Method for purifying biomass hydrolysate

Abstract

This invention relates to a novel and advantageous method for purifying biomass hydrolysates, as well as the purified hydrolysates produced by the method according to the invention and their use as a fermentation medium.

Description

[0001] This invention relates to a novel and advantageous method for purifying biomass hydrolysates, as well as the purified hydrolysates produced by the method according to the invention and their use as a fermentation medium.

[0002] Lignocellulose biomass derived from crop residues such as bagasse, rice straw, and barley straw, as well as other sugar- or polysaccharide- and protein-containing materials, are not only valuable sources of refined sugars such as monosaccharides or disaccharides, but also valuable sources of other components such as amino acids, proteins, and minerals.

[0003] Various methods exist in the prior art for separating components, particularly sugars, from sugar beets and sugarcane. Solutions obtained from these so-called "first-generation" matrices are typically relatively pure sugar solutions and can be used as in standard processes without significantly impacting process efficiency. In contrast, solutions obtained from the hydrolysis of "second-generation" matrices based on crop residues such as bagasse, straw, or barley straw are complex mixtures of proteins, minerals, and sugars. They also contain organic acids, colored particles, lignin degradation products, and other impurities. This makes these second-generation hydrolysates unsuitable for further processing, such as, for example, the preparation of polylactic acid from lactic acid. Existing processes involving this type of hydrolysate also suffer from severe scaling problems in pipes, pipelines, membranes, and other process units in the application, reducing process efficiency, necessitating more frequent cleaning and replacement of processing units, and resulting in significantly higher costs.

[0004] Therefore, there is a need for a method to prepare highly purified biomass hydrolysates containing the maximum amount of valuable compounds such as monosaccharides and disaccharides, but with only minimal impurities. This method increases the possibility of further processing and expands the potential applications of the hydrolysates.

[0005] The fundamental object of this invention is to provide a method for purifying biomass hydrolysates to prepare hydrolysates that does not exhibit the defects of methods known in the prior art.

[0006] In a first aspect, the present invention therefore provides a method for purifying biomass hydrolysis products, comprising the following steps:

[0007] a) Provide biomass hydrolysis products;

[0008] b) Adjust the temperature of the biomass hydrolysis products to a range of 50-95°C;

[0009] c) Add at least one acid to the biomass hydrolysis product;

[0010] d) Solid-liquid separation of biomass hydrolysis product-acid mixture to obtain solid and liquid phases;

[0011] e) Deionization of the liquid phase of the hydrolysis product-acid mixture after separation according to step d).

[0012] The inventors of this invention have surprisingly discovered that the combination of temperature regulation and acid addition (which even increases the ion content of the solution) leads to improved deionization in the later stages, and consequently, improved purification of the hydrolysate. Deionization is improved in two ways: the amount of salt removed during deionization is increased, and scaling in the deionization unit and surrounding components of the method is reduced. A further advantage of this invention is that the method can also be applied to cases where the biomass hydrolysate contains organic acids.

[0013] As used herein, the term "biomass" refers to any type of biomass known to those skilled in the art as suitable for the methods of this invention. Plant-derived biomass is particularly preferred. In a further preferred embodiment, the initial dry matter content of the biomass is selected from 10-100 wt.%, more preferably from 35-95 wt.%, and particularly preferably from 40-80 wt.%. The term "dry matter" (dm) refers to the mass-to-biomass ratio determined using an IR-balance after water and other volatile compounds have been removed from fresh tissue. Therefore, it is particularly preferred to select biomass such that its dry matter contains at least 25 wt.%, more preferably at least 40 wt.%, particularly preferably at least 60 wt.%, and even more preferably at least 80 wt.%. Furthermore, any suitable mixture of biomass will be included within the term "biomass."

[0014] The preferred biomass is "lignocellulose biomass".

[0015] The term "lignocellulosic biomass" refers to residues, wastes, and / or byproducts from forestry and agriculture, food processing, and papermaking, as well as municipal waste. Specifically, as used herein, the term "lignocellulosic biomass" includes hay and / or spelt wheat (such as wheat, rye, barley, oats), corn stalks, hay and / or rachis, forage grasses such as chopped broom grass, switchgrass (Panicum virgatum), elephant grass (Miscanthus; China reed), Sudan grass (Sorghum sudananse, Sorghum drummondi), reed (Arundo donax), bark, wood, wood residues, shavings and / or sawdust, fruit peels, rice straw, banana leaves, empty fruit bunches, and agave residues.

[0016] Other biomass suitable for this method includes manure from livestock sheds, herbaceous materials, ground coffee grounds and waste from oil presses such as rapeseed cake and wastewater from mills, papermaking raw materials and wastewater from paper mills, waste paper, and vegetable and fruit residues.

[0017] In a preferred embodiment of the method of the present invention, the biomass is selected from biomass containing cellulose, hemicellulose and / or lignin.

[0018] In a particularly preferred embodiment of the method of the present invention, the biomass is selected from beet pulp, bagasse, sugarcane stalks, wheat straw, wood, and mixtures thereof.

[0019] In another particularly preferred embodiment of the method of the present invention, the biomass is lignocellulose biomass derived from agricultural residues such as wheat straw, barley straw, soybean straw, bagasse, sugarcane leaves and stalks, sugarcane stalks, corn straw, barley straw, hay, and mixtures thereof.

[0020] As used in this invention, the term "biomass hydrolysis product" is understood to describe depolymerized polymers that have depolymerized through a hydrolysis reaction. "Hydrolysis reaction" is understood to mean the breaking of chemical bonds by adding water. Technically, one way to carry out hydrolysis is by adding a hydrolytic enzyme to the biomass.

[0021] In a preferred embodiment, the biomass hydrolysis product comprises at least 50 wt.-%, preferably at least 65 wt.-%, more preferably at least 75 wt.-%, even more preferably at least 85 wt.-%, and most preferably 99 wt.-%, of sugars in the form of monosaccharides and disaccharides, all relative to the dry matter (dm) of the biomass. In a further preferred embodiment, the biomass hydrolysis product comprises amino acids, oligopeptides, minerals, oligosaccharides and / or proteins, and organic acids. The mineral content is preferably at least 0.5 wt.-%, preferably at least 1 wt.-%, more preferably at least 2 wt.-%, and most preferably 3 wt.-%, of salts, all relative to the dry matter (dm) of the biomass. The biomass hydrolysis product may comprise organic acids such as formic acid, acetic acid, galacturonic acid, and lactic acid. It may also comprise the following degradation products: phenolic compounds such as 4-hydroxy-3-methoxyphenyl and 4-hydroxy-3,5-dimethoxyphenyl, ferulic acid, 4-hydroxybenzoic acid, levulinic acid, furfural, 5-hydroxymethylfurfural, tannins, and terpenes.

[0022] The biomass hydrolysis products used in the method of the present invention are preferably prepared according to the following method:

[0023] Preferably, the biomass is provided in granular form, for example, by cutting, grinding, milling, shearing, shear-dispersing, shredding, dispersing, and / or blending biomass prior to step (a). In a further embodiment, the biomass may undergo a pretreatment process.

[0024] Suitable methods for pretreatment of biomass include any kind of mechanical, biological, chemical, and / or physical pretreatment methods known to those skilled in the art. In a preferred embodiment, the pretreatment method is selected from mechanical crushing, acid and / or alkali treatment, wet oxidation, pH-controlled hydropyrolysis, and / or steam explosion.

[0025] "Steam explosion" preferably comprises pressurized hydrothermal treatment of lignocellulose-containing materials at temperatures of 60-350°C, preferably 80-300°C, particularly preferably 100-250°C, and most preferably 110-220°C, in the presence or absence of an acid (e.g., H₂SO₄, HCl, H₃PO₄) or alkali / alkaline (i.e., NH₄OH, NaOH, KOH, lime) catalyst, if present, at a concentration of 0.01-15% (wt. / wt.), preferably 0.05-12.5% ​​(wt. / wt.), more preferably 0.1-10% (wt. / wt.), and most preferably 0.25-7.5%. In preferred embodiments, the pressure is preferably selected from 1-100 bar, preferably 2-50 bar, also preferably 3-25 bar, and most preferably 5-15 bar. The reaction time during steam explosion must be selected from 10 seconds to 2 hours, preferably 1 minute to 1.5 hours, and most preferably 5 minutes to 1 hour, to provide efficient conversion of biomass components in preparation for enzymatic hydrolysis. In a particularly preferred embodiment, a "mechanical pulverization" pretreatment of the lignocellulose-containing material is carried out before or during the steam explosion pretreatment, wherein the mechanical pulverization is selected from machining, grinding, chopping, crushing, cutting, irradiation, milling, and combinations thereof.

