METHOD FOR PRODUCING A SUGAR LIQUID USING A BIOMASS CONTAINING CELLULOSE AS A RAW MATERIAL AND METHOD FOR PRODUCING A CHEMICAL PRODUCT

Abstract

 

Description

1 / 45 “METHOD FOR PRODUCING A SUGAR LIQUID USING A BIOMASS CONTAINING CELLULOSE AS RAW MATERIAL AND METHOD FOR PRODUCING A CHEMICAL PRODUCT” Technical Field

[001] The present invention relates to a method of producing a sugar liquid from a biomass containing cellulose. Background of the Invention

[002] The fermentation production process of chemical products using sugars as raw materials has been used for the production of various industrial materials. Currently, the sugars used industrially as fermentation raw materials are those derived from food materials such as sugarcane, starch, and sugar beet. However, considering the expected increase in food prices due to the future increase in the world population, or from an ethical standpoint, the fact that sugars for industrial materials may compete with sugars for food, a process for the efficient production of a sugar liquid from a non-food renewable resource, i.e., biomass containing cellulose, or a process for using the sugar liquid as a fermentation raw material to efficiently convert it into an industrial material, needs to be developed in the future.

[003] As with the state of the art for obtaining sugar from biomass, methods in which concentrated sulfuric acid is used to hydrolyze cellulose and hemicellulose contained in the biomass into monosaccharides represented by glucose and xylose (patent documents 1 and 2), and methods in which pretreatment is carried out to improve the reactivity of the biomass, followed by hydrolysis of the biomass by enzymatic reaction (patent documents 3 and 4) are generally known. In such cases, in the hydrolysis of a biomass containing cellulose, decomposition of cellulose and components of Petition 870260039364, dated 04 / 28 / 2026, page 15 / 62 2 / 45 hemicellulose etc. occurs during the decomposition reaction of produced sugars such as glucose and xylose, leading to the production of by-products such as furan compounds including furfural and hydroxymethylfurfural, and organic acids including formic acid and acetic acid, which is problematic. These compounds exhibit inhibitory actions during the fermentation step using a microorganism and cause inhibition of microorganism growth, leading to a reduced yield of the fermentation product. Therefore, these compounds are called fermentation inhibitors and were seriously problematic when a sugar liquid derived from a biomass containing cellulose was used as a fermentation feedstock. As a method for removing fermentation inhibitors in the sugar liquid production process, a method of removing fermentation inhibitors with a nanofiltration membrane or reverse osmosis membrane is known (patent document 5). State of the Art Documents Patent Documents

[004] Patent document 1: Translated Japanese patent application No. 11-506934.

[005] Patent document 2: JP 2005-229821 A.

[006] Patent document 3: JP 2001-95594 A.

[007] Patent document 4: JP 3041380 B.

[008] Patent document 5: WO2010 / 067785. Brief Description of the Invention Problems to be Solved by the Invention

[009] The present inventors have discovered that, as described above, the operation of removing fermentation inhibitors present in a sugar liquid derived from a cellulose-containing biomass using a nanofiltration membrane or reverse osmosis membrane sometimes results Petition 870260039364, dated 04 / 28 / 2026, p. 16 / 62 3 / 45 in the incomplete removal of fermentation inhibitors and found that this occurs due to the fact that unidentified fermentation inhibitors, which can only be removed with great difficulty using a nanofiltration membrane or reverse osmosis membrane, may be present in a sugar liquid derived from a cellulose-containing biomass. The present invention provides a method for producing a sugar liquid containing only a very small amount of fermentation inhibitors by removing fermentation inhibitors that have been difficult to remove using conventional methods from a sugar liquid derived from a cellulose-containing biomass. Ways to Solve the Problems

[010] As a result of intensive study, the present inventors have recently identified that fermentation inhibitors produced in the production step of a sugar liquid from a cellulose-containing biomass contain substances that have molecular weights that are equivalent to or greater than those of monosaccharides, such as coumaric acid, ferulic acid, coniferyl aldehyde and 2,3-dihydrobenzofuran, and have found that these can be efficiently removed with an ultrafiltration membrane, thus implementing the present invention.

[011] That is, the present invention consists of [1] to [6] as follows: [1] Method for producing a sugar liquid using a biomass containing cellulose as raw material, the method comprising the steps of: (1) hydrolysis of a biomass containing cellulose to produce an aqueous sugar solution; and (2) filtration of the aqueous sugar solution obtained in step (1) through an ultrafiltration membrane with a molecular weight cutoff of 600 to 2,000 Da, to remove fermentation inhibitor(s) on the side of Petition 870260039364, dated 04 / 28 / 2026, page 17 / 62 4 / 45 permeation and collecting a sugar liquid from the feed side. [2] The method of producing a sugar liquid according to [1], wherein the fermentation inhibitor(s) comprise(s) one or more substances selected from the group consisting of coumaric acid, ferulic acid and 2,3-dihydrobenzofuran. [3] The method of producing a sugar liquid according to [1] or [2], wherein in step (2), the aqueous sugar solution is filtered after adjusting the pH to 5. [4] The method of producing a sugar liquid according to any one of [1] to [3], wherein the functional layer material of the ultrafiltration membrane used in step (2) is polyethersulfone. [5] The method of producing a sugar liquid according to any one of [1] to [4], wherein the method comprises filtering the permeate obtained in step (2) containing a sugar liquid and / or fermentation inhibitor through a nanofiltration membrane and / or reverse osmosis membrane to collect a concentrated sugar liquid from the feed side. [6]Method of producing a chemical product, wherein the method comprises using, as a fermentation feedstock, a sugar liquid obtained by the method of producing a sugar liquid according to any one of [1] to [5]. Effect of the Invention

[012] By the present invention, a sugar liquid containing sugars such as glucose and xylose can be produced with high purity and high yield. Consequently, by using the purified sugar liquid obtained by the present invention as a fermentation feedstock, the fermentation production efficiency of various chemicals can be improved. Petition 870260039364, dated 04 / 28 / 2026, page 18 / 62 5 / 45 Brief Description of the Drawings

[013] Figure 1 shows the results of a fermentation test with a sugar liquid produced by concentration, using an ultrafiltration membrane or nanofiltration membrane, of an aqueous sugar solution obtained by treatment with diluted sulfuric acid of a biomass containing cellulose, the test being carried out using the glucose consumption rate as an index.

[014] Figure 2 shows the results of a fermentation test with a sugar liquid produced by concentration, using an ultrafiltration membrane or nanofiltration membrane, an aqueous sugar solution obtained by steam explosion treatment of a biomass containing cellulose, the test being carried out using the glucose consumption rate as an index.

[015] Figure 3 shows the results of improving fermentability by subjecting a biomass containing cellulose to hydrothermal treatment to obtain an aqueous sugar solution, filtering the resulting solution through an ultrafiltration membrane, and then subjecting the obtained permeate to membrane concentration, whose fermentability was analyzed using the xylose consumption rate as an index.

[016] Figure 4 shows the results of improving fermentability by subjecting a biomass containing cellulose to treatment with diluted sulfuric acid in order to obtain an aqueous sulfuric acid solution, filtering the resulting solution through an ultrafiltration membrane, and then subjecting the obtained permeate to the membrane concentration whose fermentability was analyzed using the xylose consumption rate as an index. Best Way to Carry Out the Invention Step (1)

[017] A biomass containing cellulose in the present invention means a resource that is derived from an organism and comprises at least Petition 870260039364, dated 04 / 28 / 2026, page 19 / 62 6 / 45 5% by weight of cellulose. Specific examples of biomass containing cellulose include herbaceous biomasses such as bagasse, yellow millet, maranga, Erianthus, corn hybrids, rice straw, and wheat straw; and woody biomass such as trees and construction waste. Since biomass containing cellulose contains lignin as aromatic macromolecules in addition to cellulose / hemicellulose, it is also called lignocellulose. Through the hydrolysis of cellulose and hemicellulose, which are polysaccharide components present in a biomass containing cellulose, a sugar liquid containing monosaccharides can be obtained that can be used as a fermentation feedstock for the production of a chemical product, more specifically, a sugar liquid containing xylose and glucose as top components.

[018] Specific examples of hydrolysis treatment of a biomass containing cellulose include chemical treatments, for example, acid treatment where the treatment is carried out with dilute sulfuric acid, a sulfite or similar under high temperature and high pressure; alkaline treatment where the treatment is carried out with an aqueous solution of an alkali such as calcium hydroxide or sodium hydroxide; ammonia treatment where the treatment is carried out with liquid ammonia, ammonia gas or an aqueous ammonia solution; and hydrothermal treatment where the treatment is carried out with pressurized hot water. These hydrolysis treatments can also be combined with hydrolysis treatment with a saccharifying enzyme.

