significantly smaller than the volume of the second fermentation.

[0222] In some forms, the volume of the first fermentation is 2-40%, 3-30%, 5-20%, 7.5-15%, 8-12% or about 10% of the volume of the second fermentation.

[0223] In some forms, the volume of the first fermentation is about 5, 7.5, 10, 15, 20, 25, 30, 35 or 40% of the volume of the second fermentation.

[0224] In some forms, the fermentation product is recovered by distillation.

[0225] In some embodiments, the said fermentation method comprises a lignin recovery step, such as a lignin recovery step from a distillation remnant.

[0226] In some forms, the second fermentation consists of consecutive fermentations, optionally carried out in the same fermenter.

[0227] In some embodiments, the second fermentation comprises the fermentation of both the first liquid fraction Petition 870190096087, dated 09 / 26 / 2019, page 66 / 149 60 / 116 as for C5 / C6 fiber paste.

[0228] In some forms, the fermentation product is an alcohol, organic acid, vitamin, amino acid, peptide, enzyme, or similar.

[0229] In some embodiments, the fermentation product is a C1-C4 product.

[0230] In some embodiments, the C1-C4 product is one or more of: methanol, ethanol, butanol, acetone, formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, lactic acid, malic acid, and / or any combination thereof.

[0231] In some embodiments, the C1-C4 product is EtOH.

[0232] In some embodiments, the microorganism is a eukaryotic or prokaryotic microorganism, such as a bacterium or a yeast.

[0233] In some embodiments, the microorganism is a recombinant microorganism.

[0234] In some forms, the microorganism is able to ferment C5 and C6 sugars, such as xylose and glucose.

[0235] A variety of microorganisms can be used for the fermentation of the C6+C5 product(s) into one or more fermentation products, such as the C1-C4 product as ethanol, acetone and / or organic acid(s), such as lactic or acetic acid, optionally also alone or in combination with major organic acids, such as valeric acid, caproic acid, citric acid or benzoic acid. As will be readily understood by one skilled in the art, several yeast strains are available that are suitable for converting C6 sugars as well as C5 sugars into ethanol, for example, various strains of Saccharomyces Petition 870190096087, dated 09 / 26 / 2019, p. 67 / 149 61 / 116 cerevisiae. Furthermore, for fermentations to produce lactic acid, a range of suitable microorganisms are used, such as lactic acid bacteria like Lactococcus spp., Lactobacillus spp., etc. In some embodiments, the microorganism is Lactococcus spp., Lactobacillus spp.

[0236] In some embodiments, the microorganism is a yeast, such as Saccharomyces cerevisiae, capable of or adapted to ferment xylose and glucose into EtOH.

[0237] In some embodiments, the said fermentation is carried out through the use of a microorganism, such as a recombinant microorganism capable of converting C6 sugars and C5 sugars into ethanol.

[0238] In some forms, the aforementioned fermentation is carried out through the use of a recombinant microorganism capable of converting glucose and xylose into ethanol.

[0239] In some forms, fermentation is carried out with a microorganism that is capable of fermenting at least one C5 sugar, in addition to one or more C6 sugar(s).

[0240] In some embodiments, the process is a process for the production of ethanol, whereby the process comprises fermenting a medium containing sugar(s) with a microorganism that is capable of fermenting at least one C5 sugar, in addition to one or more C6 sugar(s).

[0241] In some embodiments, the microorganism is able to ferment glucose, L-arabinose and xylose into ethanol.

[0242] In some embodiments, the microorganism that is capable of fermenting at least one C5 sugar, in addition to one or more C6 sugar(s), is a yeast. In one embodiment, the yeast belongs to the genus Saccharomyces, preferably to Petition 870190096087, dated 09 / 26 / 2019, pp. 68 / 149 62 / 116 species Saccharomyces cerevisiae. Document EP 1 499 708 describes a process for producing S. cerevisiae strains capable of producing ethanol from L-arabinose. Documents WO2003 / 062430 and WO2006 / 009434 disclose yeast strains capable of converting xylose to ethanol. These yeast strains are capable of isomerizing xylose to xylulose. In some embodiments, the microorganism is a eukaryotic microorganism as disclosed in documents EP 1 499 708, WO2003 / 062430, WO2006 / 009434 or WO2008 / 041840.

[0243] In some embodiments, the microorganism is a genetically modified yeast (e.g., Saccharomyces cerevisiae) capable of using L-arabinose and / or converting L-arabinose into L-ribulose, and / or xylulose 5-phosphate and / or into a desired fermentation product. Said microorganism may comprise the following genetic modifications: (a) a cluster consisting of the PPP genes TAL1, TKL1, RPE1, and RKI1, under the control of strong promoters; (b) a cluster consisting of the xyM gene and the XKSi gene, both under the control of constitutive promoters; (c) a cluster consisting of the araA, araB, and araD genes and / or a cluster of the xylA gene and the XKSi gene; and / or (d) deletion of an aldose reductase gene.

[0244] In one embodiment, the fermentation process is anaerobic. In another embodiment, the fermentation process is aerobic, optionally under oxygen-limited conditions.

[0245] In one embodiment, the fermentation process is under oxygen-limited conditions, such as a process in which oxygen consumption is limited by transfer. Petition 870190096087, dated 09 / 26 / 2019, pp. 69 / 149 63 / 116 of oxygen from the gas to the liquid. The degree of oxygen limitation is determined by the quantity and composition of the incoming gas stream, as well as by the actual mass transfer / mixing properties of the fermentation equipment used. Preferably, in a process under oxygen-limited conditions, the oxygen consumption rate is at least 5.5, more preferably at least 6, and even more preferably at least 7 mmol / L / h.

[0246] In a fourth aspect, the present invention relates to a method for preparing ethanol and optionally lignin from a lignocellulosic material comprising the steps of: - Supply of at least one C5 / C6 product according to the method in accordance with any of the modalities of the preceding aspects; - Fermentation of said product, at least one C5 / C6, in order to convert sugars into ethanol in the fermentation broth with a yeast; - Isolation of an ethanol-rich fraction from the fermentation broth; and optionally - Isolation of lignin.

[0247] In some embodiments, fermentation is carried out according to a method in accordance with any of the embodiments relating to the second or third aspect.

[0248] In some embodiments, lignin is isolated from spent fermentation broth or from the remnants of spent fermentation broth after isolation of the ethanol-rich fraction.

[0249] In a fifth aspect, the present invention relates Petition 870190096087, dated 09 / 26 / 2019, pp. 70 / 149 64 / 116 to lignin provided from lignocellulosic biomass, such as lignin obtained or obtainable according to any of the preceding aspects. It is believed that the lignin provided according to the present invention, in particular provided by a “V2.x” process, is different from lignin known in the art, such as lignin provided according to the “whole pulp” or V2 process. The lignin obtained is a high-value product, provided that the pretreatment is not based on the addition of acids, but, for example, carried out in the absence of added acids, as described above.

[0250] In a sixth aspect, the present invention relates to product C5 / C6 provided in accordance with any of the preceding aspects.

[0251] In a seventh aspect, the present invention relates to a fermentation substrate comprising a C5 / C6 product provided by a method in accordance with any of the preceding aspects.

[0252] In an eighth aspect, the present invention relates to a first or a second fermentation substrate provided by a method in accordance with any of the preceding aspects.

[0253] In a ninth aspect, the present invention relates to compositions comprising lignin obtained or obtainable by a method in accordance with any of the preceding aspects, including uses other than said compositions comprising lignin.

[0254] In some embodiments, the present invention relates to lignin obtained from the method according to any of the preceding aspects, such as a fraction Petition 870190096087, dated 09 / 26 / 2019, p. 71 / 149 65 / 116 solid from spent fermentation broth or from the distillation remnants of spent fermentation broth.

[0255] In some embodiments, a composition is provided comprising 0.1-99.9, or 1-90% (w / w) of lignin.

[0256] If you believe that the aforementioned lignin can be used in bitumen compositions, including asphalt compositions, such as bitumen compositions described in document WO2017 / 088892, said document being incorporated in its entirety herein.

[0257] In one embodiment, a bitumen composition comprising: a. 1-99.89% (w / w) bitumen; b. 0.1-50% (w / w) lignin; c. 0.01-20% (w / w) of plasticity-modifying agent(s); and d. 0-95% (w / w) additional component(s).

[0258] In some embodiments, the plasticity modifier is one or more plastomers, one or more thermoplastic elastomers, one or more rubbers, one or more viscosity modifiers and / or one or more reactive polymers, including any combination thereof.

[0259] In some embodiments, the aforementioned additional component(s) is / are one or more dispersing agent(s), surfactant(s), hydrotropic agent(s), emulsifier(s), preservative(s), antifoaming agent(s), viscosity modifier(s), reactive polymer(s) and any combination thereof; and / or one or more aggregate(s) and / or filler(s), such as Petition 870190096087, dated 09 / 26 / 2019, p. 72 / 149 66 / 116 natural, manufactured, recycled aggregates, including any combination thereof.

[0260] The aforementioned compositions comprising lignin can be used in a wide variety of applications. In some embodiments, the aforementioned compositions comprising lignin can be used, for example, in sealing work, road work, paving work, providing a surface layer, providing a sealing layer, providing a road and providing a pavement, providing a top layer of a road.