[0026] "Acid pretreatment" preferably constitutes a continuous treatment with dilute acid and / or weak acid, such as sulfuric acid or another organic acid such as acetic acid, formic acid, lactic acid, phosphoric acid, nitric acid, citric acid, tartaric acid, succinic acid, hydrochloric acid, or mixtures thereof. Other acids may also be used. "Weak acid treatment" is understood to be carried out at a pH of 0.1-5, preferably 2-3. In a preferred embodiment, the acid is added at a concentration of 0.01-15 wt.-% (wt. / wt.), preferably 0.05-12.5 wt.-% (wt. / wt.), more preferably 0.1-10 wt.-% (wt. / wt.), and most preferably 0.25-7.5 wt.-%. The acid is preferably sulfuric acid. The acid can be contacted with biomass at a temperature in the range of 120-280°C, preferably 135-225°C, and most preferably 150-200°C for a time period of 1-60 minutes, preferably 2-30 minutes, and most preferably 5-15 minutes. Adding a strong acid, such as sulfuric acid, can be used in a particularly preferred embodiment to remove hemicellulose.

[0027] "Chemical pretreatment" also involves treating biomass with H2O2, ozone, Lewis acids, FeCl3, (Al)2SO4 in aqueous alcohols, glycerol, dioxane, phenol, ethylene glycol, NaOH, Na2CO3, and / or ammonia. Preferred concentrations, temperatures, and durations are selected similarly to those mentioned above regarding acid pretreatment.

[0028] “Wet oxidation pretreatment” includes the use of oxidants (such as sulfite-based oxidants).

[0029] The term "mechanical pulverization" refers to any mechanical treatment that facilitates the separation and / or release of cellulose, hemicellulose, and / or lignin from biomass.

[0030] Mechanical crushing is preferably selected from machining, grinding, chopping, crushing, cutting, irradiation, and milling, such as dry milling, wet milling, and vibratory ball milling and combinations thereof.

[0031] "Biopretreatment" refers to any biological pretreatment that promotes the separation and / or release of cellulose, hemicellulose, and / or lignin from biomass. Biopretreatment technologies may include the application of lignin-dissolving microorganisms, such as actinomycetes (e.g., Streptomyces strains) or white-rotting fungi.

[0032] The pretreatment method described above is carried out in suitable apparatus known to those skilled in the art. Suitable apparatus for chemical pretreatment can be any type of vessel, such as a tank reactor or a stirred tank reactor. Suitable apparatus for steam explosion can be any type of vessel, such as a tank reactor or a stirred tank reactor, but can also be carried out in a screw reactor, preferably a continuous screw reactor, or in a plug flow reactor, preferably a continuous plug flow reactor.

[0033] The dry matter content of the pretreated biomass is preferably selected from 20-60 wt.-%, particularly preferably 35-50 wt.-%, and most preferably the biomass is pretreated by a method that does not involve the addition of any acid and / or alkali.

[0034] However, a specific advantage of the biomass hydrolysis process lies in the fact that favorable results can still be obtained using relatively large and / or untreated biomass pellets. The size of the biomass pellets is preferably such that at least 90 wt.% of the pellets have a maximum length of 200 mm, more preferably 100 mm, even more preferably 50 mm, and most preferably 25 mm. Further preferably, the size of the biomass pellets is such that at least 95 wt.% of the pellets have a maximum length of 200 mm, more preferably 100 mm, even more preferably 50 mm, and most preferably 25 mm.

[0035] The pretreated biomass is then preferably contacted with an enzyme composition comprising at least one enzyme selected from hydrolytic enzymes.

[0036] The term "contact" (or "contacting") includes any kind of contact between biomass and enzyme composition known to those skilled in the art as suitable for the methods of the present invention. In a preferred embodiment, the "contact" between biomass and enzyme composition is achieved by adding the enzyme composition to the biomass. Furthermore, it is particularly preferred that the addition of the enzyme composition is followed by or simultaneously with the mixing of the enzyme composition and biomass.

[0037] The term "enzyme composition" refers to any composition comprising at least one enzyme selected from the class of hydrolases. The at least one enzyme selected from the class of hydrolases preferably comprises 1-99.99 wt.% (relative to the weight of the enzyme composition), more preferably 5-99 wt.%, particularly preferably 10-95 wt.%, and most preferably 20-90 wt.%, and may further comprise at least one enzyme selected from the class of lysins. In embodiments where the enzyme composition comprises at least one enzyme selected from the class of lysins, the at least one enzyme selected from the class of hydrolases preferably comprises 0.01-50 wt.% (relative to the weight of the enzyme composition), preferably 0.05-20 wt.%, more preferably 0.08-5 wt.%, and most preferably 0.1-1 wt.%.

[0038] In a preferred embodiment, the enzyme composition comprises cellulase, hemicellulase, and / or pectinase.

[0039] In a particularly preferred embodiment, the enzyme composition comprises at least one cellobiase (EC 3.2.1.-) and at least one endo-,4-β-glucanase (EC 3.2.1.4).

[0040] In a particularly preferred embodiment, the enzyme composition comprises at least one cellobiase (EC 3.2.1.), at least one endo-,4-β-glucanase (EC 3.2.1.4), at least one β-glucosidase (EC 3.2.1.4), at least one glycoside hydrolase 61 (GH61 and CBM33), at least one endo-xylanase (EC 3.2.1.8), and at least one β-xylosidase (EC 3.2.1.37).

[0041] In a particularly preferred embodiment, the enzyme composition defined above further comprises a subset selected from β-glucanase (EC 3.2.1.-), acetylxylan esterase (EC 3.1.1.72), acetylgalactan esterase (3.1.1.6), α-arabinopyranosidase (3.2.1.-), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), α-glucuronase (EC 3.2.1.139), β-mannanase (EC 3.2.1.78), pectin methyl esterase (EC 3.1.1.11), pectin acetyl esterase (EC 3.1.1.-), and rhamnogalacturonase (EC 3.1.1.11). 3.2.1.-; GH28), rhamnogalacturonan acetylesterase (EC 3.1.1.86), rhamnogalacturonan endolyase (EC 4.2.2.23), rhamnogalacturonan lyase (EC 4.2.2.-), β-mannosidase (EC 3.2.1.25), polygalacturonase (EC 3.2.1.15,67,82; GH28), and pectin / pectic acid lyase (EC 4.2.2.2,6,9,10) or one or more of these enzymes.

[0042] The terms “cellulase,” “hemicellulase,” and “pectinase” refer to any blend of enzymes involved in the hydrolytic degradation (depolymerization) of polymeric cellulose, hemicellulose, and / or pectin into monosaccharides. As used herein, the terms “cellulase,” “hemicellulase,” and “pectinase” refer to both naturally occurring and non-naturally occurring blends of various enzymes, such as those produced by organisms like filamentous fungi. "Cellulase," "hemicellulase," and "pectinase" are preferably derived from fungi such as members of the subphyla Eumycota and Oomycota, including but not limited to the following genera: Aspergillus, Acremonium, Aureobasidium, Beauveria, Cephalosporium, Ceriporiopsis, Chaetomium, Chrysosporium, Claviceps, Cochiobolus, Cryptococcus, Cyathus, Endothia, etc. * *Mucor*, *Fusarium*, *Gilocladium*, *Humicola*, *Magnaporthe*, *Myceliophthora*, *Myrothecium*, *Mucor*, *Neurospora*, *Phanerochaete*, *Podospora*, *Paecilomyces*, *Pyricularia* The genera include *Rhizomucor*, *Rhizopus*, *Schizophylum*, *Stagonospora*, *Talaromyces*, *Trichoderma*, *Thermomyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trichophyton*, and *Trametes*. In a preferred embodiment, the filamentous fungi are species of the genus *Trichoderma*.

[0043] In a preferred embodiment of the enzyme-composition, the cellulase and / or pectinase are derived from a fungal source. In a particularly preferred embodiment of the enzyme-composition, this fungal source is *Trichoderma reesei*.

[0044] The term "enzyme blend" preferably refers to a blend of enzymes secreted from a single microbial source or multiple microbial sources. In some embodiments, the enzymes used in these enzyme blends may be prepared from one or more naturally occurring or engineered filamentous fungal strains. Preferred strains are those listed above. The desired ratio of enzyme components in the final blend can be achieved by altering the relative amounts of enzymes in the final blend, for example, by supplementing with purified or partially purified enzymes. In some embodiments, the final blend may be supplemented with one or more enzymes endogenously expressed by non-filamentous fungi or expressed at relatively low levels by filamentous fungi to enhance the degradation of the cellulose matrix into fermentable sugars. The supplemented enzymes may be added to the final blend as a supplement, and the enzymes may be components of the entire fermentation broth alone, or may be purified or minimally recovered and / or purified.

[0045] The term "cellulase" refers to any enzyme capable of hydrolyzing cellulose polymers into shorter oligomers and / or glucose. Preferred cellulases in the enzyme composition include cellobiase (CBH) (EC 3.2.1.-), endo-1,4-β-glucanase (EG) (EC 3.2.1.4), β-glucosidase (EC 3.2.1.4), cellobiase (EC 3.2.1.21), glycoside hydrolase 61 (GH61 and CBM33), expansin, swollenin, loosinin, and CIP protein (EC 3.1.1.-; CE15).