[019] In general, lignin is dissolved in acid treatment. Furthermore, the hemicellulose component, which has low crystallinity, is first hydrolyzed, followed by degradation of the cellulose component, which has high crystallinity. Therefore, a liquid containing a large amount of xylose derived from hemicellulose can be obtained. The number of times the treatment can be repeated is unlimited, and with the establishment of two or Petition 870260039364, dated 04 / 28 / 2026, page 20 / 62 7 / 45 more stages of acid treatment, it is possible to selectively establish suitable hydrolysis conditions for hemicellulose or cellulose, thus obtaining increased degradation efficiency and high sugar yield. The acid used in the acid treatment is not limited as long as the acid produces hydrolysis, and sulfuric acid is preferred from an economic point of view. The acid concentration is preferably 0.1 to 100% by weight, more preferably 0.5 to 15% by weight. The reaction temperature can be set within the range of 100 to 300°C, and the reaction time can be set within the range of 1 second to 60 minutes. The liquid component obtained after acid treatment comprises a large quantity of monosaccharides and their oligosaccharides obtained by hydrolysis, containing mainly components derived from hemicellulose.Specifically, hydrolysis can be achieved in a single stage using concentrated sulfuric acid at a concentration of at least 50%, more preferably at least 80%, to hydrolyze both hemicellulose and cellulose. In cases where acid treatment is followed by hydrolysis with a saccharifying enzyme, the solid content and the liquid component obtained after acid treatment can be separately subjected to hydrolysis with a saccharifying enzyme, or the mixture of solid content and liquid component can be subjected to hydrolysis without separation. Since the solid content and the liquid component obtained by acid treatment contain the acid used, the acid-treated product is preferably neutralized before carrying out the hydrolysis reaction using a saccharifying enzyme.

[020] Alkali treatment is a treatment method in which a cellulose-containing biomass is reacted in an aqueous alkaline solution, more specifically an aqueous solution of a hydroxide salt (excluding ammonium hydroxide). Through alkali treatment, lignin can be removed, which mainly inhibits the cellulose / hemicellulose reaction caused Petition 870260039364, dated 04 / 28 / 2026, page 21 / 62 8 / 45 by the saccharifying enzyme. It is preferably used as the hydroxide salt, sodium hydroxide or calcium hydroxide. The concentration of the alkali in the aqueous solution is preferably within the range of 0.1 to 60% by weight. This solution is added to the biomass containing cellulose, and the treatment is usually carried out at a temperature within the range of 100 to 200°C, preferably within the range of 110 to 180°C. The number of treatment cycles is not limited, and the treatment can be carried out one or more times. In cases where the treatment is carried out 2 or more times, the conditions for the plurality of treatment cycles may differ. As the pre-treated product obtained by the alkali treatment contains an alkali, the pre-treated product is preferably neutralized before hydrolysis with a saccharifying enzyme.

[021] Ammonia treatment is a treatment method in which an aqueous ammonia solution or 100% ammonia (liquid or gas) is reacted with a cellulose-derived biomass, and, for example, the method described in patent document JP 2008-161125 A or document JP 2008535664 A can be employed. That is, in ammonia treatment, ammonia reacts with the cellulose component to break the crystallinity of the cellulose, producing a notable increase in reaction efficiency through the saccharifying enzyme. Ammonia is usually added to the cellulose-containing biomass so that the ammonia concentration is within the range of 0.1 to 15% by weight relative to the cellulose-containing biomass, and the treatment is carried out at 4°C to 200°C, preferably 60°C to 150°C. The number of treatment cycles is not limited, and the treatment can be carried out one or more times.In cases where the previously treated product obtained by ammonia treatment is also subjected to hydrolysis using a saccharifying enzyme, it is preferable to perform ammonia neutralization or ammonia removal beforehand. Petition 870260039364, dated 04 / 28 / 2026, page 22 / 62 9 / 45

[022] Hydrothermal treatment is a treatment method in which a cellulose-derived biomass is treated with pressurized hot water at a temperature of 100 to 400°C for 1 second to 60 minutes. The treatment is usually carried out in such a way that the cellulose-containing biomass after treatment, which is insoluble in water at a normal temperature of 25°C, is present in a concentration of 0.1 to 50% by weight relative to the total weight of the cellulose-containing biomass and water. The pressure is not limited as it depends on the processing temperature and is preferably 0.01 to 10 MPa. In hydrothermal treatment, the components eluted in the hot water vary depending on the temperature of the pressurized hot water.In general, as the temperature of the pressurized hot water increases, the elution of tannin and lignin as the first group of cellulose-containing biomass occurs first, and the elution of hemicellulose as the second group occurs at a temperature of at least 140 to 150°C, followed by the elution of cellulose as the third group at a temperature above approximately 230°C. Furthermore, at the same time as elution, hydrolysis of hemicellulose and cellulose can occur. The difference in eluted components depending on the temperature of the pressurized hot water can be used to increase the reaction efficiencies of the saccharifying enzyme for cellulose and hemicellulose by performing a multi-stage treatment at different temperatures.In this case, among the fractions obtained by hydrothermal treatment, the water-soluble matter containing the components eluted in pressurized hot water is referred to as hot water-soluble matter, and the components that are not hot water-soluble matter are referred to as hot water-insoluble matter.

[023] Hot water insoluble matter is a solid matter obtained as a result of the elution of large quantities of lignin and the cellulose hemicomponent and contains mainly disaccharides and higher saccharides as the cellulose component (C6). In addition to cellulose Petition 870260039364, dated 04 / 28 / 2026, page 23 / 62 10 / 45 as the main component, the hot water insoluble matter may contain the cellulose hemicomponent and the lignin component. The ratios of these components may vary depending on the temperature of the pressurized hot water during hydrothermal treatment and the type of biomass to be processed. The water content in the hot water insoluble matter is 10% to 90%, more preferably 20% to 80%.

[024] Hot water-soluble matter is water-soluble matter in the liquid or paste state, and contains hemicellulose, lignin, tannin, and a portion of the cellulose component eluted in pressurized hot water in the liquid or paste state. Hot water-soluble matter contains a large amount of polysaccharides, oligosaccharides, and monosaccharides produced by hydrolysis. They can be used, as such or after further hydrolysis with a saccharifying enzyme, as an aqueous sugar solution.

[025] A pretreatment may be carried out prior to the hydrolysis treatment method and examples of pretreatments include pulverization treatment in which fibers are mechanically cut with a cutting mill, hammer mill or similar; fine pulverization treatment in which a ball mill or jet mill is used; mechanochemical treatment and steam explosion treatment in which a biomass containing cellulose is vaporized with water vapor for a short time interval and the pressure is then instantly released to produce pulverization due to volume expansion. This is because pulverization increases the exposed area of ​​cellulose / hemicellulose, and therefore increases the efficiency of hydrolysis with a saccharifying enzyme.

[026] The saccharifying enzyme is not limited as long as the enzyme exhibits hemicellulose or cellulose degradation activity and is preferably a saccharifying enzyme produced by a filamentous fungus. Petition 870260039364, dated 04 / 28 / 2026, p. 24 / 62 11 / 45 belonging to the genus Trichoderma. Filamentous Trichoderma fungi are microorganisms that secrete many types of saccharifying enzymes extracellularly, and the saccharifying enzyme is preferably derived from Trichoderma reesei. In addition to an enzyme exhibiting cellulose or hemicellulose degradation activity, an enzyme that supports cellulose or hemicellulose degradation is also preferably present. Examples of enzymes that support cellulose or hemicellulose degradation include cellobiohydrolase, endoglucanase, exoglucanase, β-glucosidase, xylanase, and xylosidase, and biomass swelling enzymes. The hydrolysis reaction using a saccharifying enzyme is preferably carried out at a pH of approximately 3 to 7, more preferably at a pH of approximately 5. The reaction temperature is preferably 40 to 70°C.Furthermore, hydrolysis with an enzyme is preferably followed by solid-liquid separation to remove undegraded solids. Examples of methods for removing solids include, but are not limited to, centrifugation and membrane separation. A plurality of these solid-liquid separation methods may be used in combination to avoid clogging or contamination of the ultrafiltration membrane in step (2). The aqueous sugar solution obtained in step (1) is preferably subjected to removal of solids and water-soluble macromolecules such as oligosaccharides, polysaccharides, tannin, saccharifying enzyme, and biomass-derived protein components before subjecting the solution to step (2). The method for removing these components is not limited, and preferred examples of the removal method include a method in which the aqueous sugar solution is filtered through a microfiltration membrane and / or an ultrafiltration membrane with a molecular weight cutoff greater than 2.000 Da, to remove solids and water-soluble macromolecules on the feed side. Examples of filtration methods include, but are not limited to, pressure filtration. Petition 870260039364, dated 04 / 28 / 2026, page 25 / 62 12 / 45 Vacuum filtration and centrifugal filtration. The filtration operation is not limited and can be roughly classified into constant filtration, constant flow filtration, and variable pressure / variable flow filtration. The filtration operation can be a multi-stage filtration where one or more microfiltration membrane(s) and / or ultrafiltration membrane(s) with a molecular weight cutoff greater than 2,000 Da is / are used two or more times for efficient solids removal.