[0261] In some embodiments, compositions comprising lignin may be used, e.g., in applications related to (i) agriculture, (ii) construction and industrial paving, (iii) hydraulics and erosion control, (iv) industrial, (v) paving, (vi) railways, and (vii) recreation, such as (i) disinfectants, post-coating for fences, roofing, roofing paper, paved barn flooring, barns, raw material platforms, protective tanks, vats, protection for concrete structures, paints for trees (protective); ad (ii): water and moisture barriers (above and below ground), pavement compositions, tiles, roofing, insulating fabrics, papers, stair bands, construction papers, caulking compounds, cement waterproofing compounds, glass wool compositions, insulating fabrics, felts, papers, joint filler compounds, laminated tiles, liquid roof coatings, plastic cements, tiles, acoustic blocks, compositions, felts, bricks, waterproofing coatings, compositions,. Petition 870190096087, dated 09 / 26 / 2019, p. 73 / 149 67 / 116 insulating panels, fabrics, felts, paper, masonry coverings, plasterboard, soundproofing, stucco base, panels, air-drying paints, varnishes, artificial wood, ebonized wood, insulating paints, piping, tubes, treated awnings, channel linings, seals; ad (iii): river basins, basins, dam joints, dam linings, protection, dike protection, ditches, drainage channels, structures, embankment protection, groynes, breakwaters, dike protection, dike cushions and bank protection, membrane linings, waterproofing, reservoir linings, linings, sand dune stabilization, sewage lagoons, oxidation lagoons, pools, waste lagoons, water barriers, support felts, ad (iv): aqueduct insulation, lamination, insulating panels, paint compositions, papers, tubular packaging, insulating felts, panel boards, protective coating,Battery cases, carbons, electrical insulating compounds, papers, tapes, wire coatings, junction box compound, molded aqueducts, black grease, polishing compounds, cable splicing compound, embalming, engraving compositions, extenders, explosives, cement, plasticizers, preservatives, printing inks, well drilling fluid, armored bituminous fabrics, litter impregnation, mold prevention, sawdust, cork, asphalt composition, acid-proof enamels, mastics, varnishes, acid-resistant coatings, air-drying paints, varnishes, anti-corrosive and anti-fouling paints, antioxidants and solvents, bases for solvent compositions, baked and resistant enamels, Petition 870190096087, dated 09 / 26 / 2019, pp. 74 / 149 68 / 116 heat, deck sealing compounds, japons, marine enamels, detonating fuses, briquette binders, burial vaults, casting molds, clay articles, clay doves, expansion joints, flower pots, casting cores, friction tapes, gaskets, mirrors, rubber, molded compositions, shoe soles; ad (v): airport runways, taxiways, aprons, asphalt blocks, masonry bricks, bridge deck, planing, crack fillers, building floors, warehouses, garages, highways, roads, streets, embankments, gutters, drainage ditches, parking lots, sidewalks, Portland cement concrete, protective coating, roof deck parking, pavements, pedestrian paths, soil stabilization; ad (vi) ballast treatment, dust placement, paved ballast, sub-ballast, paved crossings, freight yards, station platforms;and (vii) dance pavilions, drive-in cinemas, gymnasiums, sports arenas, playgrounds, schoolyards, running tracks, athletics tracks, skating rinks, swimming pools and children's pools, tennis courts, handball courts, synthetic playing fields and athletics tracks.; Comparison of the “paste” and “C5 derivation” methods (“V2”) with the present invention (“V2.X” alias “two-step hydrolysis and mixed sugar hydrolysis”)

[0262] Process scheme (1) (Figure 2) shows a relatively simple process configuration, such as a “full folder” process as described in document WO2015 / 014364. Petition 870190096087, dated 09 / 26 / 2019, pp. 75 / 149 69 / 116 1) Biomass, such as soft lignocellulosic biomass, is steam pretreated with low gravity (xylan number > 10%, such as 10-20%). 2) The pretreated biomass is adjusted for pH and temperature before enzymatic hydrolysis, preferably in a single-step hydrolysis process (hence the name whole-paste hydrolysis). 3) After enzymatic hydrolysis, the whole-paste hydrolysate is adjusted for pH and temperature before fermentation with a suitable microorganism. The whole-paste hydrolysate is the only substrate for microbial fermentation, such as yeast fermentation, which provides, for example, EtOH.

[0263] Process scheme (2) (Figure 3) shows a more complex process comprising a C5 bypass such as the processes described in document WO 2014 / 019589: “Methods of processing lignocellulosic biomass using singlestage autohydrolysis and enzymatic hydrolysis with C5 bypass and post-hydrolysis”: 1) Biomass, such as soft lignocellulosic biomass, is steam pretreated with low gravity (xylan number > 10%, such as 10-20%). 2) The pretreated biomass is separated (solid / liquid separation process) into a fiber fraction (A) and a liquid fraction (B), the said liquid fraction (B) comprising C5 sugars (hence the name C5 derivative). 3) The fiber fraction (A) is diluted to an appropriate dry matter content (e.g., 15-40% dry matter (DM)) and the pH and temperature are adjusted before enzymatic hydrolysis. Petition 870190096087, dated 09 / 26 / 2019, page 76 / 149 70 / 116 4) The C5 derivative (liquid fraction (B)) is added at some point to the hydrolysis fraction or hydrolyzed fiber. It is believed that, for example, hemicellulose-derived oligomers, such as the xylan oligomers of the C5 derivative, are degraded into monomers by enzymes as added in fiber hydrolysis. 5) The final hydrolysate is adjusted for pH and temperature before fermentation with a suitable microorganism. The final hydrolysate is the only substrate for microbial fermentation, such as yeast fermentation which provides, for example, EtOH.

[0264] Process scheme (3) (Figure 4) describes examples of a process according to the present invention (also referred to as V2.X (or “two-step hydrolysis and mixed sugar hydrolysis”)): 1) Lignocellulosic biomass, such as soft lignocellulosic biomass, is steam pretreated at low gravity in a single- or multi-step pretreatment process; medium or high gravities of pretreatment comprise other options according to the present invention. 2) The pre-treated biomass is separated into a first fiber fraction (“solid fraction-1”) and a first liquid fraction (“liquid fraction-1”). 3) The fiber fraction (A) is adjusted / diluted to an appropriate dry matter content (e.g., 15-40% DM) and the pH and / or temperature are adjusted before enzymatic hydrolysis of the fiber. Petition 870190096087, dated 09 / 26 / 2019, page 77 / 149 71 / 116 4) The hydrolyzed fiber fraction is separated into a first fiber fraction (“solid fraction-2”) and a liquid fraction (“liquid fraction-2). 5) The solid fraction-2 is adjusted / diluted to an appropriate dry matter content, and the pH and / or temperature are adjusted before enzymatic hydrolysis of the fiber cake. 6( “Liquid fractions-1 and -2” are combined and the pH and / or temperature adjusted before being hydrolyzed with, or without, the addition of additional enzymes (mixed sugar hydrolysis). 7) Optionally, at least a fraction of the mixed sugar hydrolysate may be subjected to ultrafiltration, with the aim of recycling at least a fraction of the enzymes and adding the recycled enzymes to the hydrolysis of the mixed sugar. 8) Optionally, the hydrolyzed fiber cake can be subjected to an additional solid / liquid separation step, providing a third fiber fraction (“fiber fraction-3”) and a third liquid fraction (“liquid fraction 3”). 9) Optionally, the hydrolysates from fiber cake hydrolysis and / or mixed hydrolysis are adjusted for pH and / or temperature before fermentation with a suitable microorganism, such as yeast, providing EtOH.

[0265] This process provides different hydrolysates with different levels of inhibitors. Thus, there is the option of feeding the fermentation from two hydrolysates with different levels of inhibitory substances formed in the pretreatment, in particular starting a fermentation with the hydrolysate with the lowest concentration of Petition 870190096087, dated 09 / 26 / 2019, pp. 78 / 149 72 / 116 inhibitors.

[0266] In addition, suitable enzyme preparations may be added either as enzyme mixtures or single enzyme activities at different process steps, such as (i) fiber hydrolysis, (ii) fiber cake hydrolysis and / or (iii) mixed sugar hydrolysis (see, e.g., Figure 1 or 4). In some embodiments, the addition of additional enzymes at any of the aforementioned steps (ii) and / or (iii) is optional – this may not be clearly represented in the figures referred to.

[0267] Adjusted / diluted to a suitable dry matter content” prior to fiber hydrolysis and / or fiber cake may comprise the addition of water, such as process water.

[0268] If available, for example, when in close proximity or in combination the suitable processing plant provides “raw juice” – i.e., a water-based liquid comprising fermentable sugars, such as a 1G EtOH processing plant, or a sugar or fruit juice producing plant – said dilution may include such raw juice. Advantages of such a combination, such as water savings and / or increased fermentation yields, are disclosed in, for example, documents WO2015 / 120859, or PCT / EP2016 / 069775, both of which are incorporated in their entirety herein by reference.

[0269] In summary, and without intending to be interpreted as limiting, the present invention can provide, inter alia, one or more of the following effects and / or advantages: 1. increased yield of the C5 / C6 product 2. reduced enzyme consumption Petition 870190096087, dated 09 / 26 / 2019, pp. 79 / 149 73 / 116 3. Addition of enzymes where they are needed. 4. Water conservation 5. financial savings, 6. Better lignin quality 7. Increased yield of fermentation product, such as the C1-C4 product, as EtOH. 8. Reducing the need for water Numbered modalities