[0046] The term "hemicellulase" refers to any enzyme that can degrade hemicellulose or support the degradation of hemicellulose. Preferred hemicellulases in the enzyme composition include β-glucanase (EC 3.2.1.-), endo-xylanase (EC 3.2.1.8), β-xylosidase (EC 3.2.1.37), acetylxylan esterase (EC 3.1.1.72), acetylgalactan esterase (3.1.1.6), acetylmannan esterase, feruloyl esterase (EC 3.1.1.73), glucuronoyl esterase (EC 3.1.1.-), α-L-arabinofuranosidase (EC 3.2.1.55), α-arabinopyranosidase (3.2.1.-), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), and α-glucuronidase (EC 3.2.1.22). 3.2.1.139), β-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), mannobiosidase (EC 3.2.1.100), arabinogalactan endoglucanase (EC 3.2.1.89), endoglucanase (EC 3.2.1.90), galactanase (EC 3.2.1.181), glucuronoarabinoxylan endoglucanase (EC 3.2.1.136), α-L-fucosidase (EC 3.2.1.51), coniferin β-glucosidase (EC 3.2.1.139), β-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), mannobiose 1,4-mannoside glycoside (EC 3.2.1.100), arabinogalactan endoglucanase (EC 3.2.1.89), endoglucanase (EC 3.2.1.90), galactan endoglucanase (EC 3.2.1.181), glucuronoarabinoxylan endoglucanase (EC 3.2.1.136), α-L-fucosidase (EC 3.2.1.51), coniferin β-glucosidase (EC 3.2.1.139), β-mannoside glycoside ... 3.2.1.126), xylan hydrolase (EC 3.2.1.150, 151, 155), xylan α-1,2-glucuronidase (EC 3.2.1.131), xylogalacturonan hydrolase (EC 3.2.1.-; GH28), α-amylase (EC 3.2.1.1), glucan 1,4-α-glucosidase (EC 3.2.1.3), galactan 1,3-galactosidase (GH43), 1,4-endogalactanase (EC 3.5.1.89; GH53), α-rhamnosidase (EC 3.2.1.40), β-rhamnosidase (EC 3.2.1.43), lignin peroxidase (EC 3.2.1.43), lignin peroxidase (EC 3.2.1.43), β-rhamnosidase (EC 3.2.1.43), lignin peroxidase (EC 3.2.1.126), xylan hydrolase (EC 3.2.1.150, 151, 155), xylan α-1,2-glucuronidase (EC 3.2.1.131), xylan α-galacturonidase (EC 3.2.1.126), xylan α-glucan ... 1.11.1.14), Mn peroxidase (EC 1.11.1.13), aryl alcohol oxidase (EC 1.1.3.7), glyoxal oxidase (EC 1.1.3.), carbohydrate oxidase (EC 1.1.3.4, 9, 10), laccase (EC 1.10.3.2), and cellobiose dehydrogenase (EC 1.1.99.18).

[0047] The term "pectinase" refers to any enzyme capable of degrading pectin or supporting the degradation of pectin. Preferred pectinases in enzyme compositions include polygalacturonase (EC 3.2.1.15, 67, 82; GH28), pectin / pectic acid lyase (EC 4.2.2.2, 6, 9, 10), pectin methyl esterase (EC 3.1.1.11), pectin acetylesterase (EC 3.1.1.-), rhamnogalacturonase (EC 3.2.1.-; GH28), rhamnogalacturon-polysaccharide acetylesterase (EC 3.1.1.86), rhamnogalacturon-polysaccharide endolyase (EC 4.2.2.23), rhamnogalacturon-polysaccharide lyase (EC 4.2.2.-), and rhamnogalacturon-polysaccharide galacturonohydrolase (EC 4.2.2.23). 3.2.1.-), xylogalacturonan hydrolase (EC 3.2.1.-), pectin methyl esterase (EC 3.1.1.11), β-arabinofuranosidase (EC 3.2.1.55), β-1,4-galactanase (EC 3.2.1.89), β-1,3-galactanase (EC 3.2.1.90), β-galactosidase (EC 3.2.1.23), α-galactosidase (EC 3.2.1.22), ferulic acid acetylesterase (EC 3.1.1.-), α-fucosidase (EC 3.2.1.51), (β-fucosidase) (EC 3.2.1.38), β-apiosidase (EC 3.2.1.-), α-rhamnosidase (EC 3.2.1.-). 3.2.1.40), β-rhamnosidase (EC 3.2.1.43), α-arabinopyranosidase (EC 3.2.1.-), β-glucuronidase (EC 3.2.1.31), α-glucuronidase (EC 3.2.1.139), β-xylosidase (EC 3.2.1.37) and α-xylosidase (EC 3.2.1.x).

[0048] Enzymes are classified according to nomenclature based on the International Union of Biochemistry and Molecular Biology's Enzyme Nomenclature and Classification (http: / / www.chem.qmul.ac.uk / iubmb / enzyme / ) or the Carbohydrate-Active Enzymes database (http: / / www.cazy.org / ).

[0049] The term "activity" of an enzyme refers to its catalytic activity under suitable conditions when it is used as a protein catalyst, converting a specific polymer or artificial substrate into a specific oligomer or monomer product. In this context, the term "suitable conditions" is well-known and applicable to those skilled in the art.

[0050] "Contact" can be accomplished by any means known to those skilled in the art as suitable for the purposes of this invention. Therefore, it is preferred that the enzyme mixture be added to the biomass while the biomass is simultaneously stirred within the container. The enzyme can also be immobilized on a carrier material.

[0051] In a preferred embodiment, the hydrolysis of biomass is carried out for a time sufficient to hydrolyze at least 20 wt.-%, preferably at least 30 wt.-%, more preferably at least 50 wt.-%, and most preferably at least 60 wt.-%. In a further preferred embodiment, the hydrolysis of biomass is carried out for a time sufficient to hydrolyze 10-100 wt.-%, preferably 20-90 wt.-%, even more preferably 30-85.0 wt.-%, and most preferably 40-75 wt.-% of the cellulose in the biomass. The term "hydrolysis" is understood to mean the conversion of the insoluble polymeric components of biomass into soluble monomers, dimers, and / or oligomers through chemical, physical, and / or enzymatic processes (such as hydrolysis).

[0052] In a particularly preferred embodiment, the hydrolysis of biomass is carried out for 1 minute to 136 hours, more preferably 30 minutes to 112 hours, particularly preferably 1 hour to 100 hours, even more preferably 4 hours to 96 hours, and also particularly preferably 12 hours to 85 hours.

[0053] In a further preferred embodiment, the hydrolysis of the biomass is carried out until the content of the remaining insoluble solids is less than 40 wt.%, preferably less than 30 wt.%, even more preferably less than 20 wt.%, and most preferably less than 15 wt.%. In a further preferred embodiment, the hydrolysis of the biomass is carried out until the content of the remaining insoluble solids is 5-40 wt.%, preferably 8-30 wt.%, and most preferably 10-25 wt.%.

[0054] In another preferred embodiment, the hydrolysis of biomass is carried out until the biomass is liquefied to at least 50%, preferably at least 60%, and most preferably at least 80%, of which 60-90% liquefaction is particularly preferred.

[0055] The reaction temperature during the hydrolysis process is preferably selected from 25-80°C, more preferably from 30-75°C, and particularly preferably from 35-65°C. In another preferred embodiment, the hydrolysis of biomass is carried out for 1-120 hours, preferably 2-110 hours, more preferably 3-100 hours, wherein the temperature is selected from 35-75°C or 45-65°C.

[0056] In another preferred embodiment, the pH during the hydrolysis process is preferably selected from 4-6.5, and particularly preferably from 4.5-5.5.

[0057] Suitable dosage levels and operating conditions will be apparent to those skilled in the art, particularly with reference to the detailed disclosure provided herein. Optimal dosage levels vary significantly depending on the matrix used and the pretreatment technique employed. The enzyme composition is preferably added to the biomass in an amount of 0.01-24 wt.-%, more preferably 0.025-12 wt.-%, particularly preferably 0.05-6 wt.-%, and most preferably 0.1-3 wt.-% of the dry matter of the biomass. The total enzyme (protein) concentration was determined using bovine serum albumin as a reference standard via the Bradford method (Bradford, M., 1976).

[0058] The hydrolysis of biomass is carried out in any type of container known to those skilled in the art as suitable for the methods of the present invention, preferably in a reactor. Suitable reactors are within the knowledge of those skilled in the art. Preferred containers / reactors include, but are not limited to, containers / reactors containing agitation tools and / or tools for pumping or recycling biomass contents within the reactor. Further preferred tools for preferred reactors include, but are not limited to, tools for temperature and / or pH control and adjustment.

[0059] According to step b) of the method of the present invention, the temperature of the biomass hydrolysis product is adjusted to a temperature selected from the range of 50-95°C, preferably the range of 60-90°C, and more preferably the range of 65-85°C. This adjustment is performed in any manner known to those skilled in the art as suitable for the method of the present invention.