[027] A microfiltration membrane means a membrane with an average pore size of 0.01 μm to 5 mm, which is called an MF membrane or for short, and the membrane is preferably used when solids present in the aqueous sugar solution must be removed. The microfiltration membrane used here can be either an inorganic membrane or an organic membrane, and examples of membrane material include organic materials such as cellulose, cellulose ester, polysulfone, polyethersulfone, chlorinated polyethylene, polypropylene, polyolefin, polyvinyl alcohol, polymethyl methacrylate, polyvinylidene fluoride and polytetrafluoroethylene; and inorganic materials such as metals including stainless steel and ceramics.

[028] The ultrafiltration membrane is that described in detail in step (2) below, and the use of an ultrafiltration membrane with a molecular weight cutoff greater than 2,000 Da is preferred for removing water-soluble macromolecules, especially the saccharifying enzyme, present in the aqueous sugar solution. Step (2)

[029] It is known that when a biomass containing cellulose is hydrolyzed in step (1), fermentation inhibitors are produced in addition to sugars. Fermentation inhibitors are compounds produced by hydrolysis of a biomass containing cellulose, and are substances with an action to produce a reduction in the amount of a chemical product produced or Petition 870260039364, dated 04 / 28 / 2026, page 26 / 62 13 / 45 accumulated or at the production rate, in the fermentation process for the production of a chemical product using a sugar liquid as raw material. The extent of fermentation inhibition by fermentation inhibitors is not limited in the present invention since the extent of microorganism inhibition varies depending on the types and quantities of fermentation inhibitors present in the aqueous sugar solution, the species of microorganism employed, and the type of chemical product to be produced.

[030] Organic acids such as acetic acid and formic acid; furan compounds such as furfural and hydroxymethylfurfural (HMF); and phenol compounds such as vanillin and 4-hydroxybenzoic acid were known until then as fermentation inhibitors, but the present inventors discovered that coumaric acid, ferulic acid, 2,3-dihydrobenzofuran and the like, in addition to those known fermentation inhibitors, can be fermentation inhibitors. In step (2), the aqueous sugar solution obtained in step (1) is filtered through an ultrafiltration membrane with a specific molecular weight cutoff (measured in Da) to remove fermentation inhibitors on the permeation side, while a sugar liquid is recovered from the feed side.

[031] The ultrafiltration membrane in the present description is a separation membrane with a molecular weight cutoff of 600 to 200,000 Da, which is also called a UF membrane or in its abbreviated form. The molecular weight cutoff is well known to those skilled in the art as an index indicating the membrane performance of an ultrafiltration membrane, as described on p. 92 of The Membrane Society of Japan ed., Membrane Experiment Series, Vol. III, Artificial Membrane, editorial committee: Shoji Kimura, Shin-ichi Nakao, Haruhiko Ohya and Tsutomu Nakagawa (1993, Kyoritsu Shuppan Co., Ltd.), where “The curve obtained by plotting the molecular weight of the solute along the abscissa and the blocking rate along the ordinate is called the molecular weight cutoff curve. The molecular weight with which the Petition 870260039364, dated 04 / 28 / 2026, page 27 / 62 "A 14 / 45 blocking rate reaching 90% is called the molecular weight cutoff of the membrane." In the technical field of separation membranes, a separation membrane with a molecular weight cutoff within the range of 600 to 1,000 Da is recognized as a membrane on the dividing line between a nanofiltration membrane and an ultrafiltration membrane. Therefore, a separation membrane that exhibits a molecular weight cutoff within the range of 600 to 1,000 Da is called a nanofiltration membrane or an ultrafiltration membrane depending on the literature. In the present description, a separation membrane with a molecular weight cutoff within the range of 600 to 200,000 Da is called an ultrafiltration membrane, and a separation membrane that exhibits a molecular weight cutoff less than 600 Da and corresponds to a membrane generally defined as "a membrane that allows the permeation of monovalent ions but blocks divalent ions" is called a nanofiltration membrane.

[032] The present invention is characterized by the fact that an ultrafiltration membrane with a molecular weight cutoff of 600 to 2,000 Da is used. The use of an ultrafiltration membrane with a molecular weight cutoff greater than 2,000 Da is not preferred since it produces permeation of both a large proportion of sugars and fermentation inhibitors on the permeation side, and the use of a membrane with a molecular weight cutoff less than 600 Da is not preferred since it leads to poor removal performance of the recently identified fermentation inhibitors, namely coumaric acid, ferulic acid and 2,3-dihydrobenzofuran, on the permeation side.

[033] Examples of ultrafiltration membrane material include, but are not limited to, organic materials such as cellulose, cellulose ester, polysulfone, sulfonated polysulfone, polyethersulfone, sulfonated polyethersulfone, chlorinated polyethylene, polypropylene, polyolefin, polyvinyl alcohol, polymethyl methacrylate, polyvinylidene fluoride and polytetrafluoroethylene; metals such as steel Petition 870260039364, dated 04 / 28 / 2026, page 28 / 62 15 / 45 stainless steel and inorganic materials such as ceramic materials. An organic membrane is especially preferred from the point of view of removal performance for hydrophobic substances. In particular, polyethersulfone is preferred. This is because a polyethersulfone membrane has been found to perform well for separating sugars of interest from fermentation inhibitors. The material is more preferably a sulfonated polyethersulfone. This is because sulfonated polyethersulfone exhibits a higher sugar blocking rate than non-sulfonated polyethersulfone.

[034] The shape of the ultrafiltration membrane is not limited and can be any type: spiral, hollow fiber, tubular, and flat membrane.

[035] Specific examples of the ultrafiltration membrane used in the present invention include the G-5 type, GH type and GK type, manufactured by DESAL; SPE1, manufactured by Synder; PM1000, PM2000, MPS-36 and SR2, manufactured by KOCH; GR95Pp and ETNA01PP, manufactured by Alfa-Laval; and NTR7450 (cutoff molecular weight, 600 to 800 Da; see WaterResearch 37(2003) 864-872) and NTR-7410 (cutoff molecular weight, 1,000 to 2,000 Da; see Collection of Papers for Sanitary Engineering Symposium, 5:246-251 (1997)), manufactured by Nitto Denko Corporation.

[036] The filtration pressure in ultrafiltration membrane filtration treatment is preferably within the range of 0.1 MPa to 8 MPa, although the filtration pressure varies depending on the concentration of the aqueous sugar solution. In cases where the filtration pressure is less than 0.1 MPa, the membrane permeation rate is low, while in cases where the filtration pressure is greater than 8 MPa, the membrane may be damaged. In cases where the filtration pressure is 0.5 MPa to 6 MPa, the membrane permeation flux is high and efficient permeation of the sugar solution is therefore possible, which is more preferred. Petition 870260039364, dated 04 / 28 / 2026, page 29 / 62 16 / 45

[037] The membrane permeation flux in filtration treatment with the ultrafiltration membrane is preferably 0.2 m / D to 2.0 m / D. This is because a membrane permeation flux of at most 0.2 m / D does not allow concentration with an ultrafiltration membrane, and a membrane permeation flux of at least 2.0 m / D causes significant membrane contamination. A filtration permeation flux of 0.5 m / D to 2.0 m / D easily allows filtration with the ultrafiltration membrane, which is more preferred.