[0270] Relevant aspects and embodiments of the present invention can also be found in the next section, entitled “numbered embodiments”. 2. A method for providing a C5 / C6 product from a lignocellulosic material comprising the steps of: a) Pre-treatment of lignocellulosic material; b) Solid / liquid separation of the pretreated lignocellulosic material from step (a) into a first solid fraction and a first liquid fraction; c) Enzymatic hydrolysis (fiber hydrolysis) of the aforementioned first solid fraction from step (b) using an enzyme composition capable of degrading lignocellulosic material, thereby providing a C5 / C6 fiber hydrolysis paste comprising C5 and / or C6 sugars; d) Solid / liquid separation of the C5 / C6 fiber hydrolysis paste from step (c) into a second solid fraction and a second liquid fraction; and optionally (e) Combination of the aforementioned first liquid fraction and the aforementioned second liquid fraction for enzymatic hydrolysis (Mixed Sugar Hydrolysis (MSH)), through which an MSH C5 / C6 product is provided. Petition 870190096087, dated 09 / 26 / 2019, pp. 80 / 149 74 / 116 3. The method according to embodiment 1, comprising step (f): enzymatic hydrolysis (fiber cake hydrolysis) of said second solid fraction of step (d) in order to obtain a C5 / C6 paste product. 4. The method according to either embodiment 1 or 2, wherein the lignocellulosic material is soft lignocellulosic biomass. 5. The method according to any of the preceding embodiments, in which the pretreatment is carried out at a dry matter (DM) content in the range of 5-80, 10-70, 20-60, 30-50%, and / or at a DM content of approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more than 80%. 6. The method, according to any of the preceding modalities, in which pretreatment is performed in low, medium or high severity; and / or under conditions that provide a xylan number of > 10%, 6-10% or <6%. 7. The method, according to any of the preceding embodiments, in which enzymatic fiber hydrolysis, fiber cake hydrolysis and / or MSH is / are carried out for a period of at least 6, 12, 24, 48 or 72h, such as 6-120h, 12-100h, or 48-96h, or approximately 12, 24, 48, 72, 96 or 120h. 8. The method, according to any of the preceding embodiments, in which enzymatic fiber hydrolysis, fiber cake hydrolysis and / or MSH is / are carried out at a pH in the range of at least pH 3.0, such as 3.0-6.0, 4.0-5.5, 4.2-5.4, and / or approximately 4.2, 4.5, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3 or 5.4. 9. The method, according to any of the preceding modalities, in which the hydrolysis of enzymatic fiber, the Petition 870190096087, dated 09 / 26 / 2019, p. 81 / 149 75 / 116 hydrolysis of fiber cake and / or MSH is / are carried out at a temperature in the range of 30-70 °C, 40-65 °C, 50-62 °C, 55-60 °C, and / or approximately 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 °C. 10. The method, according to any of the preceding embodiments, in which enzymatic fiber hydrolysis and / or fiber cake hydrolysis are carried out at a DM content, such as a DM content above 10 or 15%, such as approximately 15-45, 20-40%, 25-35%, and / or approximately 15, 20, 25, 30, 35, or 40%. 11. A method, according to any of the preceding embodiments, in which the enzyme composition capable of degrading lignocellulosic material comprises a cellulase and / or hemicellulase. 12. Method, according to any of the preceding embodiments, in which the enzyme composition capable of degrading lignocellulosic material comprises a mixture of cellulase(s) and / or hemicellulase(s). 13. A method, according to any of the preceding embodiments, in which the hemicellulase is or comprises one or more xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), and / or esterase(s), including any combination thereof. 14. The method, according to embodiment 12, in which the esterase(s) is / are or comprises one or more acetyl esterases and / or feroyl esterase. 15. The method, according to any of the preceding embodiments, in which the enzyme composition capable of degrading lignocellulosic material comprises one or more of the following activities: endocellulase, endoglucanase, exocellulase, Petition 870190096087, dated 09 / 26 / 2019, p. 82 / 149 76 / 116 exoglucanase, endoxylanase, acetylxylan esterases, xylosidase and / or β-glucosidase. 16. The method, according to any of the preceding embodiments, in which step (e) is carried out by combining said first liquid fraction and said second liquid fraction and enzymatic hydrolysis of the mixture. 17. The method, according to any of the preceding embodiments, in which the hemicellulase(s) present in step (e) comprise(s) xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), esterase(s), acetylesterases, and / or feroyl esterase(s), including any combination thereof. 18. The method, according to any of the preceding embodiments, in which all or at least a fraction of the hemicellulase(s) used in step (e) was / were added in step (c). 19. The method, according to any of the preceding embodiments, in which at least a fraction of the hemicellulase(s) used in step (e) was added in step (c). 20. The method, according to any of the preceding embodiments, in which one or more hemicellulase(s) is / are added in step (e). 21. The method, according to any of the preceding embodiments, in which one or more additional enzyme(s) is / are provided in step (e). 22. The method, according to embodiment 20, in which the additional enzyme(s) is / are not essentially Petition 870190096087, dated 09 / 26 / 2019, p. 83 / 149 77 / 116 present in the enzyme composition capable of degrading the lignocellulosic material used / provided in step (c). 23. The method, according to embodiment 20 or 21, in which the additional enzyme(s) is / are one or more of: hemicellulase(s), xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), esterase(s), acetylesterases, feroyl esterase(s) and any combination thereof. 24. The method according to any of the preceding embodiments, wherein step (e) comprises an ultrafiltration step (j) to recycle enzymes present after MSH. 25. The method, according to embodiment 23, wherein the ultrafiltration step (j) is adapted to allow the recycling of at least 30, 50, 75, 80 or 90% (w / w) of the enzymatic activity. 26. The method, according to any of the preceding embodiments, in which all the cellulase used in step (f) was added in step (c). 27. The method, according to any of the preceding embodiments, in which at least a fraction of the cellulase used in step (f) was added in step (c). 28. The method, according to any of the preceding embodiments, in which one or more cellulase(s) and optionally hemicellulase(s) is / are added in step (f). 29. The method, according to any of the preceding embodiments, in which MSH and / or fiber bolus hydrolysis are performed without the addition of one or more enzyme(s). 30. The method, according to any of the preceding embodiments, in which MSH and / or fiber cake hydrolysis are performed. Petition 870190096087, dated 09 / 26 / 2019, p. 84 / 149 78 / 116 are performed without the addition of one or more cellulases and / or one or more hemicellulases. 31. The method, according to any of the preceding modalities, additionally comprising the step of: g) Solid / liquid separation of the C5 / C6 paste product from step (f) into a third solid fraction and a third liquid fraction (liquid C5 / C6 product). 32. The method, according to any of the preceding embodiments, in which the second liquid fraction has a lower concentration of inhibitor than the first liquid fraction. 33. The method, according to any of the preceding modalities, in which the second liquid fraction has a lower concentration of inhibitor than the MSH C5 / C6 product. 34. The method, according to any of the preceding embodiments, in which the C5 / C6 paste product has a lower inhibitor concentration than the MSH C5 / C6 product. 35. The method, according to any of the preceding embodiments, additionally comprising the step of: K) Combination of at least one portion of the MSH C5 / C6 product with at least one portion of one or more of: the C5 / C6 paste product from step (f), the liquid C5 / C6 product from step (g), and / or the second liquid fraction from step (d) in order to obtain a combined C5 / C6 product. 36. The method, according to embodiment 34, in which the combined C5 / C6 product consists or essentially consists of the MSH C5 / C6 product and the C5 / C6 paste product of step (f); the MSH C5 / C6 product and the liquid C5 / C6 product of step (g); or the MSH C5 / C6 product and the second liquid fraction of step (d). Petition 870190096087, dated 09 / 26 / 2019, page 85 / 149 79 / 116 37. The method, according to any of the preceding embodiments, additionally comprising a lignin recovery step, such as water removal, compaction and / or granulation. 38. The method, according to embodiment 36, in which the aforementioned lignin recovery is carried out in the second or third solid fraction provided in steps (d) or (g). 39. The method, according to any of the preceding embodiments, in which any C5 / C6 product is a C5+C6 product, that is, a product comprising C5 and C6 carbohydrates, such as xylose and glucose, including structural analogues, isomers and / or derivatives thereof. 40. The method, according to any of the preceding embodiments, in which the C5+C6 product comprises glucose and xylose. 41. A method for providing a fermentation product, said method comprising the steps of: m) Supply of at least one C5 / C6 product according to the method of any of the preceding embodiments; en) Supply of the fermentation product by means of fermentation of said C5 / C6 product with a microorganism. 42. The method, according to embodiment 40, in which product C5 / C6 is or comprises one or more of: MSH C5 / C6 product, C5 / C6 paste product, liquid C5 / C6 product, combined C5 / C6 product, first liquid fraction or second liquid fraction, including any combination thereof. 43. The method, according to embodiment 40 or 41, in which the fermentation product is provided in a fermentation broth, additionally the said method comprising Petition 870190096087, dated 09 / 26 / 2019, page 86 / 149 80 / 116 the step(s) of: (o) recovering the said fermentation product from a fermentation broth. 44. The method, according to embodiment 40 to 42, additionally comprising the step of: (p) recovering lignin from spent fermentation broth and / or a fraction proportioned in steps (n) or (o). 45. The method, according to embodiment 43, in which fermentation is carried out in at least two fermentation steps (a first and a second fermentation step), where a first and a second fermentation substrate are fermented. 46. ​​Two-step fermentation method comprising the following steps: aa) Pre-treatment of lignocellulosic material; bb) Solid / liquid separation of the pre-treated lignocellulosic material from step (a) into a first solid fraction and a first liquid fraction; cc) Enzymatic hydrolysis (fiber hydrolysis) of the aforementioned first solid fraction from step (b) through the use of an enzyme composition capable of degrading lignocellulosic material, thereby providing a C5 / C6 fiber hydrolysis paste; dd) Solid / liquid separation of the C5 / C6 fiber hydrolysis paste from step (cc) into a second solid fraction and a second liquid fraction; (ee) Enzymatic hydrolysis (mixed sugar hydrolysis MSH) of a mixture of the first liquid fraction of step (bb) and the C5 / C6 fiber hydrolysis paste of step (cc), or the first liquid fraction of step (bb) and the second fraction Petition 870190096087, dated 09 / 26 / 2019, page 87 / 149 81 / 116 liquid from step (dd), thus providing an MSH C5 / C6 product; ff) Provision of a first fermentation substrate comprising at least a portion of the “C5 / C6 fiber hydrolysis paste” and / or the second liquid fraction; gg) Provision of a second fermentation substrate comprising at least a portion of the MSH C5 / C6 product; hh) Fermentation of the first fermentation substrate in a first fermentation with a microorganism; and ii) Fermentation of the second fermentation substrate in a subsequent second fermentation; where the step (dd) is optional. 47. The method, according to either of the modalities 44 or 45, in which the first fermentation substrate has a significantly lower inhibitor concentration than the second fermentation substrate. 48. The method, according to any of the modalities 44 to 46, in which the first fermentation is a batch or fed-batch fermentation. 49. The method, according to any of the modalities 44 to 47, in which the first fermentation is carried out by providing a first fermentation substrate comprising: x. the second net fraction provided in step (d) or (dd); y. the C5 / C6 fiber hydrolysis slurry provided in step (c) or (cc); and / or z. the C5 / C6 product obtained in step (f), that is, the liquid C5 / C6 product or the paste-like C5 / C6 product. Petition 870190096087, dated 09 / 26 / 2019, pp. 88 / 149 82 / 116 50. The method, according to any of the modalities 44 to 48, in which the first fermentation is carried out by providing a first fermentation substrate consisting essentially of: x. the second net fraction provided in step (d) or (dd); y. the C5 / C6 fiber hydrolysis slurry provided in step (c) or (cc); and / or z. the C5 / C6 product obtained in step (f), that is, the liquid C5 / C6 product or the paste-like C5 / C6 product. 51. The method, according to any of the embodiments 44 to 49, in which the first fermentation substrate comprises or consists essentially of a mixture of the second liquid fraction and the C5 / C6 product obtained in step (f), that is, the liquid C5 / C6 product or the paste-like C5 / C6 product. 52. The method, according to embodiment 50, in which the ratio of the second liquid fraction and the product C5 / C6 is in the range of 100:0.1-0.1:100, 10:0.1-0.1:10, or 10:1-1:10 (w / w); or approximately 50:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:50 (w / w). 53. The method, according to any of the modalities 44 to 51, in which the first fermentation substrate is supplied essentially without dilution with process water. 54. The method, according to any of the modalities 44 to 52, in which the second fermentation is a fed-batch fermentation or a continuous fermentation, optionally carried out in the same fermenter as the first fermentation. Petition 870190096087, dated 09 / 26 / 2019, pp. 89 / 149 83 / 116 55. The method, according to any of the modalities 44 to 53, in which fed-batch fermentation is with linear or exponential raw material. 56. The method, according to any of the modalities 44 to 54, in which the second fermentation is carried out with the same microorganisms as in the first fermentation. 57. The method, according to any of the embodiments 44 to 55, in which the second fermentation is carried out by providing a second fermentation substrate comprising or consisting essentially of a mixture of the C5 / C6 product obtained in step (f) (i.e., the liquid C5 / C6 product or the paste C5 / C6 product) and the C5 / C6 product obtained from step (e) (i.e., the MSH C5 / C6 product). 58. The method, according to embodiment 56, in which the ratio of the liquid C5 / C6 product or paste C5 / C6 product and the C5 / C6 product obtained from step (e) (i.e., MSH C5 / C6 product) is in the range of 100:0.1-0.1:100, 10:0.10.1:10, or 10:1-1:10, 5:1-1:5; 4:1-1:4, 3:1-1:3, 2.5-1:2.5, 2:1-1:2 or 1.5-1:1-1.5 ( / p); or approximately 50:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:50 (p / p). 59. The method, according to any of the embodiments 44 to 57, in which the second fermentation is carried out by providing a second fermentation substrate comprising or consisting essentially of the MSH C5 / C6 product provided in step (ee). 60. The method, according to any of the modalities 44 to 58, in which the second fermentation substrate is Petition 870190096087, dated 09 / 26 / 2019, pp. 90 / 149 84 / 116 provided essentially without dilution with process water. 61. The method, according to any of the modalities 44 to 59, in which the volume of the first fermentation is significantly smaller than the volume of the second fermentation. 62. The method, according to modality 60, in which the volume of the first fermentation is 2-40%, 3-30%, 5-20%, 7.5-15%, 8-12% or about 10% of the volume of the second fermentation. 63. The method, according to any of the modalities 40 to 61, in which the fermentation product is recovered by distillation. 64. The method, according to any of the modalities 40 to 62, in which the fermentation product is EtOH. 65. The method, according to embodiment 62 or 63, additionally comprising a step of recovering lignin from a distillation remnant. 66. The method, according to any of the modalities 44 to 64, in which the first and second fermentations are consecutive fermentations, optionally carried out in the same fermenter. 67. The method, according to any of the modalities 44 to 65, in which the second fermentation comprises the fermentation of both the first liquid fraction and the C5 / C6 fiber hydrolysis pulp. 68. The method, according to any of the modalities 40 to 66, in which the fermentation product is an alcohol, organic acid, vitamin, amino acid, peptide, enzyme or similar. Petition 870190096087, dated 09 / 26 / 2019, pp. 91 / 149 85 / 116 69. The method, according to any of the modalities 40 to 67, in which the fermentation product is a C1-C4 product. 70. The method, according to embodiment 68, in which the C1-C4 product is one or more of: methanol, ethanol, butanol, acetone, formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, lactic acid, malic acid, and / or any combination thereof. 71. The method according to either of the embodiments 68 or 69, in which the C1-C4 product is EtOH. 72. The method according to any of the embodiments 40 to 70, wherein the microorganism is a eukaryotic or prokaryotic microorganism, such as a bacterium or a yeast. 73. The method, according to any of the modalities 40 to 71, in which the microorganism is a recombinant microorganism. 74. The method, according to any of the modalities 40 to 72, in which the microorganism is capable of fermenting C5 and C6 sugars, such as xylose and glucose. 75. The method, according to any of the embodiments 40 to 73, in which the microorganism is a yeast, such as Saccharomyces cerevisiae, capable of or adapted to ferment xylose and glucose into EtOH. 76. A method for preparing ethanol and optionally lignin from a lignocellulosic material comprising the steps of: - Supply of at least one C5 / C6 product according to a method in accordance with any of the above modalities; Petition 870190096087, dated 09 / 26 / 2019, pp. 92 / 149 86 / 116 - Fermentation of said product, at least one C5 / C6, in order to convert sugars into ethanol in the fermentation broth with a yeast; - Isolation of an ethanol-rich fraction from the fermentation broth; and optionally - Lignin isolation. 77. The method, according to the modality 75 in which the fermentation is carried out according to a method, according to any of the modalities 40 to 74. 78. The method, according to either of the embodiments 75 or 76, in which lignin is isolated from spent fermentation broth or from the remnants of spent fermentation broth after isolation of the ethanol-rich fraction. 79. Lignin provided from lignocellulosic biomass according to any of the preceding embodiments. 80. A C5 / C6 product provided in accordance with any of the preceding embodiments. 81. A supply substrate comprising a C5 / C6 product provided by a method, according to any of the preceding embodiments. 82. The first or second fermentation substrate provided by a method, according to any of the preceding embodiments. 83. Use of lignin, according to embodiment 78, in a bitumen composition, such as asphalt. 84. A composition comprising 0.1-99.9% (w / w) lignin, according to embodiment 78. 85. A bitumen composition comprising: a. 1-99.89% (w / w) bitumen; Petition 870190096087, dated 09 / 26 / 2019, pp. 93 / 149 87 / 116 b. 0.1-50% (w / w) lignin, according to modality 78; c. 0.01-20% (w / w) of plasticity-modifying agent(s); and d. 0-95% (w / w) additional component(s). 86. The bitumen composition according to embodiment 84, wherein the plasticity modifier is one or more plastomers, one or more thermoplastic elastomers, one or more rubbers, one or more viscosity modifiers and / or one or more reactive polymers, including any combination thereof. 87. The bitumen composition according to embodiment 84 or 85, wherein the additional component(s) is / are one or more dispersing agent(s), surfactant(s), hydrotropic agent(s), emulsifier(s), preservative(s), antifoaming agent(s), viscosity modifier(s), reactive polymer(s) and any combination thereof; and / or one or more aggregate(s) and / or filler(s), such as natural, manufactured, recycled aggregates, including any combination thereof. 88. A composition comprising 0.1-99.9% (w / w) lignin, according to embodiment 78. Use of a composition according to any of the modalities 83 to 87, in sealing work, road work, paving work, providing a surface layer, providing a sealing layer, providing a road and providing a pavement, providing a top layer of a road. 90. Use of a composition according to any of embodiments 83 to 87, in applications relating to (i) agriculture, (ii) industrial construction and paving, Petition 870190096087, dated 09 / 26 / 2019, pp. 94 / 149 88 / 116 (iii) hydraulics and erosion control, (iv) industrial, (v) paving, (vi) railways, and (vii) recreation, such as (i) disinfectants, post-coating for fences, roofing, roofing paper, paved barn flooring, barns, raw material platforms, protective tanks, vats, protection for concrete structures, paints for trees (protective);ad (ii): water and moisture barriers (above and below ground), pavement compositions, tiles, roofing, insulating fabrics, papers, stair bands, construction papers, caulking compounds, cement waterproofing compounds, glass wool compositions, insulating fabrics, felts, papers, joint filler compounds, laminated tiles, liquid roof coatings, plastic cements, tiles, acoustic blocks, compositions, felts, bricks, waterproofing coatings, compositions, insulating panels, fabrics, felts, paper, masonry coatings, soundproofing gypsum boards, stucco base, panels, air-drying paints, varnishes, artificial wood, ebonized wood, insulating paints, piping, pipes, treated awnings, channel linings, sealants;ad (iii): river basins, basins, dam joints, dam linings, protection, dike protection, ditches, drainage channels, structures, embankment protection, groynes, breakwaters, dike protection, dike cushions and bank protection, membrane linings, waterproofing, reservoir linings, linings, sand dune stabilization, sewage lagoons, oxidation lagoons, pools, waste lagoons, water barriers, support felts, ad (iv):; Petition 870190096087, dated 09 / 26 / 2019, pp. 95 / 149 89 / 116 aqueduct insulation, lamination, insulating panels, paint compositions, papers, tubular packaging, insulating felts, panel boards, protective coating, battery boxes, carbons, electrical insulating compounds, papers, tapes, wire coatings, junction box compound, molded aqueducts, black grease, polishing compounds, cable splicing compound, embalming, engraving compositions, extenders, explosives, cement, plasticizers, preservatives, printing inks, well drilling fluid, armored bituminous fabrics, burlap impregnation, mold prevention, sawdust, cork, asphalt composition, acid-proof enamels, mastics, varnishes, acid-resistant coatings, air-drying paints, varnishes, anti-corrosive and anti-fouling paints, antioxidants and solvents, bases for solvent compositions, baked and heat-resistant enamels, deck sealing compounds, Japan, marine enamels,detonating fuses, briquette binders, burial vaults, casting molds, clay articles, clay doves, expansion joints, flower pots, casting cores, friction tapes, gaskets, mirrors, rubber, molded compositions, shoe soles; ad (v): airport runways, taxiways, aprons, asphalt blocks, masonry bricks, bridge deck, leveling, crack fillers, building floors, warehouses, garages, highways, roads, streets, embankments, gutters, drainage ditches, parking lots, sidewalks, Portland cement concrete, protective coating, rooftop parking decks, pavements, pedestrian paths, soil stabilization; ad (vi) treatment of, Petition 870190096087, dated 09 / 26 / 2019, pp. 96 / 149 90 / 116 ballast, dust placement, paved ballast, sub-ballast, paved crossings, loading yards, station platforms; and (vii) dance pavilions, drive-in cinemas, gymnasiums, sports arenas, playgrounds, schoolyards, running tracks, athletics tracks, skating rinks, swimming pools and children's pools, tennis courts, handball courts, synthetic playing fields and athletics tracks. EXAMPLES General methods and materials used in the examples