[0060] In a particularly preferred embodiment, step b) of the method of the present invention is performed for 1 minute to 120 minutes, more preferably 2 minutes to 90 minutes and particularly preferably 3 minutes to 75 minutes, and even more preferably 30 minutes to 90 minutes and 45 minutes to 75 minutes.

[0061] According to step c) of the method of the present invention, at least one acid is added to the biomass hydrolysis product to obtain a biomass hydrolysis product-acid mixture. The at least one acid can be an organic or inorganic acid. In a preferred embodiment, the at least one acid is preferably selected from sulfuric acid, phosphoric acid, hydrochloric acid, nitric acid, acetic acid, formic acid, lactic acid, galacturonic acid, citric acid, succinic acid, and mixtures thereof. In a preferred embodiment, the at least one acid is selected from acids with a pKa value below 5.0, preferably from acids with a pKa value below 3.5, therefore a pKa value of -4.0 to 5.0 is particularly preferred and a pKa value of -3.0 to 5.0 is most preferred.

[0062] In a further preferred embodiment, at least one acid is added to the biomass hydrolysate until the biomass hydrolysate reaches a pH of 1.5-4.5, preferably 2.0-4.0 and most preferably 2.5-3.5.

[0063] In a further preferred embodiment, the temperature is selected from the range of 65-85°C, and the pH is selected from the range of 2.0-3.5. In a further preferred embodiment, the temperature is increased to 70°C and the pH is set to 2.5.

[0064] In another preferred embodiment, steps b) and c) of the method of the present invention are performed at least partially simultaneously. Therefore, it is particularly preferred that at least one acid is added during the process of adjusting the temperature of the biomass hydrolysis product from 50°C, and more preferably from 60°C, to a temperature selected from the range of 65-90°C. Preferably, at least one acid is added during the process of adjusting the temperature of the biomass hydrolysis product from 50°C, and more preferably from 60°C, to a temperature selected from the range of 65-85°C.

[0065] In another preferred embodiment, the temperature of at least one acid is selected from the range of 5-50°C, preferably 10-40°C and most preferably 15-30°C, and the at least one acid is added to the biomass hydrolysate at a biomass hydrolysate temperature selected from the range of 50-95°C, preferably 65-85°C. Therefore, it is particularly preferred that the temperature difference between the at least one acid and the biomass hydrolysate is selected from the range of 35-95%, preferably 40-90%.

[0066] The scope of this invention also includes step c) of the method of the invention performed before step b).

[0067] The composition obtained after adding at least one acid to the biomass hydrolysate and adjusting the temperature of the biomass hydrolysate is referred to as a "biomass hydrolysate-acid mixture" within the scope of this application.

[0068] According to step (d) of the method of the present invention, the solid phase and liquid phase are separated from the biomass hydrolysis product-acid mixture. The separation of the solid and liquid phases of the hydrolysis product-acid mixture (hereinafter, "liquid phase" or "liquid phase of hydrolysis product" is used synonymously with "liquid phase of hydrolysis product-acid mixture") can be carried out by any means known to those skilled in the art as suitable for the purposes of the present invention, and is preferably carried out by filtration, centrifugation, decantation, or pressing (e.g., by a screw press). A filter press is preferred, and a membrane filter press is most preferred. In a preferred embodiment, the filter cloth of the filter press has a filter cloth concentration of 2-10 L / dm³. 2 / min of cloth air permeability. Filter aids such as diatomaceous earth or kieselguhr or perlite may also be added during the filtration process, preferably at a concentration of 0.1 wt.-% to 10 wt.-%, more preferably 0.5 wt.-% to 5 wt.-%, and most preferably 1 wt.-% to 3 wt.-%.

[0069] After the separation of the solid and liquid phases, deionization of the liquid phase according to step (e) is performed. Deionization is preferably carried out by electrodialysis, capacitive deionization, membrane capacitive deionization, nanofiltration, reverse osmosis, chromatographic separation such as ion exchange chromatography, hydrophobic interaction chromatography, and / or size exclusion chromatography, or any combination of two or more of these methods. "Membrane capacitive deionization" is performed by inserting cation exchange membranes and anion exchange membranes into the capacitive deionization unit.

[0070] In a particularly preferred embodiment, deionization is performed by standard electrodialysis or by electrodialysis using at least one bipolar membrane, and is particularly preferred to be followed by capacitive deionization, membrane capacitive deionization, or ion exchange chromatography.

[0071] When deionization is performed using standard electrodialysis or electrodialysis using at least one bipolar membrane, the ions removed from the solution are preferably recovered in a liquid called a "concentrate". In this regard, it is particularly preferred that the liquid be added to the compartment of the electrodialysis apparatus before deionization begins. In a further preferred embodiment, this liquid is not replaced after a given amount of deionization is stopped, but the concentrate is reused in repeated deionization cycles for at least two cycles, more preferably at least four cycles, particularly preferably six cycles, and most preferably ten cycles.

[0072] In this invention, "electrodialysis using at least one bipolar membrane" is understood to include the use of a membrane suitable for removing ions such as Na+ present in the liquid phase. + K + Mg 2+ Ca 2+ SO4 2- PO3 3- Cl -Any technique using three different types of membranes to remove salts by decomposing H2O. Electrodialysis using at least one bipolar membrane preferably includes the use of a cation exchange membrane, an anion exchange membrane, and a catalytic intermediate layer, a so-called "bipolar membrane," to be able to decompose water in the liquid phase into protons and hydroxide ions. By combining the selective removal of salts via cation and anion exchange membranes with the simultaneous water dissociation on the catalytic intermediate layer, acid and base components are formed.

[0073] In a preferred embodiment, at least one cation exchange membrane, at least one anion exchange membrane, and at least one catalytic intermediate or bipolar membrane are used. In a further preferred embodiment, at least two sets of these membranes are arranged in series, preferably at least four sets, even more preferably at least six sets, and most preferably at least ten sets. In a particularly preferred embodiment, all three types of membranes or all sets of membranes defined above are arranged in a single device.

[0074] Deionization is preferably carried out at a temperature within the range of 5°C-80°C, more preferably 10°C-75°C, and most preferably 15°C-70°C. The pressure drop across the electrodialysis unit is preferably less than 1 bar, more preferably less than 0.5 bar. In a further particularly preferred embodiment, deionization is carried out until the conductivity of the solution decreases to at least 10 mS / cm, more preferably at least 6 mS / cm, particularly preferably at least 4 mS / cm, and most preferably at least 2 mS / cm.

[0075] In a further preferred embodiment, electrodialysis deionization using at least one bipolar membrane is followed by capacitive deionization. Capacitive deionization is preferably applied as so-called "membrane capacitive deionization," i.e., by inserting cation exchange membranes and anion exchange membranes into the capacitive deionization unit. If membrane capacitive deionization is performed after electrodialysis using at least one bipolar membrane, electrodialysis is preferably continued until the conductivity of the solution is reduced to at least 10 mS / cm, more preferably at least 6 mS / cm, particularly preferably at least 4 mS / cm, and most preferably at least 2 mS / cm before switching to membrane capacitive deionization. The membrane capacitive deionization of the subsequent electrodialysis is then used to further reduce the conductivity of the solution to preferably at least 8 mS / cm, more preferably at least 6 mS / cm, particularly preferably at least 4 mS / cm, and most preferably at least 2 mS / cm.

[0076] In a further preferred embodiment, ion exchange chromatography is performed following deionization using at least one bipolar membrane via electrodialysis. If ion exchange chromatography is performed after electrodialysis using at least one bipolar membrane, electrodialysis is preferably performed until the conductivity of the solution is reduced to at least 10 mS / cm, more preferably at least 6 mS / cm, particularly preferably at least 4 mS / cm, and most preferably at least 2 mS / cm before switching to membrane capacitance deionization. The subsequent ion exchange chromatography is then used to further reduce the conductivity of the solution to preferably at least 8 mS / cm, more preferably at least 6 mS / cm, particularly preferably at least 4 mS / cm, and most preferably at least 2 mS / cm.

[0077] In this invention, "ion exchange" is defined as the exchange of ions between a solution containing at least one ion and a solid polymer or mineral ion exchange material, wherein ions dissolved in the solution are exchanged and replaced by ions of the same charge through contact with the ion exchange material.