[038] The pH of the aqueous sugar solution in ultrafiltration membrane filtration treatment is not limited and, in view of permeability to fermentation inhibitors, the pH is preferably at most 5, more preferably at most 4. As in cases where the pH is at most 1, a large amount of acid is required for pH adjustment, the lower limit of the pH is preferably 1 from an economic point of view. The effect of adjusting the pH of the aqueous sugar solution is especially noticeable in cases where a substance such as coumaric acid or ferulic acid, which is an aromatic compound with a carboxylic group, is present as a fermentation inhibitor.

[039] The sugar liquid recovered from the feed side in the filtration treatment with an ultrafiltration membrane can be used as if it were raw material in the fermentation step described later, or the sugar solution can also be subjected to the filtration treatment described in patent document WO2010 / 067785 using a nanofiltration membrane and / or reverse osmosis membrane to concentrate sugars on the feed side, followed by the use of the resulting concentrated sugar liquid in the fermentation step described later.

[040] In filtration treatment with an ultrafiltration membrane, sugars may be partially lost on the permeation side, Petition 870260039364, dated 04 / 28 / 2026, page 30 / 62 17 / 45 and in such a case, the permeate recovered from the permeation side, containing fermentation inhibitors, can be subjected to the filtration treatment described in patent document WO2010 / 067785 using a nanofiltration membrane and / or reverse osmosis membrane, to recover a concentrated sugar liquid from the retention side. The concentrated sugar liquid obtained by this process is also used as raw material in the fermentation step subsequently described. Note that the concentrated sugar liquid obtained by filtration treatment with a nanofiltration membrane and / or reverse osmosis membrane showed a tendency to have a higher fermentation performance in the fermentation step subsequently described in cases where the filtration treatment is carried out with an ultrafiltration membrane with a molecular weight cutoff of 600 to 2.000 Da, compared to cases where filtration treatment was not performed or cases where filtration treatment was performed with an ultrafiltration membrane with a molecular weight cutoff greater than 2,000 Da. It is concluded that this is due to the fact that an aqueous sugar solution derived from a biomass containing cellulose contains a small amount of unknown fermentation inhibitors with molecular weights of approximately 2,000 Da, and that such inhibitors are concentrated with the nanofiltration membrane and / or reverse osmosis membrane. Fermentation stage

[041] The sugar liquid obtained in step (2) comprises glucose and / or xylose as carbon source(s) for the growth of microorganisms and cultured cells that can produce chemicals as metabolites, while the contents of fermentation inhibitors such as coumaric acid, ferulic acid and 2,3-dihydrobenzofuran are very small, so the sugar liquid can be effectively used as a fermentation feedstock, especially as a source of Petition 870260039364, dated 04 / 28 / 2026, page 31 / 62 18 / 45 carbon for the production of a chemical product. The fermentation step can be carried out according to the fermentation step described in patent document WO2010 / 067785.

[042] The chemical produced by the fermentation step is not restricted as long as it is a substance produced in a culture liquid by the above microorganisms or cells. Specific examples of the chemical include alcohols, organic acids, amino acids, and nucleic acids, which are substances mass-produced in the fermentation industry. Examples of alcohols include ethanol, butanol, 1,3-propanediol, 2,3-butanediol, 1,4-butanediol, and glycerol; examples of organic acids include acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, and citric acid; examples of nucleic acids include nucleosides such as inosine and guanosine, and nucleotides such as inosinic acid and guanylic acid; and diamine compounds such as cadaverine. In addition, the present invention can also be applied to the production of substances such as enzymes, antibiotics, and recombinant proteins. Examples Example of Reference 1 Method for Measuring Monosaccharide Concentrations

[043] Monosaccharide concentrations (glucose concentration and xylose concentration) present in the sugar liquid obtained in each of the examples and comparative examples were analyzed by HPLC under the following conditions and quantified based on a comparison with standard samples: Column: Luna NH2 (manufactured by Phenomenex, Inc.) Mobile phase: ultrapure water: acetonitrile = 25:75 (flow rate, 0.6 mL / min.) Reaction liquid: none Petition 870260039364, dated April 28, 2026, pages 32 / 62 19 / 45 Detection method: RI (differential refractive index) Temperature: 30 °C. Example 2: Reference method for measuring fermentation inhibitor concentrations.

[044] The concentrations of furan-based fermentation inhibitors (HMF and furfural) and phenol-based fermentation inhibitors (coumaric acid, ferulic acid, and 2,3-dihydrobenzofuran), among fermentation inhibitors present in the sugar liquid, were analyzed by HPLC under the following conditions and quantified based on comparison with standard samples: Column: Synergi HidroRP 4.6 mm*250 mm (manufactured by (Phenomenex, Inc.) Mobile phase: Acetonitrile - 0.1 wt% H3PO4 (flow rate, 1.0 mL / min.) Detection method: UV (283 nm) Temperature: 40 °C.

[045] Organic acids (acetic acid and formic acid), among fermentation inhibitors present in the sugar liquid, were analyzed by HPLC under the following conditions and quantified based on comparison with standard samples: Column: Shim-Pack SPR-H and Shim-Pack SCR101H (manufactured by Shimadzu Corporation) which were arranged linearly. Mobile phase: 5 mM p-Toluenesulfonic acid (flow rate, 0.8 mL / min.) Reaction liquid: 5 mM p-Toluenesulfonic acid, 20 mM BisTris, 0.1 mM EDTA-2Na (flow rate, 0.8 mL / min.) Detection method: electrical conductivity Temperature: 45 °C. Petition 870260039364, dated 04 / 28 / 2026, page 33 / 62 20 / 45 Example of reference 3 Biomass Stage Containing Cellulose Through Acid Treatment Diluted Sulfuric Acid / Enzyme Treatment

[046] Rice straw was used as the cellulose-containing biomass. The cellulose-containing biomass was immersed in a 1% aqueous sulfuric acid solution and processed using an autoclave (manufactured by Nitto Koatsu Co., Ltd.) at 150°C for 30 minutes. Then, solid-liquid separation was performed to separate the sulfuric acid-treated cellulose from the aqueous sulfuric acid solution. Subsequently, the sulfuric acid-treated cellulose was mixed with diluted sulfuric acid treatment liquid under stirring so that the solids concentration was 10% by weight, and the pH was adjusted to approximately 5 with sodium hydroxide. “Accellerase Duet” (manufactured by Danisco Japan), a saccharifying enzyme derived from Trichoderma reesei, was added to this mixture. The resulting mixture was stirred at 50°C for 1 day to carry out the hydrolysis reaction.Next, centrifugation (3000 G) was performed to separate and remove undegraded cellulose and lignin in order to obtain an aqueous sugar solution treated with dilute sulfuric acid. The compositions of fermentation inhibitors and monosaccharides present in the aqueous sugar solution treated with dilute sulfuric acid were as presented in Tables 1 to 3. Table 1 Table 1 Quantification of fermentation inhibitors 1 Unit [g / L] Formic acid Acetic acid HMF Furfural Aqueous sugar solution treated with dilute sulfuric acid 0.1 2.4 0.125 0.875 Table 2 Table 2 Quantification of fermentation inhibitors 2 Unit [g / L] Coumaric acid Ferulic acid 2,3-Dihydrobenzofuran Aqueous sugar solution treated with dilute sulfuric acid 0.15 0.075 0.01 Petition 870260039364, dated 04 / 28 / 2026, page 34 / 62 21 / 45 Table 3 Table 3 Quantification of monosaccharides Unit [g / L] Glucose Xylose Aqueous sugar solution treated with dilute sulfuric acid 25 12 Example of Reference 4: Hydrolysis Stage of Biomass Containing Cellulose by Steam Explosion Treatment / Enzyme Treatment

[047] Rice straw was used as biomass containing cellulose. In a 2-L steam explosion tester (Nihon Dennetsu Co., Ltd.), 100 g of cellulose-containing biomass were placed, and steam was then injected. The pressure was maintained at 2.5 MPa for 2.5 minutes, and the atmosphere in the container was then rapidly released to perform the explosion treatment, followed by sample recovery. The temperature inside the container was 225°C at this time. The water content of the processed product was 84.4%. Water was added to the product so that the solid concentration was 10% by weight, and 1 N of aqueous sodium hydroxide solution was added to the resulting mixture to adjust the pH to 5.0. Then, “Accellerase Duet” was added to the mixture as a saccharifying enzyme, and the resulting mixture was left to stand at 50°C for 1 day to allow the reaction to proceed. The composition of the resulting aqueous sugar solution is presented in Tables 4 to 6. Table 4 Table 4 Quantification of fermentation inhibitors 1 Unit [g / L] Formic acid Acetic acid HMF Furfural Steam-exploded treated aqueous sugar solution 1.7 2.3 0.29 0.24 Table 5 Table 5 Quantification of fermentation inhibitors 2 Unit [g / L] Coumaric acid Ferulic acid 2,3-Dihydrobenzofuran Aqueous sugar solution treated with steam explosion 0.15 0.11 0.08 Petition 870260039364, dated 04 / 28 / 2026, page 35 / 62 22 / 45 Table 6 Table 6 Quantification of monosaccharides Unit [g / L] Glucose Xylose Aqueous sugar solution treated with steam explosion 34 5 Example of Reference 5 Hydrolysis Step of Biomass Containing Cellulose by Treatment With Ammonia / Enzyme Treatment