[0271] In this section, the general methods and materials used for the examples presented in this application are described. If you deviate from the general methods and materials, this will be specified in the example. Pre-treatment

[0272] Pretreatment was carried out at the Inbicon pilot plant, Sk$rb$k, Denmark. Wheat straw (WS) was soaked in water, pH > 4.0, prior to pretreatment to approximately 40% dry matter (DM). About 50 kg DM / h of biomass was pretreated at temperatures of 180–200 °C with a residence time of approximately 18 minutes. The biomass was introduced into the reactor using a sluice system (WO2010 / 058285) and the pretreated material was discharged again using a sluice system. The pressure inside the pressurized pretreatment reactor corresponded to the saturated steam pressure at the temperature used. The pretreated biomass was subjected to solid / liquid separation using a screw press, producing a liquid fraction (C5 derivative, first liquid fraction) and a solid fraction (first solid fraction) with Petition 870190096087, dated 09 / 26 / 2019, pp. 97 / 149 91 / 116 a DM content of approximately 60%. The pretreatment process is further described in Petersen et al. (2009). Analytical measurement of pretreatment fractions

[0273] Raw materials were analyzed for carbohydrates according to the methods described in Sluiter et al. (2005) and Sluiter et al. (2008) using a Dionex Ultimate 3000 HPLC system equipped with a Phenomenex Rezex Monosaccharide H+ column.