[0078] Deionization via electrodialysis using at least one bipolar membrane reduces waste generated during deionization and process costs in other applications. The use of at least one bipolar membrane in electrodialysis results in the production of an alkaline fraction, which can be used as a pH agent, for example, for enzyme production, biomass hydrolysis, or biomass pretreatment. In a preferred embodiment, the produced alkaline fraction has a pH of 9-14, more preferably 12-13. The use of at least one bipolar membrane in electrodialysis also results in the production of an acidic fraction, which can be used, for example, for biomass hydrolysis or biomass pretreatment, or step c) of the method of the present invention. In a preferred embodiment, the produced acidic fraction has a pH of 1-5, more preferably 2-4. It is particularly preferred that the acidic fraction be used for steam explosion. Most preferably, the acidic fraction is added to the biomass hydrolysis product in step c) of the method of the present invention. Deionization via electrodialysis is preferably carried out in the liquid phase at a temperature in the range of 5°C-80°C, more preferably 10°C-75°C, and most preferably 15°C-70°C. The pressure drop across the electrodialysis unit is preferably less than 1 bar, more preferably less than 0.5 bar. In a further, particularly preferred embodiment, deionization is performed by standard electrodialysis or by electrodialysis using at least one bipolar membrane until the conductivity of the solution decreases to 10 mS / cm, more preferably 6 mS / cm, particularly preferably 4 mS / cm, and most preferably 2 mS / cm. In a further preferred embodiment, deionization is then further continued by capacitive deionization, membrane capacitive deionization, or ion exchange chromatography.

[0079] In a preferred embodiment, the ion exchange resin used in the ion exchange chromatography step is a cation exchange resin and an anion exchange resin. In a further preferred embodiment, the anion exchange resin and the cation exchange resin are used in a subsequent ion exchange step. Particularly preferred anion exchange resins are those having tertiary amine functional groups. The anion exchange resin matrix is ​​preferably a styrene-divinylbenzene copolymer or a cross-linked acrylic gel structure. Further preferred are anion exchange resins in the OH- form. Particularly preferred cation exchange resins are cation exchange resins having sulfonate or carboxylic acid functional groups. The cation exchange resin matrix is ​​preferably a styrene-divinylbenzene copolymer or a cross-linked acrylic structure. Further preferred are cation exchange resins in the H+ form. In a preferred embodiment, the ion exchange resin has a capacity of at least 0.5 eq / L resin, more preferably at least 1 eq / L resin, and most preferably at least 2 eq / L resin. 1 eq is defined as 1 mol of ions to be exchanged through the resin divided by the valence of that ion.

[0080] When cation exchange resin is brought into contact with a liquid, the liquid should be at a temperature of 5°C to 135°C, preferably 10°C to 70°C. When anion exchange resin is brought into contact with a liquid, the liquid should be at a temperature of 5°C to 75°C, preferably 10°C to 60°C.

[0081] In a preferred embodiment, the contact time between the ion exchange resin and the liquid for each contact should be between 0.1 and 300 min, more preferably between 0.2 and 100 min, and most preferably between 0.3 and 10 min.

[0082] Cation exchange resins are regenerated using acid, preferably sulfuric acid, nitric acid, phosphoric acid, or hydrochloric acid. The acid used should be concentrated, preferably between 0.05 and 20 M, and most preferably between 0.5 and 10 M. Regeneration of the cation exchange resin is preferably carried out at at least 15°C. Anion exchange resins are regenerated using alkali, preferably sodium hydroxide, sodium carbonate, or ammonium carbonate. The alkali used should be concentrated, preferably between 0.05 and 20 M, and most preferably between 0.5 and 10 M. Regeneration of the anion exchange resin is preferably carried out at at least 15°C. The contact time between the ion exchange resin and the alkali or acid should preferably be at least 5 minutes, more preferably at least 15 minutes. Both cation and anion exchange resins are preferably subjected to at least 500 deionization-regeneration cycles, more preferably at least 1500 cycles.

[0083] In a preferred embodiment, ion exchange chromatography is performed in a fixed bed or loose bed within a chromatographic column. However, the ion exchange according to the invention is not performed in a simulated moving bed apparatus. Simulated moving bed apparatuses are only used when substances with very similar properties must be separated from each other, for example, in the case of separating glucose and xylose, two sugar monomers. However, in the method of the invention, salts are removed from the liquid, i.e., ions (charged substances) are separated from the remainder of the component (uncharged substances). When the two types of components to be separated are significantly different in their fundamental physical properties, simulated moving bed apparatuses are unsuitable for the method of the invention. Furthermore, since simulated moving bed apparatuses are rather complex and expensive, the preferred embodiment using a fixed bed or loose bed within a chromatographic column offers further advantages.

[0084] In a further preferred embodiment, the anion exchange resin and the cation exchange resin are in two different columns and are not mixed. In a further preferred embodiment, the liquid to be deionized is first contacted with the cation exchange resin and then with the anion exchange resin. In a further preferred embodiment, the liquid is contacted with the cation exchange resin, then with the anion exchange resin, and then with a new cation exchange resin or the cation exchange resin already used in the first step. In a further preferred embodiment, the cation exchange resin and anion exchange resin cycle is repeated. The ion exchange resin used in the repeated cycles can be a new ion exchange resin or an ion exchange resin already used in the previous cycle. The number of repeated contact cycles between the ion exchange resin and the liquid is preferably 1-10, most preferably 2-5.

[0085] When the liquid is brought into contact with the ion exchange resin in the column, the flow rate should preferably be 1-200 bed volumes / hour, more preferably 2-80 bed volumes / hour.

[0086] In a further preferred embodiment, ion exchange chromatography is performed in a stirred tank.

[0087] In a further particularly preferred embodiment, at least one adsorbent is added before or during any of steps (b), (c), or (d). The at least one adsorbent is preferably selected from bentonite, charcoal, activated carbon, diatomaceous earth or loose diatomaceous earth, perlite, bleaching clay, clay minerals, polymer resins, and mixtures thereof.

[0088] Steps a)-e) of the method of the present invention result in a composition referred to as "purified hydrolysis product" within the scope of this application.

[0089] Another aspect of the invention relates to purified hydrolysate prepared according to the method of the invention as defined herein. The salt content of the purified hydrolysate is preferably up to 80%, more preferably up to 60%, more preferably up to 40%, more preferably up to 20%, and most preferably up to 10%, all relative to the salt content after hydrolysis of the matrix.

[0090] The present invention further relates to the use of purified hydrolysis products prepared according to the method of the present invention as fermentation media.

[0091] Valuable organic compounds produced by bacterial fermentation of purified hydrolysates include, but are not limited to, organic acids (such as acetic acid, lactic acid, succinic acid, itaconic acid, fumaric acid, propionic acid, and glucuronic acid), amino acids (such as glutamic acid, leucine, lysine, threonine, aspartic acid, phenylalanine, and cysteine), caprolactams (such as α-amino-caprolactam), antibiotics (such as bleomycin, virginicin, lincomycin, monensin, blastomycin, and tetracycline), vitamins (such as vitamins B2, B12, and C), enzymes, nucleotides / nucleosides (such as NADH, ATP, cAMP, FAD, and coenzyme A), biogases, biopolymers (such as polyhydroxybutyrate and polyamide / silk protein), proteins, polysaccharides (such as xanthan gum and dextran), glycosaminoglycans (such as hyaluronic acid), as well as organic solvents and biofuels (such as acetone, ethanol, butanol, and propylene glycol).

[0092] Valuable organic compounds produced by yeast fermentation of purified hydrolysates include, but are not limited to, organic solvents (e.g., ethanol, propanol), nucleotides (e.g., RNA), biosurfactants (e.g., sophorolipids), enzymes, and biopolymers (e.g., spider silk proteins).

[0093] Valuable organic compounds produced by fungal fermentation of purified hydrolysates include organic acids (such as citric acid, fumaric acid, and itaconic acid), antibiotics (such as penicillin and cephalosporins), enzymes, and polysaccharides (such as chitin).

[0094] In a further preferred embodiment of this method, the organic compound is selected from alcohols, organic acids, biopolymers, antibiotics, amino acids, caprolactams, polysaccharides, organic solvents, biofuels, glycosaminoglycans, nucleotides / nucleosides, vitamins, biosurfactants, enzymes, and mixtures thereof.

[0095] The following describes particularly preferred embodiments of the method of the present invention, which are not to be construed as limiting the invention in any way.

[0096] Particularly preferred embodiment 1

[0097] A particularly preferred method is for purifying biomass hydrolysis products, comprising the following steps:

[0098] a) Provide biomass hydrolysis products;

[0099] b) Adjust the temperature of the biomass hydrolysis products to a range of 50-95°C, preferably 30-90°C, particularly preferably 45-75°C, and most preferably up to 70°C.

[0100] c) Add at least one acid to the biomass hydrolysis product;

[0101] d) Solid-liquid separation of biomass hydrolysis product-acid mixture to obtain solid and liquid phases;

[0102] e) Deionization of the liquid phase of the hydrolysis product-acid mixture after separation according to step d);

[0103] The biomass is a lignocellulose matrix, preferably grain straw or sugarcane bagasse, and especially preferably pretreated grain straw or sugarcane bagasse.

[0104] Particularly preferred embodiment 2

[0105] For the method defined in the particularly preferred embodiment 1, step b) is performed for 1-90 minutes, preferably 2-75 minutes.

[0106] Particularly preferred embodiment 3

[0107] As defined in particularly preferred embodiment 1 or 2, the acid is an organic acid, preferably sulfuric acid, and the pH of the hydrolysis product is adjusted to 2.0-3.0.

[0108] Particularly preferred embodiment 4

[0109] For example, in the method defined by any of the particularly preferred embodiments 1-3, step c) is performed after step b).