[048] Rice straw was added as biomass containing cellulose. The cellulose-containing biomass was placed in a compact reactor (manufactured by Taiatsu Techno Corporation, TVS-N2 30 mL) and cooled with liquid nitrogen. Ammonia gas at a concentration of 100% was circulated in this reactor, and the sample was completely immersed in 100% liquid ammonia. The reactor lid was closed, and the reactor was allowed to stand at room temperature for approximately 15 minutes. Then, the reactor was processed in an oil bath at 150°C for 1 hour. Afterwards, the reactor was removed from the oil bath, and the ammonia gas was immediately released into a fume hood, followed by vacuum aspiration from the inside of the reactor at 10 Pa using a vacuum pump, thus drying the cellulose-containing biomass.Processed cellulose-containing biomass was mixed with pure water under stirring to achieve a solid concentration of 15% by weight, and the pH was adjusted to approximately 5 with sulfuric acid. Accellerase Duet was added as a saccharifying enzyme, and the hydrolysis reaction was carried out under stirring at 50°C for 3 days. Subsequently, centrifugation (3000 G) was performed to separate and remove undegraded cellulose and lignin to obtain an aqueous sugar solution from which undegraded cellulose and lignin were removed. The inhibitor compositions... Petition 870260039364, dated 04 / 28 / 2026, page 36 / 62 23 / 45 fermentation and monosaccharides present in the aqueous sugar solution were as shown in tables 7 to 9. Table 7 Table 7 Quantification of fermentation inhibitors 1 Unit [g / L] Formic acid Acetic acid HMF Furfural Ammonia-treated aqueous sugar solution 1.1 0.5 0.012 0.005 Table 8 Table 8 Quantification of fermentation inhibitors 2 Unit [g / L] Coumaric acid Ferulic acid 2,3-Dihydrobenzofuran Aqueous sugar solution treated with ammonia 0.03 0.008 0.005 Table 9 Table 9 Quantification of monosaccharides Unit [g / L] Glucose Xylose Aqueous sugar solution treated with ammonia 40 24 Example of Reference 6: Hydrolysis Step of Biomass Containing Cellulose by Hydrothermal Treatment / Enzyme Treatment

[049] Rice straw was used as the cellulose-containing biomass. The cellulose-containing biomass was immersed in water and processed using an autoclave (manufactured by Nitto Koatsu Co., Ltd.) at 180°C for 20 minutes. The pressure at this time was 10 MPa. Then, centrifugation (3000 G) of the solution component and the processed biomass component was performed to achieve solid-liquid separation. The pH of the solution component was 4.0. The pH of the solution component was then adjusted to 5.0 with sodium hydroxide. “Accellerase Duet” was added to the mixture as a saccharifying enzyme, and the resulting mixture was stirred at 50°C for 1 day to carry out a hydrolysis reaction in order to obtain a hydrothermally treated liquid. Petition 870260039364, dated 04 / 28 / 2026, page 37 / 62 The compositions of fermentation inhibitors and monosaccharides present in the hydrothermally treated liquid were as shown in Tables 10 to 12. Table 10 Table 10 Quantification of fermentation inhibitors 1 Unit [g / L] Formic acid Acetic acid HMF Furfural Hydrothermally treated liquid 1.1 2.2 0.12 0.5 Table 11 Table 11 Quantification of fermentation inhibitors 2 Unit [g / L] Coumaric acid Ferulic acid 2,3- Dihydrobenzofuran Hydrothermally treated liquid 0.2 0.13 0.03 Table 12 Table 12 Quantification of monosaccharides Unit [g / L] Glucose Xylose Hydrothermally treated liquid 7 15 Example of Reference 7 Fermentation Evaluation Method

[050] Using a yeast strain (Pichia stipitis, NBRC1687), a fermentation test was performed. A medium to be used for fermentation was prepared by dilution to a glucose concentration of 25 g / L and addition of additives to the resulting dilution so that the composition shown in Table 13 was obtained, followed by sterilization with a filter (Millipore, Stericup 0.22 μm). The culture was carried out by inoculation of the yeast in a quantity of 0.5%, and agitation of the flask at 150 rpm at 28°C for 72 hours. The degree of fermentation inhibition was analyzed based on the glucose consumption rate of the yeast strain. The method for evaluating the glucose consumption rate of the yeast strain was as follows: the medium component was removed on a clean bench under sterile conditions at 16, 24, 40, 48, Petition 870260039364, dated 04 / 28 / 2026, page 38 / 62 25 / 45 and 72 hours after the start of the culture, the medium was centrifuged and filtered, followed by quantification of the glucose concentration by HPLC according to reference example 1. Table 13 Composition: Concentration of Composition: Glucose 25 g / L, Bacto yeast extract 10 g / L, Peptone 20 g / L Example 1

[051] The aqueous sugar solution treated with dilute sulfuric acid described in reference example 3 was filtered through a microfiltration membrane with a pore size of 0.08 μm, and a permeate from the microfiltration membrane was filtered through an ultrafiltration membrane. The following ultrafiltration membranes were used: “NTR-7450” (manufactured by Nitto Denko Corporation; material: sulfonated polyethersulfone, cutoff molecular weight: 600 to 800 Da), “NTR-7410” (manufactured by Nitto Denko Corporation; material: sulfonated polyethersulfone, cutoff molecular weight: 1,000 Da), “SPE1” (manufactured by Synder; material: polyethersulfone; cutoff molecular weight: 1,000 Da), GH series manufactured by GE Osmonics (material: polyethylene glycol; cutoff molecular weight: 1,000 Da), “GR95Pp” (manufactured by Alfa-Laval; material: polyethersulfone; cutoff molecular weight: 2,000 Da), or GK series manufactured by GE (material: polyethylene glycol; cutoff molecular weight: 2,000 Da).For each membrane, 1.5 L of permeate obtained by filtering the saccharified liquid treated with diluted sulfuric acid through the microfiltration membrane was provided, and the filtration treatment was carried out using a "SEPA-II" flat membrane filtration unit (manufactured by GE Osmonics) at a linear speed of... Petition 870260039364, dated 04 / 28 / 2026, pp. 39 / 62 26 / 45 membrane surface area of ​​20 cm² / second and a filtration pressure of 3 MPa until the volume of liquid collected from the feed side was 0.5 L. The results are illustrated in Table 14. Consequently, it was found that monosaccharides are concentrated by ultrafiltration membrane treatment, but that formic acid, acetic acid, HMF, and furfural, which are low molecular weight substances, are not concentrated, and furthermore, that coumaric acid, ferulic acid, and 2,3-dihydrobenzofuran are quite concentrated. A small amount of the sugar liquid was collected from the feed side of the ultrafiltration membrane (A to C) and subjected to a fermentation test under the conditions of reference example 7. The results are illustrated in Figure 1. Comparative example 1