[0274] Samples of liquid and solid fractions were collected after three hours of continuous pretreatment, and samples were collected three times over three hours to ensure that a sample was obtained from the steady-state pretreatment.

[0275] The solid fractions were analyzed for carbohydrates according to the methods described in Sluiter et al. (2008) using a Dionex Ultimate 3000 HPLC system equipped with a Phenomenex Rezex Monosaccharide H+ column.

[0276] The liquid fractions were analyzed for carbohydrates and degradation products according to the methods described in Sluiter et al. (2006) using a Dionex Ultimate 3000 HPLC system equipped with a Phenomenex Rezex Monosaccharide H+ column.

[0277] The total solids content (TS), hereinafter referred to as dry matter, was measured by drying for approximately 24 hours to constant weight at 105 °C. Suspended solids (SS) were analyzed using a method adapted from the methods described in Weiss et al. (2009) by analyzing the TS of the sample and the TS in a sample filtered through Petition 870190096087, dated 09 / 26 / 2019, pp. 98 / 149 92 / 116 a paper filter and calculating the amount of SS.

[0278] Mass balances were established as described in Petersen et al. (2009) and cellulose and hemicellulose recoveries were determined. Hydrolysis

[0279] The hydrolysis experiments were carried out at the Inbicon pilot plant, Sk^rb^k, Denmark, on two scales.

[0280] Fiber hydrolysis experiments were performed on a 10 kg balance in a free-fall reactor, as described in document WO2006 / 056838. The reactor was designed to perform experiments with a suspended dry matter content above 20%. The reactor consists of a horizontally placed drum divided into 6 chambers, each 24 cm wide and 50 cm high. A horizontal rotating shaft mounted with three blades in each chamber is used for mixing / agitation. A 1.1 kW motor is used as a drive, and the rotation speed is adjustable within the range of 2.5 and 16.5 rpm. The direction of rotation is programmed to change every two minutes between clockwise and counterclockwise. A water-heated jacket on the outside of the chambers allows temperature control up to 80 °C.

[0281] Hydrolysis experiments are carried out by adding the fiber fraction corresponding to 2.2 kg of suspended solids to a chamber and then adding water or liquid fraction until the desired degree of separation of dissolved solids between the fiber fraction and the liquid is obtained, in order to simulate a full-scale process. The temperature is set to 50 °C. The pH is adjusted to the optimum pH for the enzyme used by using Ca(OH)2 beforehand. Petition 870190096087, dated 09 / 26 / 2019, pp. 99 / 149 93 / 116 of the addition of enzymes. The enzymes are added. Agitation is performed at 6 rpm. After liquefaction, the experiments were transferred to shaken flasks. The experiments are sampled after 4 hours and every 24 hours by sampling and tenfold dilution and analysis according to Kristensen et al. (2009) with a Dionex Ultimate 3000 HPLC system equipped with a Phenomenex Rezex Monosaccharide H+ column. Separation between fiber hydrolysis and fiber cake hydrolysis

[0282] After hydrolysis, the pulp was separated into a second fiber fraction, fiber fraction - 2 and a second liquid fraction, liquid fraction - 2, by pressing in a filter chamber press using a cassette with Tetex Mono V05-1001-SK025 polypropylene fabric filter at 60 to 65 °C for ten minutes at 5 bar inlet pressure and 13 bar pressure during pressing. Materials

[0283] The materials used are listed below in Table 1. Table 1: List of materials Compound Manufacturer: Pretreated wheat straw fibers and liquid fraction. Enzyme: Cellic® CTec3 Ca(OH)2. Example 1 - Comparison of conversion method V2 X and method V2 (derivation

[0284] An example of process V2 of process 3 (Figure 4). The principle Inbicon Novozymes Sigma total carbohydrates in the io of C5) X is shown in the scheme al hypothesis behind the Petition 870190096087, dated 09 / 26 / 2019, pp. 100 / 149 94 / 116 The formation of the V2.X process is – without wanting to be tied to any theory – that significant and probably major cellulase components will follow the fibers and major hemicellulase components will follow the liquid phase. The cellulases will be reused in a second fiber hydrolysis step and the hemicellulases will be reused in the mixed sugar hydrolysis for the hydrolysis of xylo- and other hemicellulose oligomers found in liquid fraction 1 and liquid fraction 2. As an option, ultrafiltration (UF) can be used to concentrate the enzymes in the mixed sugar hydrolysis and / or to improve the hydrolysis yield. The fermentation process benefits from and is becoming more efficient by using the two hydrolysates with different levels of inhibitory substances in the ideal way.

[0285] The differences between the V2.X process (e.g., process scheme 3) and the V2 or C5 derivation process (process scheme 2, Figure 3) include the introduction of a two-step hydrolysis and / or that the hydrolysis of mixed sugar is carried out without fibers.

[0286] In an experimental study, the conversions of glucan and xylan in the V2.X method and the C5 derivation method were compared (Figure 5). The first 72 hours of fiber hydrolysis are the same for the V2.X method and the C5 derivation method. The first 72 hours of fiber hydrolysis were carried out on a 10 kg scale in a free-fall mixer. After 72 hours of fiber hydrolysis, the slurry was divided into two fractions, one fraction to be used to continue with the V2.X method and another fraction to be used to continue with the C5 derivation method. Petition 870190096087, dated 09 / 26 / 2019, pp. 101 / 149 95 / 116

[0287] Fiber hydrolysis for the V2.X method and for the C5 derivation method:

[0288] The fiber fraction was added to the chambers of the free-fall reactor and water was added to achieve a suspended dry matter content of 22% by weight, giving a total dry matter content of 25% by weight. The pH was adjusted to 5.3 and the temperature to 50 °C. The agitator in the free-fall mixer was set to 6 rpm. Five chambers were used to compare the V2.X method and the C5 derivation method.

[0289] Table 2 shows the enzyme dosages used. After 72 hours at 50 °C and pH adjusted in the range of 4.8 - 5.3, fiber hydrolysis was stopped. Table 2: Enzyme and SS dosages for fiber hydrolysis experiments Experiment Enzyme Dosage Suspended Dry Matter [g of Cellic® CTec3 / kg of [% by weight of glucan in FH] SS] 16-13-R6-2 50 22 16-13-R6-3 40 22 16-13-R6-4 75 22 16-13-R6-5 50 22 16-13-R6-6 40 22 C5 derivation method

[0290] The fraction to continue with the C5 derivation method was transferred to shake flasks, which were placed in a shake incubator for 24 hours at 50 °C. After 24 hours, the C5 derivation (see section pre Petition 870190096087, dated 09 / 26 / 2019, pp. 102 / 149 96 / 116 treatment above) was added and hydrolysis was continued for 50 hours without the addition of enzymes. Table 3 shows the enzyme dosages and the suspended dry matter (% by weight of SS) used. Table 3: Enzyme and suspended dry matter (SS) dosages for mixed sugar hydrolysis (MSH) using the V2 method or C5 Derivation Experiment Enzyme Dosage Suspended Dry Matter [g of Cellic® CTec3 / kg of glucan in FH] [% by weight of SS] 16-13-R6-2-FE-12-3 50 17 16-13-R6-3-FE-12-4 40 17 16-13-R6-4-FE-12-5 75 17 16-13-R6-4-FE-12-6 75 17 16-13-R6-5-FE-12-7 50 17 16-13-R6-6-FE-12-8 40 17 Method V2.X

[0291] The pulp fraction that continued in the V2.X method was pressed into a fiber cake and a filtrate as described above. The fiber cakes were allocated into six shaker flasks and resuspended in water.

[0292] Fiber cake hydrolysis was performed for 72 hours with enzyme and SS % dosages as shown in Petition 870190096087, dated 09 / 26 / 2019, pp. 103 / 149 97 / 116 Table 4. The filtered fraction was transferred to shake flasks and the C5 derivatization was added to initiate mixed sugar hydrolysis, which had a retention time of 48 hours. Table 5 shows the enzyme dosage and % SS in MSH. Both fiber cake hydrolysis and MSH were performed at 50 °C and pH 5.0-5.3. The agitation for fiber cake hydrolysis and MSH was set to 250 rpm in the shaker incubator; see also Figure 5 for an overview of the setup. Table 4: Enzyme and SS dosages for fiber cake hydrolysis using the V2.X method. Experiment: Enzyme dosage / Suspended dry matter [g of Cellic® CTec3 / kg of glucan in FH] [% by weight SS] of 16-13-R6-2-FE-13-14 50 19 16-13-R6-3-FE-13-17 40 19 16-13-R6-4-FE-13-20 75 18 16-13-R6-5-FE-13-23 50 19 16-13-R6-6-FE-13-26 40 18 Table 5: Enzyme and SS dosages for MSH using the V2.X method Experiment Enzyme dosage Suspended dry matter [g of Cellic® CTec3 / kg of glucan in FH] of [% by weight SS] of Petition 870190096087, dated 09 / 26 / 2019, pp. 104 / 149 98 / 116 16-13-R6-2-FE- 13-28 50 0 16-13-R6-3-FE- 13-29 40 0 ​​16-13-R6-4-FE- 13-30 75 0 16-13-R6-5-FE- 13-31 50 0 16-13-R6-6-FE- 13-32 40 0 Results

[0293] After all hydrolysis had been carried out, sugar concentrations were measured and mass balances were calculated. Glucan, xylan, and arabinan conversions were calculated as the sum of glucose, xylose, and arabinose after mixed sugar hydrolysis and fiber cake hydrolysis divided by the sum of glucan, xylan, and arabinan respectively in the fiber fraction and C5 derivative. The glucan conversion calculated based on the total amount of glucan from the pretreatment increases by 11-17% (relatively) when using the V2.X method compared to the C5 derivation method, see Figure 6. The xylan conversion calculated based on the total amount of xylan from the pretreatment is similar for the V2.X method and the C5 derivation method, respectively, which confirms that most xylanases follow the filtrate after pressing the paste from fiber hydrolysis (Figure 7).The conversion of arabinan, calculated based on the total amount of arabinan from the pretreatment, increases by 7 to 19% (relatively) when using the V2.X method. Petition 870190096087, dated 09 / 26 / 2019, pp. 105 / 149 99 / 116 comparison with the C5 derivation method, which shows that other hemicellulases besides xylanases follow the filtrate (Figure 8). Conclusion

[0294] Hydrolysis experiments were performed on 10 kg balances, using relevant industrial dry matter, with three different enzyme dosages in order to compare the V2 method (C5 derivation method) and the V2.X method. In all experiments, better yields were obtained for the overall yield of monomeric carbohydrates in the V2.X method. The average increase observed was 8% more absolute conversion of glucan to glucose, no significant change in the conversion of xylan to xylose was observed, and 7% more absolute conversion of arabinan to arabinose. Example 2 - Comparison of carbohydrate conversion of the fiber fraction in the V2.X method and in the V2 method (C5 derivation) with multiple pretreatments and biomasses.