[0110] Particularly preferred embodiment 5

[0111] For the method defined in any of the particularly preferred embodiments 1-4, the solid-liquid separation is carried out by a filter press, preferably by a membrane filter press.

[0112] Particularly preferred embodiment 6

[0113] For example, in the method defined in any of the particularly preferred embodiments 1-5, deionization is performed by electrodialysis.

[0114] Particularly preferred embodiment 7

[0115] For the method defined in any of the particularly preferred embodiments 1-6, deionization is performed by electrodialysis followed by an ion exchange chromatography step or by membrane capacitance deionization.

[0116] Particularly preferred embodiment 8

[0117] For example, in the method defined in any of the particularly preferred embodiments 1-7, deionization is carried out by electrodialysis using at least one bipolar membrane.

[0118] Particularly preferred embodiment 9

[0119] For example, in the method defined in any one of the particularly preferred embodiments 1-6 or 8, deionization is performed by electrodialysis using at least one bipolar membrane followed by an ion exchange chromatography step or membrane capacitance deionization.

[0120] Particularly preferred embodiment 10

[0121] For the method defined in any of the particularly preferred embodiments 5-9, the deionization by electrodialysis is preferably carried out at a temperature in the range of 5°C-80°C, more preferably 10°C-75°C, and most preferably 15°C-70°C.

[0122] Particularly preferred embodiment 11

[0123] For the method defined in any of the particularly preferred embodiments 5-10, deionization is carried out by electrodialysis, and the pressure drop across the electrodialysis unit is preferably less than 1 bar, more preferably less than 0.5 bar.

[0124] Particularly preferred embodiment 12

[0125] For the method defined in any of the particularly preferred embodiments 1-4, deionization is performed by ion exchange chromatography, preferably by cation exchange followed by anion exchange.

[0126] Particularly preferred embodiment 13

[0127] A particularly preferred method is for purifying biomass hydrolysis products, comprising the following steps:

[0128] a) Providing biomass hydrolysis products from pretreated grain straw or bagasse;

[0129] b) Adjust the temperature of the biomass hydrolysis products to a temperature selected from the range of 30-90°C for 2-75 minutes;

[0130] c) Add at least one organic acid to the biomass hydrolysis product to adjust the pH to 2.0-3.0;

[0131] d) Solid-liquid separation of biomass hydrolysis product-acid mixture by membrane filter press to obtain solid and liquid phases;

[0132] e) Deionization of the liquid phase of the hydrolysis product-acid mixture after separation according to step d), wherein the deionization is carried out by electrodialysis followed by an ion exchange chromatography step or membrane capacitance deionization.

[0133] Particularly preferred embodiment 14

[0134] A particularly preferred method is for purifying biomass hydrolysis products, comprising the following steps:

[0135] a) Providing biomass hydrolysis products from pretreated grain straw or bagasse;

[0136] b) Adjust the temperature of the biomass hydrolysis products to a temperature selected from the range of 30-90°C for 2-75 minutes;

[0137] c) Add at least one organic acid to the biomass hydrolysis product to adjust the pH to 2.0-3.0;

[0138] d) Solid-liquid separation of biomass hydrolysis product-acid mixture by membrane filter press to obtain solid and liquid phases;

[0139] e) Deionization of the liquid phase of the hydrolysis product-acid mixture after separation according to step d).

[0140] Deionization is performed by ion exchange chromatography, which involves cation exchange followed by anion exchange.

[0141] Particularly preferred embodiment 15

[0142] A particularly preferred method is for purifying biomass hydrolysis products, comprising the following steps:

[0143] a) Providing biomass hydrolysis products from pretreated grain straw or bagasse;

[0144] b) Adjust the temperature of the biomass hydrolysis products to a temperature selected from the range of 30-90°C for 2-75 minutes;

[0145] c) Add at least one organic acid to the biomass hydrolysis product to adjust the pH to 2.0-3.0;

[0146] d) Solid-liquid separation of biomass hydrolysis product-acid mixture by membrane filter press to obtain solid and liquid phases;

[0147] e) Deionization of the liquid phase of the hydrolysis product-acid mixture after separation according to step d).

[0148] Deionization is performed by ion exchange chromatography, which involves cation exchange followed by anion exchange.

[0149] Particularly preferred embodiment 16

[0150] A particularly preferred method is for purifying biomass hydrolysis products, comprising the following steps:

[0151] a) Providing biomass hydrolysis products from pretreated grain straw or bagasse;

[0152] b) Adjust the temperature of the biomass hydrolysis products to a temperature selected from the range of 30-90°C for 2-75 minutes;

[0153] c) Add at least one organic acid to the biomass hydrolysis product to adjust the pH to 2.0-3.0;

[0154] d) Solid-liquid separation of biomass hydrolysis product-acid mixture by membrane filter press to obtain solid and liquid phases;

[0155] e) Deionization of the liquid phase of the hydrolysis product-acid mixture after separation according to step d).

[0156] Deionization is performed via ion-exchange chromatography involving cation exchange followed by anion exchange, and

[0157] The additive is added before adjusting the temperature according to step b).

[0158] Particularly preferred embodiment 17

[0159] A particularly preferred method is for purifying biomass hydrolysis products, comprising the following steps:

[0160] a) Providing biomass hydrolysis products from pretreated grain straw or bagasse;

[0161] b) Adjust the temperature of the biomass hydrolysis products to a temperature selected from the range of 30-90°C for 2-75 minutes;

[0162] c) Add at least one organic acid to the biomass hydrolysis product to adjust the pH to 2.0-3.0;

[0163] d) Solid-liquid separation of biomass hydrolysis product-acid mixture by membrane filter press to obtain solid and liquid phases;

[0164] e) Deionization of the liquid phase of the hydrolysis product-acid mixture after separation according to step d).

[0165] Deionization is performed via ion-exchange chromatography involving cation exchange followed by anion exchange, and

[0166] The additive is added after adjusting the pH according to step c).

[0167] Examples and figures

[0168] The present invention will now be described with reference to the following embodiments and accompanying drawings. The embodiments and drawings are for illustrative purposes only and should not be construed as limiting the invention.

[0169] Figure 1 The results show a relative increase in salt removal after ion exchange chromatography of the untreated hydrolysate (left column) and the treated hydrolysate (right column) after ion exchange chromatography (the method of the present invention: heating to 70°C, followed by pH change to 2.5) when the method of the present invention is carried out according to Example 1.

[0170] Figure 2 The results show the relative increase in the weight of anion exchange resin after ion exchange chromatography of the untreated hydrolysate (left column) and the treated hydrolysate (method of the present invention: heating to 70°C, followed by pH change to 2.5) (right column) when the method of the present invention is carried out according to Example 1.

[0171] Figure 3 The results show a relative increase in salt removal when the method of the present invention is carried out according to Example 2, after ion exchange chromatography of the untreated hydrolysate (left column) and after ion exchange chromatography of the treated hydrolysate (the method of the present invention: adding bentonite, heating to 70°C, and then changing the pH to 2.5) (right column).

[0172] Figure 4 The results show the relative increase in the weight of anion exchange resin after ion exchange chromatography of the untreated hydrolysate (left column) and the treated hydrolysate (right column) after ion exchange chromatography of the hydrolysate (method of the present invention: addition of bentonite, heating to 70°C, followed by pH change to 2.5) when the method of the present invention is carried out according to Example 2.

[0173] Figure 5 The results show a relative increase in salt removal when the method of the present invention is carried out according to Example 3, after ion exchange chromatography of the untreated hydrolysate (left column) and after ion exchange chromatography of the treated hydrolysate (the method of the present invention: heating to 70°C, followed by pH change to 2.5 and addition of loose diatomaceous earth) (right column).

[0174] Figure 6 The results show the relative increase in the weight of anion exchange resin after ion exchange chromatography of the untreated hydrolysate (left column) and the treated hydrolysate (right column) after ion exchange chromatography (the method of the present invention: heating to 70°C, followed by pH adjustment to 2.5 and addition of loose diatomaceous earth) when the method of the present invention is carried out according to Example 3.

[0175] Figure 7This shows the relative amount of xylose consumed after 16 hours of fermentation in Pachysolen tannophilus when using the hydrolysate treated according to the present invention as described in Example 4.

[0176] Figure 8 The fermentation yield is shown as g itaconic acid produced per g sugar after 100 h of Aspergillus terreus fermentation using the hydrolysate treated according to the present invention, as described in Example 5.

[0177] Figure 9 The fermentation yield is shown as g itaconic acid produced per g sugar after 100 h of Aspergillus terreus fermentation using the hydrolysate treated according to the present invention, as described in Example 6.