[052] The same filtration treatment as in example 1 was performed using an ultrafiltration membrane with a higher molecular weight cutoff, “SPE3” (manufactured by Synder; material: polyethersulfone; molecular weight cutoff: 3,000 Da), or a nanofiltration membrane “UTC60” (manufactured by Toray Industries, Inc.; material: piperazine polyamide), HL series (manufactured by GE Osmonics; material: composite membrane) or DK series (manufactured by GE Osmonics; material: composite membrane). The results are illustrated in Table 14. It was found that the use of the ultrafiltration membrane with a molecular weight cutoff of 3,000 Da results in an extreme reduction in the monosaccharide concentration rate.In terms of concentration with nanofiltration membranes, coumaric acid, ferulic acid, and 2,3-dihydrobenzofuran were concentrated, although the concentration of the concentrate varied slightly, and also in the fermentation test (D), the glucose consumption rate was lower than in the cases of example 1, in which ultrafiltration membranes (A to C) were used. Petition 870260039364, dated April 28, 2026, pp. 40-62 Table 14 Filtration treatment of aqueous sugar solution treated with diluted sulfuric acid. Unit [g / L] Membrane Type Material Molecular Weight Cutoff (Da) Glucose Xylose Formic Acid Acetic Acid HMF Furfural Coumaric Acid Ferulic Acid 2,3-Dihydrobenzofuran Example 1 (Fermentation Test A) NTR-7450 s-PES 600 to 800 73 30 0.1 2.4 0.12 0.75 0.2 0.09 0.015 Example 1 NTR-7410 s-PES 1000 65 25 0.1 2.4 0.12 0.75 0.18 0.08 0.01 Example 1 (Fermentation Test B) SPE1 (Synder) PES 1000 66 25 0.1 2.4 0.12 0.75 0.18 0.08 0.01 Example 1 GH (GE) PEG 1000 65 25 0.1 2.4 0.12 0.75 0.2 0.085 0.012 Example 1 (Fermentation Test C) GR95Pp (Alpha) PES 2000 50 20 0.1 2.4 0.12 0.75 0.15 0.075 0.01 Example 1 GK (GE) PEG 2000 48 20 0.1 2.4 0.12 0.75 0.18 0.08 0.01 Comparative Example 1 SPE3 (Synder) PES 3000 27 12 0.1 2.4 0.12 0.75 0.15 0.075 0.01 Comparative Example 1 (Fermentation Test D) UTC-60 PPA Less than 600 (NF Membrane) 75 35 0.1 2.6 0.13 0.78 0.45 0.235 0.025 Comparative Example 1 HL Composite Membrane Less than 600 (NF Membrane) 74 33 0.1 2.4 0.12 0.765 0.43 0.23 0.025 Comparative Example 1 DK Composite Membrane Less than 600 (NF Membrane) 75 36 0.1 2.8 0.15 0.82 0.45 0.235 0.025. 27 / 45 Petition 870260039364, dated April 28, 2026, pp. 41-62 28 / 45 Example 2

[053] The same filtration treatment as in example 1 was performed for the permeate obtained by filtering the steam-exploded aqueous sugar solution described in reference example 4 through the microfiltration membrane. The results are illustrated in table 15. In addition, the fermentation results performed by the method of reference example 7 (E to G) are shown in figure 2. Comparative Example 2

[054] The permeate obtained by filtering the steam-exploded treated saccharified liquid through a microfiltration membrane was subjected to filtration treatment using the same membranes as in comparative example 1. The results on the liquid composition are shown in Table 15, and the results of the fermentation test are shown in Figure 2. Similar to the comparison results between Example 1 and comparative example 1, the use of the ultrafiltration membrane with a molecular weight cutoff of 3,000 Da resulted in an extreme reduction in the concentration rate of monosaccharides. In terms of concentration with the nanofiltration membranes, coumaric acid, ferulic acid, and 2,3-dihydrobenzofuran were concentrated, although the concentration of the concentrate varied slightly, and also in the fermentation test (H), the glucose consumption rate was lower than that in the cases of Example 2, in which ultrafiltration membranes were used. Petition 870260039364, dated April 28, 2026, pages 42 / 62 Table 15 FILTRATION TREATMENT OF AQUEOUS SUGAR SOLUTION TREATED WITH STEAM EXPLOSION Unit [g / L] Membrane Type Material Molecular Weight Cutoff (Da) Glucose Xylose Formic Acid Acetic Acid HMF Furfural Coumaric Acid Ferulic Acid 2,3-Dihydrobenzofuran Example 2 (Fermentation Test E) NTR-7450 s-PES 600 to 800 98 10 1.7 2.3 0.28 0.22 0.04 0.025 0.008 Example 2 NTR-7410 s-PES 1000 to 2000 90 8 1.7 2.3 0.28 0.22 0.03 0.023 0.008 Example 2 (Fermentation Test F) SPE1 (Synder) PES 1000 92 9 1.7 2.3 0.28 0.22 0.03 Example 2 GH (GE) PEG 1000 90 8 1.7 2.3 0.28 0.22 0.03 0.022 0.008 Example 2 (Fermentation Test G) GR95Pp (Alpha) PES 2000 84 7 1.7 2.3 0.28 0.22 0.03 0.022 0.008 Example 2 GK (GE) PEG 2000 80 7 1.7 2.3 0.28 0.22 0.03 0.022 0.008 Comparative Example 2 SPE3 (Synder) PES 3000 40 5 1.7 2.3 0.28 0.22 0.03 0.022 0.008 Comparative Example 2 (Fermentation Test H) UTC-60 PPA Less than 600 (NF Membrane) 102 14 1.7 2.4 0.29 0.22 0.08 0.062 0.024 Comparative Example 2 HL Composite Membrane Less than 600 (NF Membrane) 100 14 1.7 2.3 0.28 0.22 0.07 0.06 0.022 Comparative example 2 DK Composite membrane Less than 600 (NF membrane) 102 15 1.7 2.6 0.31 0.24 0.08 0.064 0.024. 29 / 45 Petition 870260039364, dated April 28, 2026, pp. 43-62 30 / 45 Example 3

[055] The same concentration test as in example 1 was performed for the permeate obtained by filtration of the aqueous sugar solution treated with ammonia described in reference example 5 through the microfiltration membrane. The results are shown in table 16. Comparative example 3

[056] The permeate obtained by filtration of the aqueous sugar solution treated with ammonia through the microfiltration membrane was subjected to filtration treatment using the same membranes as in comparative example 1. The results on the liquid composition are shown in Table 16. Similar to the comparison results between example 1 and comparative example 1, the use of the ultrafiltration membrane with a molecular weight cutoff of 3,000 Da resulted in an extreme reduction in the concentration rate of monosaccharides. In terms of concentration with the nanofiltration membranes, coumaric acid, ferulic acid and 2,3-dihydrobenzofuran were concentrated, although the concentration of the concentrate varied slightly. Petition 870260039364, dated April 28, 2026, pp. 44 / 62 Table 16 Filtration Treatment of Ammonia-Treated Aqueous Sugar Solution Unit [g / L] Membrane Type Material Molecular Weight Cutoff (Da) Glucose Xylose Formic Acid Acetic Acid HMF Furfural Coumaric Acid Ferulic Acid 2,3-Dihydrobenzofuran Example 3 NTR-7450 s-PES 600 to 800 110 58 1.1 0.5 0.012 0.004 0.04 0.008 0.005 Example 3 NTR-7410 s-PES 1000 to 2000 106 52 1.1 0.5 0.012 0.004 0.03 0.008 0.005 Example 3 SPE1 (Synder) PES 1000 105 51 1.1 0.5 0.012 0.004 0.03 0.008 Example 3 GH (GE) PEG 1000 100 48 1.1 0.5 0.012 0.004 0.03 0.008 0.005 Example 3 GR95Pp (Alfa) PES 2000 82 42 1.1 0.5 0.012 0.004 0.03 0.008 0.005 Example 3 GK (GE) PEG 2000 80 40 1.1 0.5 0.012 0.004 0.03 0.008 0.005 Comparative Example 3 SPE3 (Synder) PES 3000 60 30 1.1 0.5 0.012 0.004 0.03 0.008 0.005 Comparative example 3 UTC-60 PPA Less than 600 (NF membrane) 119 70 1.1 0.6 0.014 0.005 0.088 0.024 0.007 Comparative example 3 HL Composite membrane Less than 600 (NF membrane) 118 68 1.1 0.5 0.013 0.005 0.078 0.022 0.006 Comparative Example 3 DK Composite Membrane Less than 600 (NF Membrane) 120 71 1.1 0.6 0.015 0.005 0.089 0.024 0.008. / 45 Petition 870260039364, dated April 28, 2026, pages 45 / 62 32 / 45 Example 4