[0295] The V2.X process was tested with different batches of wheat straw and different pretreatments (see also Table 6). The comparison between the V2.X method and the V2 or C5 derivation method was based on the enzymatic conversion of the fiber fractions. The total enzyme dose for the fiber fraction was similar; 75 g of Cellic® CTec3 / kg of total glucan in FH. The enzyme was added in one portion to the fiber hydrolysis in the C5 derivation method, being added in two distributed steps for the hydrolysis of fiber and fiber cake in the V2.X method. In both cases, no mixed sugar hydrolysis was performed. The comparison of mixed sugar hydrolysis in both methods is described in Example 5. Petition 870190096087, dated 09 / 26 / 2019, pp. 106 / 149 100 / 116

[0296] Fiber hydrolysis for the C5 derivation method:

[0297] The fiber fraction was added to the chambers of the free-fall reactor and water was added to achieve a SS of 22% by weight, giving a total dry matter content of 25% by weight. The pH was adjusted to 5.3 and the temperature to 50 °C. The agitator in the free-fall mixer was set to 6 rpm. After approximately 100 hours at 50 °C and pH adjusted in the range of 4.8 - 5.3, fiber hydrolysis was stopped. In one case (16-13-R6-4), the fiber mass was removed from the free-fall reactor after 72 h and fiber hydrolysis was continued in agitated flasks for another 24 h.

[0298] Table 6: Enzyme dosage for fiber hydrolysis experiment using the C5 derivatization method Wheat straw batch Hydrolysis experiment Enzyme dosage [g of Cellic® CTec3 / kg of glucan in FH] of pre-treatment WS F WS F 20150729 15-71-R6-1 75 WS F ws F 20150902 15-71-R6-2 75 WS F ws F 20150923 16-4-R6-4 75 WS F ws F 20140828 16-4-R6-2 75 WS H ws H 20160203 16-13-R6-4 75 Fiber hydrolysis and fiber cake for the V2.X method:

[0299] The fiber fraction was added to the chambers of the free-fall reactor and water was added to achieve a SS of 22% by weight, giving a total dry matter content of 25%. Petition 870190096087, dated 09 / 26 / 2019, pp. 107 / 149 101 / 116 by weight. The pH was adjusted to 5.3 and the temperature to 50 °C. The enzyme dosage for this experiment is given in Table 7. The stirrer in the free-fall mixer was set to 6 rpm. After approximately 72 hours at 50 °C and the pH adjusted in the range of 4.8–5.3, fiber hydrolysis was stopped. The paste was pressed into a fiber cake and filtered as previously described. The fiber cakes were allocated into five shaker flasks and resuspended in water, and the second portion of enzymes was added. Fiber cake hydrolysis was carried out for 68–72 hours at 50 °C and pH 5.0–5.3. The stirring for fiber cake hydrolysis was set to 250 rpm in the shaker incubator. Table 7: Enzyme dosages for fiber and fiber cake hydrolysis using the V2.X method. Dosage Batch of straw Pre-treatment experiment Dosage of enzyme, Enzyme fiber hydrolysis, Wheat cake hydrolysis treatment Hydrolysis [g of CTec3 / kg in FH] of Cellic® glucan fiber WS_F WS_F_20150729 15-78-R6-1-FE-48-1 / 2 / 11 / 12 50 25 WS_F WS_F_20150902 15-78-R6-2-FE-48-3 / 4 50 25 WS_F WS_F_20150923 15-78-R6-4-FE-48-5 / 6 50 25 WS F WS F 20140828 15-78-R6-5-FE-50 25 Petition 870190096087, dated 09 / 26 / 2019, pp. 108 / 149 102 / 116 48-7 / 8 WS_H WS_H_20160203 16-13-R6-2 / 5- 50 25 FE-13-4 / 13 Results

[0300] At the end of the hydrolysis, the sugar concentrations in all streams were measured and mass balances were established. The glucan and xylan conversions of the fibers from the pretreatment were calculated based on the monomeric sugar concentrations in the fiber slurry (C5 derivation method) or as the sum of the filtrate and the slurry of the fiber cake. The glucan and xylan conversions are shown in Figures 9 and 10. Conclusion

[0301] The hydrolysis yield of fibers after pretreatment was compared for two different batches of wheat straw and five different pretreatment dates. In all trials, a significantly better glucan conversion was achieved, 13% (relatively) more in V2.X compared to V2, of the fibers when performing two-stage hydrolysis (V2.X). Xylan conversion also showed, in most cases, better performance with two-stage hydrolysis (V2.X), giving an average increase of 8% (relatively) greater xylose from V2.X compared to V2.

[0302] The conclusion is that V2.X is yielding more than the C5 derivation method (V2) over a series of experiments with varying biomass composition and repeated pretreatment experiments with approximately 500 kg of pretreated wheat straw processed in each experiment. Petition 870190096087, dated 09 / 26 / 2019, pp. 109 / 149 103 / 116 Example 3 - Comparison of two-stage hydrolysis with enzyme dose splitting

[0303] In practical experiments, it has been shown that two-stage hydrolysis yields are higher than single-stage hydrolysis yields, but the effect is not high if all the enzyme is added in fiber hydrolysis. A significantly higher yield is obtained with two-stage hydrolysis when the enzyme dose is divided between both stages.

[0304] Three fiber hydrolyses were carried out in the free-fall reactor on a 10 kg balance, one with 50 g CTec3 / kg glucan and 22 wt% SS and two others with 75 g standard CTec3 / kg glucan; one with 22 wt% SS and the other with 18 wt% SS corresponding to the final dry matter of a two-stage hydrolysis, with 22 wt% SS in both fiber hydrolysis and fiber cake hydrolysis. Otherwise, standard hydrolysis conditions.

[0305] After 44 hours of fiber hydrolysis, the pulps were pressed into a filtrate and a fiber cake, as previously described. The fiber cake was resuspended in water to 22% SS in shake flasks. For the chamber with 50 g CTec3 / kg glucan in fiber hydrolysis, the remaining enzyme up to a total enzyme dose of 75 g CTec3 / kg of original glucan was added to the fiber cake hydrolysis. No enzyme was added to the fiber cake for the assay with 75 g CTec3 / kg glucan in fiber hydrolysis. Results

[0306] Figure 11 shows the results of one- and two-stage hydrolysis and dry matter dependence. A Petition 870190096087, dated 09 / 26 / 2019, pp. 110 / 149 The lowest conversion (71%) is obtained by conducting a one-stage hydrolysis at 22% SS. The yield is improved in a one-stage hydrolysis to 74% if the SS in the hydrolysis is reduced from 22% to 18% by weight SS. The water consumption in a one-stage hydrolysis at 18% by weight SS is equal to the water consumption in a two-step hydrolysis at 22% by weight SS (because of the two steps at 22% SS). Thus, it is not unexpected that the yield from a one-stage hydrolysis at 18% SS is yielding comparable conversion to a two-step hydrolysis at 22% by weight SS, although a small increase in yield is expected due to less product inhibition in the fiber cake hydrolysis. The yield increases from 74% to 76% when going from a one-stage hydrolysis to a two-stage hydrolysis, while maintaining the same water consumption in the overall process.A significantly higher yield is obtained with two-stage hydrolysis, where the enzyme dose is split and dosed in both stages. By adding only two-thirds of the enzyme to the fiber hydrolysis and the remaining (one-third) of the enzyme to the fiber cake hydrolysis, the glucan conversion increases from 76% to 82%, see Figure 11. Conclusion.

[0307] Going from one-stage to two-stage hydrolysis increased glucan conversion by 6% (relatively) while maintaining the same water consumption in the process. It is advantageous to add some of the enzyme to the fiber hydrolysis and the remainder of the enzyme to the fiber cake hydrolysis. This enzyme dosing method will increase the effect of the two-step hydrolysis, giving an increase in glucan conversion of 16% (relatively) instead of just 6% (relatively). Petition 870190096087, dated 09 / 26 / 2019, pp. 111 / 149 105 / 116 Example 4 - Comparison of mixed sugar hydrolysis (MSH) with fibers (C5 derivation method) and without fibers (V2.X method)

[0308] In the V2.X process, process scheme (3), the MSH is a mixture of sugar juice from fiber hydrolysis and liquid fraction - 1. The MSH is mixed in a volume ratio of approximately one part liquid fraction 1 and two parts liquid fraction 2. No enzymes are added to the mixed sugar hydrolysis. The enzymes are one part of the liquid fraction - 2. The enzymes added to the fiber hydrolysis and remaining in solution will follow the liquid fraction - 2 after the liquid-solid separation of the fiber hydrolysis.

[0309] The experimental work was set up to prove whether there is a difference in the conversion efficiency of xylo-oligomer in MSH (without fibers) and in MSH including fibers (C5 derivation process).