[0178] Example 1:

[0179] Grain straw with a dry matter content of 45 wt.-% was pretreated by steam explosion (220°C). After steam explosion, the treated grain straw (“substrate”) was introduced into a mixing tank (Labfors, Infors AG, Switzerland). Containing 91.3 wt.-% An enzyme composition consisting of cellulase (C2730 Sigma) from *Trichoderma reesei* ATCC 26921 and 8.7 wt.-% glucosidase (49291 Sigma) was added to the matrix at an enzyme-to-solid ratio of 0.5 wt.-% to hydrolyze the matrix to obtain a slurry. Hydrolysis was carried out at 50°C and pH 5.0 with stirring at 50 rpm for 72 hours. After hydrolysis, the slurry was heated to 70°C for 1 hour with stirring at 200 rpm, and then the pH was set to 2.5 using 1 M H₂SO₄. The slurry thus treated was then used at a constant pressure of 3 bar with a flow rate of 5 L / dm³. 2 The filter cloth with a permeability of 5 mL / min was filtered using a filter press to obtain a liquid phase and a solid phase. 200 mL of the liquid phase was then deionized using ion exchange resin: the liquid was pumped at a rate of 5 mL / min and at room temperature to a solution containing 30 g of cation exchange resin (…). S8528 (Lanxess) glass column (XK16, GE Healthcare). After the cation exchange column, the resulting liquid phase was pumped at a rate of 5 mL / min to a container containing 30 g of anion exchange resin (S8528, Lanxess). S6368 A, Lanxess) in a glass column (XK16, GE Healthcare). The same deionization was performed using hydrolysates without a heating step and a pH change treatment to pH 2.5 (i.e., prior art methods). The improved purification process was demonstrated in two ways: (1) deionization efficiency and (2) scaling on the IEX resin.

[0180] The deionization efficiency in both experiments was determined by measuring the amount of salt removed from the liquid phase of the hydrolysis products. The results are shown in... Figure 1 The comparison showed that salt removal from the liquid phase of the hydrolysate treated with heating and pH shift was significantly increased compared to salt removal from the untreated liquid phase of the hydrolysate (a prior art method).

[0181] In both experiments, scaling of the anion exchange resin was determined by comparing the increase in resin weight before and after deionization. Results were shown in... Figure 2 The comparison of this value between the two tests showed that the resin in contact with untreated hydrolysis products (generated according to existing methods) had 30.3% more scaling.

[0182] Example 2:

[0183] Grain straw with a dry matter content of 45 wt.-% was pretreated by steam explosion (220°C). After steam explosion, the treated grain straw (“substrate”) was introduced into a mixing tank (Labfors, Infors AG, Switzerland). Containing 91.3 wt.-% An enzyme composition consisting of cellulase (C2730 Sigma) from *Trichoderma reesei* ATCC 26921 and 8.7 wt.-% glucosidase (49291 Sigma) was added to the matrix at an enzyme-to-solid ratio of 0.5 wt.-% to hydrolyze the matrix to obtain a slurry. Hydrolysis was carried out at 50°C and pH 5.0 with stirring at 50 rpm for 72 hours. After hydrolysis, 2 wt.-% bentonite was added to the slurry. 210FF, Clariant Produkte (Deutschland) GmbH), and the mixture was stirred at 200 rpm for 1 h at room temperature. The slurry was then heated to 70°C for 1 h while stirring at 200 rpm, and the pH was then set to 2.5 using 1 M H₂SO₄. The slurry thus treated was then used at a constant pressure of 3 bar with a flow rate of 5 L / dm³. 2 The filter cloth with a permeability of 5 mL / min was filtered using a filter press to obtain a liquid phase and a solid phase. 200 mL of the liquid phase was then deionized using ion exchange resin: the liquid was pumped at a rate of 5 mL / min and at room temperature to a solution containing 30 g of cation exchange resin (…). S8528, Lanxess, glass column (XK16, GE Healthcare). After the cation exchange column, the resulting liquid phase was pumped at a rate of 5 mL / min to a container containing 30 g of anion exchange resin at room temperature. In a glass column (XK16, GE Healthcare) of S6368A (Lanxess). The same deionization was performed using hydrolysates without bentonite, heating steps, and pH shift treatment to pH 2.5 (i.e., prior art methods). The improved purification process was demonstrated in two ways: (1) deionization efficiency and (2) scaling on the IEX resin.

[0184] The deionization efficiency in both experiments was determined by measuring the amount of salt removed from the liquid phase of the hydrolysis products. The results are shown in... Figure 3 The comparison showed that salt removal from the liquid phase of the hydrolysis product treated with bentonite, a heating step, and a pH change was significantly increased compared to salt removal from the untreated liquid phase of the hydrolysis product (using prior art methods).

[0185] In both experiments, scaling of the anion exchange resin was determined by comparing the increase in resin weight before and after deionization. Results were shown in... Figure 4 The comparison of this value between the two tests showed that the resin in contact with untreated hydrolysis products (produced according to existing methods) had 26.5% more scaling.

[0186] Example 3:

[0187] Grain straw with a dry matter content of 45 wt.-% was pretreated by steam explosion (220°C). After steam explosion, the treated grain straw (“substrate”) was introduced into a mixing tank (Labfors, Infors AG, Switzerland). Containing 91.3 wt.-% An enzyme composition consisting of cellulase (C2730 Sigma) from *Trichoderma reesei* ATCC 26921 and 8.7 wt.-% glucosidase (49291 Sigma) was added to the matrix at an enzyme-to-solid ratio of 0.5 wt.-% to hydrolyze the matrix to obtain a slurry. Hydrolysis was carried out at 50 °C and pH 5.0 with stirring at 50 rpm for 72 hours. After hydrolysis, the slurry was heated to 70 °C for 1 hour with stirring at 200 rpm, and then the pH was set to 2.5 using 1 M H₂SO₄. Then, 2 wt.-% dispersed diatomaceous earth was added to the slurry. 200 (Eaton), and stirred at 200 rpm for 1 hour at room temperature. The slurry thus treated was then used at a constant pressure of 3 bar with a flow rate of 5 L / dm³. 2The filter cloth with a permeability of / min was filtered using a filter press to obtain a liquid phase and a solid phase. The liquid phase was then deionized using an ion exchange resin: the liquid was poured into a glass stirred tank (Multifors, Infors AG) and 15 wt.% cation exchange resin was added at room temperature. S8528, Lanxess). The mixture was stirred at 200 rpm for 1 h. Then, the cation exchange resin was removed by filtering the mixture through a paper filter (Black ribbon 589 / 1, Whatman). The resulting liquid phase was poured back into a glass stirred tank (Multifors, Infors AG) and 15 wt.% anion exchange resin was added at room temperature. S6368 A, Lanxess). The mixture was stirred at 200 rpm for 1 h. Then, the anion exchange resin was filtered to remove the mixture using a paper filter (Black ribbon 589 / 1, Whatman). The same deionization was performed using a pH transition to pH 2.5 without heating, followed by the addition of hydrolysate treated with diatomaceous earth (“prior art” method). The improved purification process was demonstrated in two ways: (1) deionization efficiency and (2) scaling on the IEX resin.

[0188] The deionization efficiency in both experiments was determined by measuring the amount of salt removed from the liquid phase of the hydrolysis products. The results are shown in... Figure 5 The comparison showed that salt removal of the liquid phase of the hydrolysate treated with a heating step and a pH change to pH 2.5 followed by the addition of diatomaceous earth was significantly increased compared to salt removal of the liquid phase of the untreated hydrolysate (using prior art methods).

[0189] In both experiments, scaling of the anion exchange resin was determined by comparing the increase in resin weight before and after deionization. Results were shown in... Figure 6 The comparison of this value between the two tests showed that the resin in contact with untreated hydrolysis products (produced according to existing technology methods) had 26.4% more scaling.

[0190] Example 4:

[0191] Grain straw with a dry matter content of 45 wt.-% was pretreated by steam explosion (220°C). After steam explosion, the treated grain straw (“substrate”) was introduced into a mixing tank (Labfors, Infors AG, Switzerland). Containing 91.3 wt.-% An enzyme composition consisting of cellulase (C2730 Sigma) from *Trichoderma reesei* ATCC 26921 and 8.7 wt.-% glucosidase (49291 Sigma) was added to the matrix at an enzyme-to-solid ratio of 0.5 wt.-% to hydrolyze the matrix to obtain a slurry. Hydrolysis was carried out at 50 °C and pH 5.0 with stirring at 50 rpm for 72 hours. After hydrolysis, the slurry was heated to 70 °C for 1 hour with stirring at 200 rpm, and then the pH was set to 2.5 using 1 M H₂SO₄. The slurry thus treated was then used at a constant pressure of 3 bar with a flow rate of 5 L / dm³. 2 Filtering with a filter cloth having a permeability of [missing value] / min using a filter press yielded liquid and solid phases. The liquid phase was then deionized by electrodialysis using a bipolar membrane (ED64004, PCCell) with a membrane stack consisting of 10 bipolar membranes (PCCell), 10 anion exchange membranes (PC200D, PCCell), and 9 cation exchange membranes (PC SK, PCCell). Electrodialysis was performed at 32°C for 2 hours with a pumping rate of 50 L / h for both diluent and concentrate. After 2 hours, the conductivity decreased by 83%. Weighing of the deionized electrodialysis membranes showed that these membranes had a lower weight compared to membranes used for untreated hydrolysate (produced according to prior art methods). Scale formation on the membranes used for treated hydrolysate was therefore reduced compared to electrodialysis with untreated hydrolysate (prior art).