[057] A comparison A was made between the case where, prior to filtration treatment of the hydrothermally treated aqueous sugar solution prepared in reference example 6, using the ultrafiltration membrane “NTR-7450” or “NTR-7410”, filtration treatment was performed using a second ultrafiltration membrane with a molecular weight cutoff of 10,000 Da (manufactured by Applied Membranes, Inc.; material: polyethersulfone), and the case where no filtration treatment was performed using the second ultrafiltration membrane. The results are shown in Table 17. It was found that in the cases where treatment with the second ultrafiltration membrane was performed, the membrane permeation flux during ultrafiltration membrane treatment with “NTR-7450” or “NTR-7410” (in terms of average over processing time) greatly increased and the monosaccharide concentration rate on the feed side improved. Petition 870260039364, dated April 28, 2026, pages 46 / 62 Table 17 Comparison Between Concentrate Compositions Prepared With or Without Second Membrane Treatment Ultrafiltration Unit [g / L] Membrane Type Membrane Pretreatment Permeation Flux Glucose Xylose Formic Acid Acetic Acid HMF Furfural Coumaric Acid Ferulic Acid 2,3-Dihydrobenzofuran Example 4 NTR-7450 No 0.5 m / D 18 30 1.1 2.2 0.12 0.48 0.22 0.15 0.03 Example 4 NTR-7450 Yes 1.5 m / D 21 40 1.2 2.4 0.15 0.5 0.23 0.15 0.03 Example 4 NTR-7410 No 0.64 m / D 14 25 1.1 2.2 0.12 0.47 0.19 0.13 0.03 Example 4 NTR-7410 Yes 2.0 m / D 17 30 1.1 2.3 0.13 0.48 0.2 0.13 0.03 33 / 45 Petition 870260039364, dated April 28, 2026, pages 47 / 62 34 / 45 Example 5

[058] Similarly to Example 1, 1.5 L of permeate obtained by filtration of the hydrothermally treated aqueous sugar solution prepared in Reference Example 6 through a microfiltration membrane was subjected to filtration treatment using an “NTR-7410” ultrafiltration membrane (manufactured by Nitto Denko Corporation; material: sulfonated polyethersulfone; molecular weight cutoff: 1,000 Da). The compositions of fermentation inhibitors and monosaccharides in the concentrate on the feed side (0.5 L) and the filtrate on the permeation side (1.0 L) obtained are shown in Table 18. Subsequently, the filtrate was filtered through a “UTC-60” nanofiltration membrane (manufactured by Toray Industries, Inc.; material: piperazine polyamide). The compositions of fermentation inhibitors and monosaccharides in the concentrate on the feed side (0.33 L) are shown in Table 19.Reagents were added to this concentrate to obtain the composition shown in Table 20. The same fermentation test as in reference example 7 was performed, and the xylose consumption rate was measured. The results are shown in Figure 3 (see J in Fig. 3). Comparative Example 4

[059] Table 19 shows the compositions of fermentation inhibitors and monosaccharides in 0.75 L of the concentrate on the feed side obtained by filtration treatment using a “UTC-60” nanofiltration membrane, and 1.5 L of the permeate obtained by filtration of the hydrothermally treated aqueous sugar solution prepared in reference example 6 through a microfiltration membrane. Similarly to example 5, reagents were added to this. Petition 870260039364, dated April 28, 2026, pages 48 / 62 35 / 45 concentrated so that the composition shown in Table 20 was obtained, and the resulting mixture was subjected to a fermentation test. The results (xylose consumption rates) were as shown in Figure 3 (see J in Fig. 3).

[060] It was found that although the sugar liquid obtained in Example 5 contained slightly higher concentrations of coumaric acid, ferulic acid, and 2,3-dihydrobenzofuran, the fermentability of the sugar liquid was better than in comparative Example 4 in terms of the xylose consumption rate. This is believed to be due to the presence in the aqueous sugar solution of unidentified fermentation inhibitors to which an ultrafiltration membrane with a molecular weight cutoff of 600 to 2,000 Da is impermeable. Furthermore, from Example 5, it was found that not only the sugar liquid on the feed side of the ultrafiltration membrane with a molecular weight cutoff of 600 to 2,000 Da, but also the second concentrated sugar liquid obtained by filtering the filtrate on the permeation side through a nanofiltration membrane and / or reverse osmosis membrane and by collecting the sugar liquid from the feed side, are sugar liquids with optimal fermentability. Petition 870260039364, dated April 28, 2026, pp. 49-62 Table 18 Composition of hydrothermally treated concentrated and filtered liquid obtained with an ultrafiltration membrane. Unit [g / L] Type of membrane Liquid treated Glucose Xylose Formic acid Acetic acid HMF Furfural Coumaric acid Ferulic acid 2,3-Dihydrobenzofuran Example 5 NTR-7410 Concentrate 14 25 1.1 2.2 0.12 0.47 0.2 0.13 0.03 Example 5 NTR-7410 Filtrate 3.5 10 1.1 2.2 0.12 0.51 0.18 0.11 0.03 36 / 45 Petition 870260039364, dated April 28, 2026, pages 50 / 62 Table 19 Comparison Between Concentrate Obtained by Nanofiltration Membrane Treatment of Raw Hydrothermally Treated Liquid and Concentrate Obtained by Ultrafiltration Membrane Treatment of Raw Hydrothermally Treated Liquid Followed by Nanofiltration Membrane Treatment of the Resulting Filtrate Unit [g / L] Type of membrane Liquid treated Concentration rate Glucose Xylose Formic acid Acetic acid HMF Furfural Coumaric acid Ferulic acid 2,3-Dihydrobenzofuran Comparative example 4 (fermentation test 1) UTC-60 Raw liquid 2-fold 14 30 1.1 2.4 0.12 0.49 0.4 0.26 0.06 Example 5 (fermentation test J) UTC-60 Raw filtrate material 3-fold 10 30 1.1 2.5 0.12 0.5 0.54 0.33 0.09 37 / 45 Petition 870260039364, dated April 28, 2026, pages 51 / 62 38 / 45 Table 20 Composition: Concentration of composition: Glucose 15 g / L, Xylose 25 g / L, Bacto yeast extract 10 g / L, Peptone 20 g / L Example 8: Evaluation of Capacities in Removing Fermentation Inhibitors from Aqueous Sugar Solutions with Different pHs

[061] Using the hydrothermally treated liquid described in reference example 6, after adjusting the pH to various values, the permeation rates of fermentation inhibitors present in the aqueous sugar solution through an ultrafiltration membrane were compared and studied. The permeation rate of each fermentation inhibitor was represented as the ratio (%) calculated by dividing the concentration of the component on the filtrate side by the concentration of the component on the feed side in the membrane treatment and multiplying the resulting value by 100. Since the addition of dilute sulfuric acid or sodium hydroxide to the hydrothermally treated liquid leads to the production of precipitates, centrifugation and subsequent microfiltration membrane treatment were then carried out. An ultrafiltration membrane “NTR-7410” (manufactured by Nitto Denko Corporation; material: sulfonated polyethersulfone; molecular weight cutoff: 1) was then used.A sample of 000 Da was placed in a “SEPA-II” flat membrane filtration unit (manufactured by GE Osmonics), and filtration treatment was performed at a linear membrane surface velocity of 20 cm / second at a filtration pressure of 2 MPa. Since the concentration on the filtrate side does not become stable within a short time interval, the filtrate obtained from 20 minutes of filtration was returned to the feed side, and a sample of stable filtrate was taken 20 minutes later. As a result of the permeation rate calculations, it was found, as shown in Table 21, that by adjusting the pH to a maximum of 5, the removal performance of coumaric acid and ferulic acid, which are aromatic fermentation inhibitors with a carboxylic group, increased considerably. Petition 870260039364, dated April 28, 2026, pages 52 / 62 Table 21 Permeation Rates of Aqueous Sugar Solution (Hydrothermally Treated Liquid) Through a Membrane Ultrafiltration at Different pHs (Unit: %) Glucose Xylose Formic acid Acetic acid HMF Furfural Coumaric acid Ferulic acid 2,3-Dihydrobenzofuran pH 3 9 30 110 105 100 105 100 100 95 pH 4 15 37 110 105 100 110 89 75 95 pH 5 17 42 100 100 102 110 68 49 100 pH 6 18 45 90 84 104 110 15 7 100 pH 7 17 43 88 80 110 115 10 5 100 pH 9 17 46 85 78 105 115 10 5 100 39 / 45 Petition 870260039364, dated April 28, 2026, pages 53 / 62 40 / 45 Example 6