[0310] Standard fiber hydrolysis was performed in a free-fall reactor on a 10 kg balance. After 72 hours of fiber hydrolysis, half of the slurry was pressed and the other half was retained as a slurry. The filtrate (liquid fraction - 2) and the slurry were transferred to individual stirring flasks and liquid fraction - 1 was added to all stirring flasks. MSH was performed for 96 hours under standard conditions. Results

[0311] In Figure 12, it can be seen that the liquid fraction - 1 with added MSH (without fiber) is giving a xylan conversion similar to that of the paste with added MSH (with fiber). These data indicate that most of the relevant enzymes (xylanases) in Cellic® Ctec3 are following the filtrate (liquid fraction - 2) after fiber hydrolysis. Petition 870190096087, dated 09 / 26 / 2019, pp. 112 / 149 106 / 116

[0312] In MSH, approximately 40% of the xylan is converted to monomeric xylose at time zero. Very rapidly (< 10 h) 60% of the xylose potential is converted to xylose. After 10 hours, the conversion rate is very slow. At 48 h of MSH, 63-67% conversion is achieved. By adding large amounts of Cellic®Ctec3, xylan conversion rates of up to 90% were obtained in 48 h (see, for example, Figure 13). It is further proven in drilling experiments (data not shown) that monomeric sugars (glucose and xylose) do not inhibit xylan conversion, but pretreatment inhibitors and the concentration of oligomers in mixed sugar hydrolysis have a significant inhibitory effect on xylan conversion. Conclusion

[0313] MSH is equally efficient with and without fibers, meaning that enzymes important for the hydrolysis of hemicellulose fragments and xylo-oligomers are soluble and follow the aqueous phase. Example 5 - Dose Response in Mixed Sugar Hydrolysis

[0314] Fiber hydrolysis was carried out in a free-fall reactor on a 10 kg balance. After 72 hours of fiber hydrolysis, the paste was pressed. The filtrate (liquid fraction - 2) and liquid fraction - 1 were heated to 80 °C for 20 min to deactivate enzyme activity. 66 g of the filtrate (liquid fraction - 2) from the press were transferred to 12 shake flasks and 33 g of liquid fraction - 1 was added to all shake flasks. A different amount of Cellic®CTec3 was added and MSH was carried out for 48 hours under standard conditions. The concentration in the mixture was measured and converted to zero. Petition 870190096087, dated 09 / 26 / 2019, pp. 113 / 149 107 / 116 The addition of enzyme to heat-treated liquid was calculated. A MSH with unheated liquids and without enzyme addition was performed in conjunction with the other shake flasks, showing the conversion due to enzymes following the filtrate from fiber hydrolysis. Results

[0315] Figure 13 shows the total xylose conversion as a function of enzyme dosage. If all the enzyme added to fiber hydrolysis had been added to MSH, this would have corresponded to 240 g of Cellic®Ctec3 / kg of sugar. The result without the addition of active enzyme indicates that only a relatively small portion of the enzyme in the filtrate stream is active. However, it can also be seen that with a sufficiently high enzyme concentration and hydrolysis time, a total xylan conversion of up to 90% can be achieved.

[0316] Since the high enzyme concentration leads to almost complete and / or faster conversion of xylan, enzyme recycling in MSH could be of great advantage. Ultrafiltration (UF) is a standard unit operation for recovering enzymes from the fermentation broth. In this V2.X process, UF can recover enzymes after MSH and recycle them to MSH. This will lead to a high enzyme concentration in MSH over time, which will improve the hydrolysis of xylo-oligomers.

[0317] It is also thought that only some enzyme activities are lacking due to instability and, thus, loss of activity during the first 100 hours of reaction or adsorption to fibers or soluble compounds, such as organic degradation products or carbohydrates. The addition of Petition 870190096087, dated 09 / 26 / 2019, pp. 114 / 149 108 / 116 unique activities such as β-xylosidase can, therefore, lead to a large increase in conversion and may be advantageous compared to adding large amounts of enzyme mixtures. Conclusion

[0318] Enzymatic hydrolysis of oligomeric hemicellulose is possible in the liquid fractions from the V2.X process. A conversion of 67% is obtained by hydrolyzing the enzymatic activity transferred through the filtrate to the mixed sugar hydrolysis. However, up to 90% can be achieved by adding more enzyme, Cellic®CTec3 or other commercial enzyme mixtures or isolated activities such as βxylosidase or others. Increased conversion can also be obtained by enzyme recirculation, for example, by increasing the concentration through ultrafiltration. Example 6 - addition of β-xylosidase to MSH

[0319] It is believed that the addition of β-xylosidase will significantly increase xylan conversion, such as by at least 80 or 90% in MSH.

[0320] Fiber hydrolysis was carried out in a 10 kg free-fall reactor using 75 g CTec3 / kg of glucan. The paste is pressed after 72 hours of fiber hydrolysis. Then, 66 g of the filtrate (liquid fraction - 2) and 33 g of liquid fraction - 1 are transferred to 27 shaken flasks (three groups in triplicate). In the first group, βxylosidase is added at a concentration corresponding to 1, 5, 10, 20 and 40% of the total enzyme protein added with CTec3 for fiber hydrolysis. In the second group, CTec3 is added at a concentration corresponding to 10 and 40% of CTec3 added to fiber hydrolysis. The third group Petition 870190096087, dated 09 / 26 / 2019, pp. 115 / 149 109 / 116 is a control group with no additional enzymes added. The xylose concentration in the mixture is measured by HPLC and the xylan conversion is calculated for each treatment.

[0321] β-xylosidase can be, for example, from Bacillus pumilus, such as a high-purity recombinant β-xylosidase obtainable from Megazyme (EC 3.2.1.37; CAZy Family: GH43; CAS: 9025-53-0; in 3.2 M ammonium sulfate; provided at ~75 U / mL, with a specific activity of ~18 U / mg (35 °C, pH 7.5 in p-nitrophenylβ-D-xylopyranoside).

[0322] Samples with extra β-xylosidase reveal a significantly higher xylan conversion, such as at least 80 or 90% xylan conversion after MSH, in contrast to the control without enzyme and / or with CTec3 addition, with a xylan conversion of about 66%. Example 7 - Comparison of fermentation substrates using the V2 and V2.X methods.

[0323] The fermentation process in the V2.X process (see for example Figures 1 and 4) differs from the fermentation process in the C5 derivation process (V2), process scheme (2) (see for example Figure 3), inter alia, in the way fermentation is introduced from more than one substrate (hydrolysate from fiber cake hydrolysis and mixed sugar hydrolysis). Furthermore, the fiber cake hydrolysate (C6+C5 paste product) may undergo an additional liquid / solid separation step. In the C5 derivation process there is only one substrate (see Figure 3) and fermentation requires dilution with water at the beginning of the feed batch due to high concentrations of acetic acid and other yeast-inhibiting substances such as Petition 870190096087, dated 09 / 26 / 2019, pp. 116 / 149 110 / 116 as furfural. Otherwise, the time required for, for example, the conversion of sugar into ethanol would be significantly prolonged. The composition of the hydrolysates for fermentation in the V2.X process can be seen in Table 8.

[0324] Table 8: Composition of hydrolysates from hydrolysis of mixed sugar MSH and hydrolysis of fiber cake (see, for example, Figure 1, steps (e), and step (f), respectively). Hydrolysate from step f) [g / kg wet] Hydrolysate from step e) Glucose 97 57 Xylose 20 44 Acetic acid 2.3 9.7 Furfural 0.3 1.2 5-HMF 0.1 0.3 5-HMF: 5-(hydroxymethyl)furfural

[0325] As can be seen from Table 8, the hydrolysate from step (f) (hydrolysate - 1) contains significantly lower concentrations of inhibitor (acetic acid, furfural and 5-HMF) than the hydrolysate from step (e) (hydrolysate - 2).

[0326] Surprisingly and unexpectedly, the inventors realized that the two hydrolysates with different inhibitor concentrations could be used in a new and advantageous fermentation process. Commonly, at the beginning of fermentation, the hydrolysate is diluted with water in order to reduce the inhibitor concentration to an acceptable level. According to the present invention, a hydrolysate, Petition 870190096087, dated 09 / 26 / 2019, pp. 117 / 149 111 / 116 which is poor in inhibitor – for example, the C5 / C6 paste product or the liquid C5 / C6 product obtained after a solid-liquid separation of said C5 / C6 paste product – can be used in an initial phase of a microbial fermentation, generally a batch fermentation. In this way, dilution water can be avoided or reduced (if any dilution is still necessary), fermentation time can be reduced, and production costs can be reduced, since less water needs to be removed from the fermentation product, in addition to the aforementioned time savings. It is also conceivable that a faster fermentation, as provided, for example, according to the present invention, will reduce the risk of contamination, i.e., the growth of undesirable microorganisms, resulting in lower yields of the fermentation product.

[0327] When performing fermentation, batch feeding can provide one or more of the following benefits: Furans and / or other inhibitory compounds inhibit different microorganisms, including yeasts, and consequently, these need to be controlled and / or reduced to an appropriately low level to improve growth and / or fermentation product formation, such as yeast growth and EtOH production. By using a fed-batch approach, this can be achieved, as the yeast removes, for example, furans present in the initial batch phase. During the fed-batch phase, the yeast continuously removes the furans. Petition 870190096087, dated 09 / 26 / 2019, pp. 118 / 149 112 / 116 so the detected level is very low or even close to zero. ii. Acetic acid is another inhibitor, and by choosing an initial discontinuous phase with a low level, the production strain will have an easier start, beginning to grow and produce the product more quickly. iii. In batch feeding, it is possible to control the addition of raw material so that the glucose concentration is kept below approximately 10 g / kg wet weight, improving the conversion of xylose into C5 GMO yeast.

[0328] In all cases, it may also be important to choose the ideal starting volume in the initial batch phase compared to the total volume, and an optimal (small) amount of yeast inoculum.

[0329] One advantage of having two hydrolysate qualities in terms of inhibitor concentration, as in Version 2.X, is that it is possible to conduct fed-batch fermentations without dilution in the initial batch phase, while still being able to convert essentially all the added xylose in a suitable time period, even with a very high acetic acid concentration (such as approximately 10 g / kg), and also with a relatively high overall furfural concentration (such as about 1 g / kg of wet hydrolysate). Example 8 - Improved fermentation in the V2 process

[0330] For a fermentation in the V2 process, all the C5 by-product liquid is added to the “post-hydrolysis”, thus providing only a single substrate for fermentation (see for example, Figure 3). It is believed Petition 870190096087, dated 09 / 26 / 2019, pages 119 / 149 113 / 116 that the post-hydrolysis-related terms used in some examples correspond to mixed sugar hydrolysis (MSH). Similarly, the terms related to C5 derivation or C5 derivation liquid fraction are believed to correspond to liquid fraction 1 or first liquid fraction. Typically, it is necessary to dilute the post-hydrolysis substrate with water in the initial batch phase of a fed-batch fermentation in order to reduce the concentration of inhibitors, such as to provide an efficient and / or reliable fermentation, such as in terms of microorganism growth and / or fermentation product yield. Typically, fermentations are yeast fermentations, aiming at the production of 2G EtOH; however, it is believed that other microorganisms can also be used, thus also providing different fermentation products.