[0192] Following electrodialysis, the treated hydrolysate was used as a substrate for the fermentation of *Braunschweig*. Fermentation was carried out in a glass stirred tank (Multifors, Infors AG, Switzerland) equipped with temperature and pH controls. Fermentation was initiated by adding 10% (wt. / wt.) of *Braunschweig* seed culture (DSMZ No. 70352, Braunschweig) to 750 mL of the treated hydrolysate after electrodialysis. Fermentation was carried out in batches at 30°C and pH 6.0 with stirring at 200 rpm for 100 hours. Compared to the untreated hydrolysate, xylose consumption was significantly increased when using the hydrolysate according to the method of the present invention, thus significantly accelerating the fermentation process, increasing productivity, and reducing costs. Results are shown in… Figure 7 middle.

[0193] Example 5:

[0194] Grain straw with a dry matter content of 45 wt.-% was pretreated by steam explosion (220°C). After steam explosion, the treated grain straw (“substrate”) was introduced into a mixing tank (Labfors, Infors AG, Switzerland). Containing 91.3 wt.-% An enzyme composition consisting of cellulase (C2730 Sigma) from *Trichoderma reesei* ATCC 26921 and 8.7 wt.-% glucosidase (49291 Sigma) was added to the matrix at an enzyme-to-solid ratio of 0.5 wt.-% to hydrolyze the matrix to obtain a slurry. Hydrolysis was carried out at 50°C and pH 5.0 with stirring at 50 rpm for 72 hours. After hydrolysis, the slurry was heated to 70°C for 1 hour with stirring at 200 rpm, and then the pH was set to 2.5 using 1 M H₂SO₄. The slurry thus treated was then used at a constant pressure of 3 bar with a flow rate of 5 L / dm³. 2 Filtering with a filter cloth having a permeability of [missing value] / min using a filter press yielded liquid and solid phases. The liquid phase was then deionized by electrodialysis using a bipolar membrane (ED64004, PCCell) consisting of a membrane stack of 10 bipolar membranes (PCCell), 10 anion exchange membranes (PC 200D, PCCell), and 9 cation exchange membranes (PC SK, PCCell). Electrodialysis was performed at 32°C for 2 hours with a pumping rate of 50 L / h for both diluent and concentrate. After 2 hours, the conductivity decreased by 83%. Weighing of the deionized electrodialysis membranes showed that these membranes had a lower weight compared to membranes used for untreated hydrolysate (produced according to prior art methods). Scale formation on the membranes used for treated hydrolysate was therefore reduced compared to electrodialysis with untreated hydrolysate (prior art).

[0195] Following electrodialysis, the treated hydrolysate was used as a substrate for Aspergillus terreus fermentation. Fermentation was carried out in 50 mL shake flasks placed in an incubator (Multitron, Infors AG, Switzerland). Fermentation was initiated by adding 10% (wt. / wt.) of Aspergillus terreus (ATCC 32359) seed culture to 10 mL of the treated hydrolysate after electrodialysis. Fermentation was carried out in batches at 35°C and pH 3.0, with stirring at 250 rpm for 100 hours at 80% relative humidity. Although Aspergillus terreus fermentation in untreated hydrolysate did not show significant growth and significant itaconic acid production, the hydrolysate treated according to the present invention allowed for significant cell growth and significant itaconic acid production. Fermentation yields in g itaconic acid / g sugar are shown in... Figure 8 middle.

[0196] Example 6:

[0197] Grain straw with a dry matter content of 45 wt.-% was pretreated by steam explosion (220°C). After steam explosion, the treated grain straw (“substrate”) was introduced into a mixing tank (Labfors, Infors AG, Switzerland). Containing 91.3 wt.-% An enzyme composition consisting of cellulase (C2730 Sigma) from *Trichoderma reesei* ATCC 26921 and 8.7 wt.-% glucosidase (49291 Sigma) was added to the matrix at an enzyme-to-solid ratio of 0.5 wt.-% to hydrolyze the matrix to obtain a slurry. Hydrolysis was carried out at 50 °C and pH 5.0 with stirring at 50 rpm for 72 hours. After hydrolysis, the slurry was heated to 70 °C for 1 hour with stirring at 200 rpm, and then the pH was set to 2.5 using 1 M H₂SO₄. The slurry thus treated was then used at a constant pressure of 3 bar with a flow rate of 5 L / dm³. 2 Filtering with a filter cloth having a permeability of [missing value] / min using a filter press yielded liquid and solid phases. The liquid phase was then deionized by electrodialysis using a bipolar membrane (ED64004, PCCell) with a membrane stack consisting of 10 bipolar membranes (PCCell), 10 anion exchange membranes (PC200D, PCCell), and 9 cation exchange membranes (PC SK, PCCell). Electrodialysis was performed at 32°C for 2 hours with a pumping rate of 50 L / h for both diluent and concentrate. After 2 hours, the conductivity decreased by 83%. Weighing of the deionized electrodialysis membranes showed that these membranes had a lower weight compared to membranes used for untreated hydrolysate (produced according to prior art methods). Scale formation on the membranes used for treated hydrolysate was therefore reduced compared to electrodialysis with untreated hydrolysate (prior art).

[0198] After electrodialysis, 200 mL of this treated hydrolysate was used. Explorer (GE Healthcare) unit with 30g of ion exchange resin in glass column XK16 ( S6368 A, Lanxess) contact. The flow rate was 1 mL / min and the contact was carried out at 21 °C.

[0199] After electrodialysis and ion-exchange chromatography, the treated hydrolysate was used as a substrate for Aspergillus terreus fermentation. Fermentation was carried out in 50 mL shake flasks placed in an incubator (Multitron, Infortr AG, Switzerland). Fermentation was initiated by adding 10% (wt. / wt.) of Aspergillus terreus (ATCC 32359) seed culture to 10 mL of the treated hydrolysate after electrodialysis and ion-exchange chromatography. Fermentation was carried out in batches at 35 °C and pH 3.0, with stirring at 250 rpm for 100 hours at 80% relative humidity. Although Aspergillus terreus fermentation in untreated hydrolysate did not show significant growth and significant itaconic acid production, the hydrolysate treated according to the present invention allowed for significant cell growth and significantly improved itaconic acid production. Fermentation yields in g itaconic acid / g sugar are shown in... Figure 9 middle.

Claims

1. A method for purifying biomass hydrolysis products, comprising the following steps: a) Provide biomass hydrolysis products from hydrolytic enzymes; b) Adjust the temperature of the biomass hydrolysis product to a temperature selected from the range of 50-95°C; c) Add at least one acid to the biomass hydrolysate until the pH of the biomass hydrolysate reaches 2.0-4.5; d) Solid-liquid separation of the biomass hydrolysis product-acid mixture to obtain a solid phase and a liquid phase; e) Deionization of the liquid phase of the biomass hydrolysis product-acid mixture after separation according to step d).

2. The method of claim 1, wherein the temperature in step b) is selected from the range of 65-90°C.

3. The method according to claim 1 or 2, wherein the at least one acid is selected from acids with a pKa value of -4.0 to 5.

0.

4. The method according to claim 1 or 2, wherein at least one adsorbent is added before or during either step b) or d).

5. The method according to claim 4, wherein the at least one adsorbent is selected from bentonite, charcoal, activated carbon, diatomaceous earth, bleaching clay, clay minerals, polymer resins, and mixtures thereof.

6. The method of claim 5, wherein the diatomite is dispersed diatomite.

7. The method of claim 1 or 2, wherein steps b) and c) are performed at least partially simultaneously, wherein the at least one acid is added during the process of adjusting the temperature of the hydrolysis product upward from a temperature of 50°C to a temperature selected from the range of 65-90°C.

8. The method according to claim 1 or 2, wherein the temperature of the at least one acid is selected from 5-45°C and the at least one acid is added to the biomass hydrolysate at a temperature selected from 70-95°C.

9. The method of claim 8, wherein the temperature difference between the at least one acid and the biomass hydrolysis product is selected from the range of 35-95%.

10. The method of claim 1 or 2, wherein the deionization is performed by electrodialysis, membrane capacitance deionization, nanofiltration, reverse osmosis, chromatographic separation, or any combination thereof.

11. The method of claim 10, wherein the chromatographic separation is selected from at least one of ion exchange chromatography, hydrophobic chromatography, and size exclusion chromatography.

12. The method of claim 11, wherein deionization is performed by electrodialysis followed by membrane capacitance deionization, or by electrodialysis followed by ion exchange chromatography.

13. The method of claim 10, wherein deionization is carried out by electrodialysis using at least one bipolar membrane.

14. The method of claim 1 or 2, wherein step c) is performed before step b).

15. The method of claim 10, wherein step c) is performed before step b).

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