[062] The aqueous sulfuric acid solution obtained in reference example 3 was neutralized to pH 4.0 with ammonia and subjected to microfiltration membrane treatment. Similarly, as in example 1, 1.5 L of the obtained permeate were filtered through an “NTR-7450” ultrafiltration membrane (manufactured by Nitto Denko Corporation; material: sulfonated polyethersulfone; molecular weight cutoff: 600 to 800 Da). The compositions of fermentation inhibitors and monosaccharides present in the concentrate on the feed side (0.5 L) and the filtrate on the permeation side (1.0 L) were as shown in Table 22. The filtrate was filtered through a “UTC 60” nanofiltration membrane (manufactured by Toray Industries, Inc.; material: piperazine polyamide). The compositions of fermentation inhibitors and monosaccharides in the concentrate on the feed side (0.33 L) are shown in Table 23.Reagents were added to this concentrate so that the composition shown in Table 24 was obtained, and the resulting mixture was subjected to the same fermentation test as in reference example 7. The results of the xylose consumption rate measurement are shown in Figure 4 (see L in Figure 4). Comparative Example 5

[063] The aqueous sulfuric acid solution obtained in reference example 3 was neutralized to pH 4.0 with ammonia and subjected to microfiltration membrane treatment. Filtration treatment of 1.5 L of the obtained permeate was performed using a “UTC-60” nanofiltration membrane. The compositions of fermentation inhibitors and monosaccharides present in 0.75 L of the concentrate on the feed side were as follows Petition 870260039364, dated April 28, 2026, pages 54 / 62 41 / 45 shown in Table 22. Similarly, as in Example 6, reagents were added to this concentrate so that the composition shown in Table 24 was obtained, and the resulting mixture was subjected to a fermentation test. The results (xylose consumption rates) are shown in Figure 4 (see K in Figure 4).

[064] It was found that although the sugar liquid obtained in Example 6 contained slightly higher concentrations of coumaric acid, ferulic acid, and 2,3-dihydrobenzofuran, the sugar liquid demonstrated higher fermentability than that of comparative Example 5 in terms of the xylose consumption rate. This is due to the presence in the aqueous sugar solution of unidentified fermentation inhibitors for which an ultrafiltration membrane with a molecular weight cutoff of 600 to 2,000 Da is impermeable. Furthermore, from Example 6, it was found that not only the sugar liquid on the feed side of the ultrafiltration membrane with a molecular weight cutoff of 600 to 2,000 Da, but also the second concentrated sugar liquid obtained by filtering the filtrate on the permeation side through a nanofiltration membrane and / or reverse osmosis membrane and collecting the sugar liquid from the feed side, are sugar liquids with optimal fermentability. Petition 870260039364, dated April 28, 2026, pages 55 / 62 Table 22 Concentrate and filtrate compositions obtained by ultrafiltration membrane treatment of a solution. Aqueous sulfuric acid Unit [g / L] Type of membrane Liquid treated Glucose Xylose Formic acid Acetic acid HMF Furfural Coumaric acid Ferulic acid 2,3-Dihydrobenzofuran Example 6 NTR-7450 concentrate 5 36 0.6 3.4 0.08 0.2 0.15 0.1 0.03 Example 6 NTR-7450 filtered 1 12 0.6 3.4 0.08 0.2 0.13 0.09 0.03 42 / 45 Petition 870260039364, dated April 28, 2026, pages 56 / 62 Table 23 Comparison Between Concentrate Obtained by Nanofiltration Membrane Treatment of Raw Aqueous Sulfuric Acid Solution and Concentrate Obtained by Ultrafiltration Membrane Treatment of Raw Aqueous Sulfuric Acid Solution Followed by Nanofiltration Membrane Treatment of the Resulting Filtrate Unit [g / L] Type of membrane Liquid treated Concentration rate Glucose Xylose Formic acid Acetic acid HMF Furfural Coumaric acid Ferulic acid 2,3-Dihydrobenzofuran Comparative example 5 (Fermentation test K) UTC-60 Raw liquid 2 times 6 40 0.6 3.4 0.08 0.2 0.15 0.1 0.03 Example 6 (Fermentation test L) UTC-60 Raw filtrate material 3.3 times 2.7 40 0.7 3.6 0.1 0.25 0.16 0.11 0.04 43 / 45 Petition 870260039364, dated April 28, 2026, pages 57 / 62 44 / 45 Table 24 Composition: Concentration of composition: Glucose 6 g / L, Xylose 40 g / L, Bacto yeast extract 10 g / L, Peptone 20 g / L, pH 6.5 Industrial Applicability

[065] By the present invention, fermentation inhibitors can be efficiently removed from an aqueous sugar solution derived from a biomass containing cellulose, and, on the other hand, a purified sugar liquid containing monosaccharides such as glucose and xylose can be produced with high purity and high yield so that the use of the purified sugar liquid as a fermentation feedstock allows increasing the efficiency of fermentation production of various chemicals. Description of the Symbols

[066] A - liquid sugar obtained by filtration treatment of an aqueous sugar solution treated with dilute sulfuric acid using an ultrafiltration membrane “NTR-7450”.

[067] B - liquid sugar obtained by filtration treatment of an aqueous sugar solution treated with dilute sulfuric acid using an ultrafiltration membrane “SPE1”.

[068] C - liquid sugar obtained by filtration treatment of an aqueous sugar solution treated with dilute sulfuric acid using a “GR95Pp” ultrafiltration membrane.

[069] D - liquid sugar obtained by filtration treatment of an aqueous sugar solution treated with dilute sulfuric acid using a “UTC-60” nanofiltration membrane. Petition 870260039364, dated April 28, 2026, pages 58 / 62 45 / 45

[070] E - liquid sugar obtained by steam-exploded filtration treatment of an aqueous sugar solution using an ultrafiltration membrane “NTR-7450”.

[071] F- liquid sugar obtained by steam-exploded filtration treatment of an aqueous sugar solution with an ultrafiltration membrane “SPE1”.

[072] G - liquid sugar obtained by steam-exploded filtration treatment of an aqueous sugar solution with a “GR95Pp” ultrafiltration membrane.

[073] H - liquid sugar obtained by steam-exploded filtration treatment of an aqueous sugar solution with a “UTC-60” nanofiltration membrane.

[074] I - concentrated sugar liquid obtained by filtering a hydrothermally treated aqueous sugar solution with a “UTC-60” nanofiltration membrane.

[075] J - concentrated sugar liquid obtained by filtration treatment of a hydrothermally treated aqueous sugar solution with an ultrafiltration membrane “NTR-7410” followed by filtration treatment of the obtained permeate with a nanofiltration membrane “UTC-60”.

[076] K - concentrated sugar liquid obtained by filtration treatment of an aqueous sulfuric acid solution with a “UTC-60” nanofiltration membrane.

[077] L - concentrated sugar liquid obtained by filtration treatment of an aqueous sulfuric acid solution with an ultrafiltration membrane “NTR-7450” followed by filtration treatment of the obtained permeate with a nanofiltration membrane “UTC-60”. Petition 870260039364, dated April 28, 2026, pages 59 / 62

Claims

1 / 2 Claims 1. METHOD FOR PRODUCING A SUGAR LIQUID USING A BIOMASS CONTAINING CELLULOSE AS A RAW MATERIAL, characterized by comprising the steps of: (1) hydrolyzing a biomass containing cellulose to produce an aqueous sugar solution; and (2) filtering said aqueous sugar solution obtained in step (1) through an ultrafiltration membrane with a molecular weight cutoff of 600 to 2,000 Da, to remove a fermentation inhibitor(s) on the permeation side and collect a sugar liquid on the feed side wherein said fermentation inhibitor(s) comprise(s) one or more substance(s) selected from the group consisting of coumaric acid, ferulic acid and 2,3-dihydrobenzofuran.

2. METHOD, according to claim 1, characterized in that, in step (2), said aqueous sugar solution is filtered after adjusting the pH to a maximum of 5.

3. METHOD, according to any one of claims 1 to 2, characterized in that the material of the functional layer of said ultrafiltration membrane used in said step (2) is polyethersulfone.

4. METHOD, according to any one of claims 1 to 3, characterized by comprising filtering the permeate obtained in step (2) containing a sugar liquid and / or fermentation inhibitor through a nanofiltration membrane and / or reverse osmosis membrane, to collect a concentrated sugar liquid from the feed side.

5. METHOD FOR PRODUCING A CHEMICAL PRODUCT, characterized by comprising the use, as a fermentation feedstock, of a sugar liquid obtained by the method of producing a sugar liquid, as defined in any one of claims 1 to 4, comprising the steps of: (1) hydrolyzing a biomass containing cellulose to produce an aqueous sugar solution; and (2) filtering said aqueous sugar solution obtained in step (1) through an ultrafiltration membrane with a molecular weight cutoff of 600 to 2,000 Da, to remove a fermentation inhibitor(s) on the permeation side and collect a sugar liquid on the feed side.

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