[0331] Surprisingly and unexpectedly, the inventors realized that process V2 could also be adapted in view of the above findings relating to hydrolysates or product streams with different inhibitor concentrations. See Figure 14 for an example of a main process configuration for this improved fermentation. Consequently, in order to avoid (or reduce) dilution at the start of fermentation, it is believed to be possible to use the hydrolysate from the first hydrolysis (e.g., from step (c)) as the material to start the fermentation. This hydrolysate is much lower in yeast inhibitor concentration than the Post-Hydrolysate. Since the amount of hydrolysate from step (c) that is needed for the initial discontinuous phase of Petition 870190096087, dated 09 / 26 / 2019, pp. 120 / 149 114 / 116 fermentation having been removed, the remaining hydrolysate can be mixed with the C5 derivative liquid fraction in order to ensure the hydrolysis of the C5 oligomers present in the C5 liquid. The post-hydrolysis step will then have a smaller fraction of hydrolysate from step (c), compared to the C5 liquid than in the original arrangement. The GMO yeast indicated in Figure 14 is optional; other suitable microorganisms could be used, thus allowing the provision of other fermentation products besides, for example, alcohol / EtOH. Furthermore, it is believed that such a process will work reliably with different DM concentrations and different xylan numbers. A two-step fermentation scheme is shown in Figure 15. An initial fermentation is carried out using only a fraction, such as, for example, about 5%, 10% or 20% of the fermenter volume, followed by a second fermentation, in which the fermenter is full.The first fermentation could, for example, be a batch fermentation, and the second fermentation, a fed-batch fermentation.

[0332] The ratio between the liquid fraction C5 and hydrolysate from step (c) will be higher in post-hydrolysis (see Figure 3) in the proposed improved V2 process. It was tested whether hydrolysis in step (d) would be negatively affected. Below is a description of how this was tested and that hydrolysis is not negatively affected. The results are shown in Figure 16. Materials and methods

[0333] Fiber hydrolysis (step (c)) was carried out in a vertical pilot reactor on a 240 kg scale using 75 g of CTec3 / kg of glucan with a dry matter of 22% of Petition 870190096087, dated 09 / 26 / 2019, pp. 121 / 149 115 / 116 suspended solids. After 117 hours of fiber hydrolysis, the paste was pressed. The filtrate and the liquid fraction of C5 were mixed in different ratios of 1:2, 1:1, and 2:1 in shaken flasks with a total volume of 100 grams. Post-hydrolysis was carried out in shaken incubators for 48 hours under standard conditions; pH 5.0-5.3; 50 °C. The xylose concentration in the mixture was measured by HPLC, and the xylan conversion of the liquid fraction of C5 was calculated for each ratio. To calculate the xylan conversion of the liquid fraction of C5, it was assumed that the filtrate from the hydrolysis step (c) did not contribute to the increase in xylose concentration as a function of time. Figure 16 shows changes in xylan conversion in increasing proportions of liquid C5 in post-hydrolysis. The filtrate is the liquid fraction after the hydrolysis step (c).Only very limited effects on hydrolysis efficiency are seen in Figure 16 (a 1:1 mixture is a much higher proportion of liquid C5 than would be the case for the improved fermentation setup). LIST OF REFERENCES: .Kristensen, J.B., C. Felby, and H. Jorgensen, Determining Yields in High Solids Enzymatic Hydrolysis of Biomass. Appl. Biochem. Biotechnol., 2009. 156: pg. 557 - 562. .Petersen, M.0., J. Larsen, and M.H. Thomsen, Optimization of hydrothermal pretreatment of wheat straw for production of bioethanol at low water consumption without addition of chemicals. Biomass and Bioenergy, 2009. 33: pg. 834 - 840. .Weiss, ND, et al., A Simplified Method for the Measurement of Insoluble Solids in Pretreated Biomass Slurries. Appl. Biochem. Biotechnol., 2009. 162(4): pg. 975 - 987. Petition 870190096087, dated 09 / 26 / 2019, pages 122 / 149 116 / 116 .Sluiter, A., et al., Determintation of Structural Carbohydrates and Lignin in Biomass (NREL / TP-510-42618). 2008, revisto em agosto de 2012, National Renewable Energy Laboratory. .Sluiter, A., et al., Determination of Sugars, Byproducts, and Degradation Products in Liquid Fraction Process Samples. 2005, NREL - Biomass Program. 6. Faulds e Williamson, Appl. Microbiol. Biotechnol. nov de 1995; 43(6): 1082 - 7) 7. Sorensen et al. (2005) “Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial fermentation residue” (Enzyme and Microbial Technology 36 (2005) 773 - 784) 8. Nishitani, K.; Nevins, D.J. (1988). Enzymic analysis of feruloylated arabinoxylans (Feraxan) derived from Zea mays cell walls. I. Purification of novel enzymes capable of dissociating Feraxan fragments from Zea mays coleoptile cell wall. Plant Physiol. 87: 883—890.) 9. Rasmussen (2016) “Carbohydrate degradation mechanisms and compounds from pretreated biomass” Doctoral Thesis, Technical University of Denmark. Petition 870190096087, dated 09 / 26 / 2019, pp. 123 / 149

Claims

1 / 6 CLAIMS 1. A method for providing a C5 / C6 product from a lignocellulosic material, characterized in that it comprises the steps of: a) Autohydrolysis pretreatment of the lignocellulosic material; b) Solid / liquid separation of the pretreated lignocellulosic material from step (a) into a first solid fraction and a first liquid fraction; c) Enzymatic hydrolysis of the fiber from said first solid fraction of step (b) using an enzyme composition capable of degrading lignocellulosic material, thereby providing a C5 / C6 fiber paste comprising C5 and / or C6 sugars; d) Solid / liquid separation of at least a portion of the C5 / C6 fiber paste from step (c) into a second solid fraction and a second liquid fraction;(ee) Combination of at least one portion of said first liquid fraction and at least one portion of said second liquid fraction for enzymatic hydrolysis of mixed sugar (MSH), whereby an MSH C5 / C6 product is provided, in which the xylose yield of the MSH C5 / C6 product of step (e) is at least 60% of the theoretical xylose yield.

2. Method according to claim 1, characterized in that the pretreatment is carried out: a. at a dry matter content (DM) in the range of 580%; Petition 870260019266, dated 02 / 03 / 2026, page 26 / 84 2 / 6 b. under conditions that provide a xylan number of 6-10%.

3. Method according to claim 1, characterized in that the enzymatic hydrolysis of the fiber and / or MSH is / are carried out: a. during a period of 6h, 12h, 24h, 48h, 72h, 96h or 120h; b. at a pH in the range of 3.0-6.0; c. at a temperature in the range of 30-70 °C; and at a DM content of 10-40%.

4. Method according to claim 1, characterized in that the enzyme composition capable of degrading lignocellulosic material comprises: a. a cellulase or a hemicellulase; b. a mixture of cellulase(s) and hemicellulase(s); or c. one or more xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), esterase(s) and any combination thereof; and wherein the esterase(s) comprise one or more acetylsterases and / or feroyl esterases; and / or one or more of endocellulase(s), endoglucanase(s), exocellulase(s), exoglucanase(s), endoxylanase(s), acetylxylan esterase(s), xylosidase(s), ε-glucosidase(s) and any combination thereof.

5. Method according to claim 4, characterized in that the hemicellulase(s) is / are also present in step (e); and wherein the hemicellulase(s) present in step (e) comprises xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), esterase(s), acetylesterases, feroyl esterase(s) or any combination thereof.

6. Method according to claim 5, characterized in that all or at least a fraction of the hemicellulase(s) present in step (e) was added in step (c); and / or one or more hemicellulase(s) is / are added in step (e); and / or where one or more additional enzyme(s) is / are added in step (e) and where the additional enzyme(s) is / are not present in the enzyme composition capable of degrading lignocellulosic material added in step (c); and / or the additional enzyme(s) is / are one or more of: hemicellulase(s), xylanase(s), xylosidase(s), arabinoxylanase(s), xyloglucanase(s), glucuronoxylanase(s), glucomannanase(s), esterase(s), acetylesterases, feroyl esterase(s), and any combination thereof.

7. Method according to claim 1, characterized in that step (e) is conducted by combining at least one portion of said first liquid fraction and at least one portion of said second liquid fraction and by enzymatically hydrolyzing the mixture.

8. Method according to claim 1, characterized in that step (e) comprises an ultrafiltration step (j) for recycling enzymes present after MSH.

9. Method according to claim 1, characterized in that MSH is performed without the addition of one or more enzyme(s).

10. Method according to claim 1, Petition 870260019266, dated 02 / 03 / 2026, page 28 / 84 4 / 6 characterized in that a. the second liquid fraction has a lower concentration of fermentation inhibitor than the first liquid fraction; and / or b. the second liquid fraction has a lower concentration of fermentation inhibitor than the MSH C5 / C6 product.

11. Method according to claim 1, characterized in that it further comprises a lignin recovery step, wherein said lignin recovery is carried out on the second solid fraction provided in step (d).

12. Method according to claim 1, characterized in that the method further comprises k. combining at least one portion of the MSH C5 / C6 product with at least one portion of the second liquid fraction from step (d) to obtain a combined C5 / C6 product.

13. Method according to claim 12, characterized in that the combined C5 / C6 product consists of the MSH C5 / C6 product from step (e) and the second liquid fraction from step (d).

14. Method according to claim 13, characterized in that said method further comprises the steps of: m. Providing a C5 / C6 product for fermentation by combining at least one portion of one or more of: (i) the first liquid fraction of step (b); (ii) the C5 / C6 fiber paste of step (c); (iii) the second liquid fraction of step (d); (iv) the C5 / C6 MSH product of step (e); and Petition 870260019266, dated 02 / 03 / 2026, page 29 / 84 5 / 6 (v) the combined C5 / C6 product of step (k); and n. Fermenting said C5 / C6 product with a microorganism to provide a fermentation product.

15. Method according to claim 14, characterized in that a fermentation product is provided in a fermentation broth, wherein said method further comprises the step(s) of: o. Recovering said fermentation product from the fermentation broth; and / or p. Recovering lignin from spent fermentation broth, and / or from at least a portion of the fermentation product provided from steps (n) or (o).

16. Method according to claim 14, characterized in that the fermentation is carried out in at least a first and a second fermentation step, wherein a first fermentation substrate comprising at least a portion of the C5 / C6 fiber paste provided in step (c) or the second liquid fraction provided in step (d); and a second substrate comprising at least a portion of the C5 / C6 MSH product obtained in step (e) are fermented.

17. Method according to claim 1, characterized in that the glucose yield of the C5 / C6 fiber paste from step (c) is at least 60% of the theoretical glucose yield.

18. Method according to claim 1, characterized in that the method further comprises f. enzymatic hydrolysis of the fiber cake of said second solid fraction of step (d) to obtain a C5 / C6 slurry product, wherein the xylose yield of the C5 / C6 fiber slurry of step (c) and / or the C5 / C6 slurry product of step (f) is at least 60% of the theoretical xylose yield. Petition 870260019266, dated 02 / 03 / 2026, page 30 / 84 6 / 6

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