Method for producing organic compound, and composition for culturing microorganism

Abstract

Provided is a method for producing an organic compound, preferably ethanol, which includes bringing a liquid containing glucose into contact with a microorganism, wherein the liquid containing glucose is a hydrolysate of cellulose obtained by bringing a liquid containing cellulose and water into contact with a solid acid catalyst, and when the liquid containing glucose is subjected to LC analysis including a differential refractive index detector, the total area value of peaks derived from components having a lower molecular weight than glucose is 0.01-2.0 times and preferably 0.01-0.2 times the area value of peaks derived from glucose.

Description

Method for producing organic compounds and composition for culturing microorganisms

[0001] The present invention relates to a method for producing an organic compound and a composition for culturing a microorganism.

[0002] In recent years, the synthesis and production of various organic compounds using biomass, a renewable resource, has been explored from the perspectives of global environmental protection and the effective utilization of waste. For example, bioethanol has been widely explored as a raw material for low-carbon footprint materials toward achieving carbon neutrality. In particular, the production of bioethanol from edible biomass, followed by olefination and subsequent polymerization via dehydration, has been investigated. The conversion of edible biomass to bioethanol is well established and is considered a promising method for producing raw materials for plastic products. However, competition with edible biomass exists as a food source, and in recent years, efforts have been made to switch from edible biomass to nonedible biomass as a raw material for bioethanol production. Cellulose, a nonedible biomass, can be hydrolyzed to glucose using enzymatic or sulfuric acid methods, and the glucose can then be converted to ethanol via microbial fermentation. However, the decomposition to glucose is generally difficult due to the chemically stable structure of cellulose. For example, even if cellulose can be converted to glucose, the resulting glucose is not sufficiently pure, and even if it is purified, ethanol cannot be efficiently obtained. Therefore, in a method for producing ethanol using cellulose, which is a non-edible biomass, as a starting material, there is a need for a method for efficiently promoting alcoholic fermentation by microorganisms or a method for obtaining a decomposition product of cellulose.

[0003] Since the importance of environmental conservation due to climate change and carbon neutrality have been advocated, various methods for producing ethanol using biomass as a raw material have been investigated. For example, Patent Document 1 describes a method for producing ethanol by fermenting monosaccharides obtained by hydrolyzing cellulose through three processes: a hot-compressed water reaction, a saccharification enzyme reaction, and a solid acid catalyst reaction. Specifically, Patent Document 1 describes a "method for producing ethanol by fermenting monosaccharides obtained by a monosaccharide production process including: a hot-compressed water reaction process in which compressed hot water is applied to biomass to selectively decompose hemicellulose contained in the biomass; a beating process in which the solid residue after the hot-compressed water reaction process is beaten; a primary saccharification process in which a saccharification enzyme is applied to the solid residue after the beating process; and a secondary saccharification process in which a product from the primary saccharification process is applied with a solid acid catalyst." Furthermore, Patent Document 2 describes a "bioethanol production method for producing ethanol from lignocellulose, characterized in that biomass is pulverized in a mill, the pulverized biomass powder is mixed with a catalyst and preheated steam in a preheater to produce a biomass powder slurry, and the biomass powder slurry supplied from the preheater is heated in a hydrolysis tower with heating steam."

[0004] JP 2013-141415 A JP 2008-297229 A

[0005] In recent years, the production of organic compounds using biomass as a raw material has attracted attention. Among these, the industrialization of bioethanol production has progressed and it is used as a carbon-neutral fuel. It is known that ethanol fermentation typically involves not only the main reaction, which converts glucose present in cellulose hydrolysate to ethanol, but also various side reactions. Therefore, the amount (yield) and purity of ethanol decrease due to by-products produced in the side reactions. In particular, the amount of by-products produced during ethanol fermentation tends to increase as the fermentation time (reaction time) elapses.

[0006] Acetobacter bacteria convert ethanol into acetic acid. In ethanol production, acetic acid bacteria should be avoided because they reduce the yield and purity of ethanol. However, acetic acid bacteria are widely present in nature as normal bacteria and are prone to contamination. For example, acetic acid bacteria are found suspended in the air and also naturally present in the environment or reaction site where sugars or plant carbohydrates are fermented by yeast to produce ethanol. As described above, to solve the problems caused by acetic acid bacteria, it is important to prevent their presence in the environment and reaction site of ethanol fermentation. However, it is not easy to eliminate acetic acid bacteria, which are normal bacteria, from the environment and reaction site. Acetobacter bacteria are unavoidable contaminants, especially in ethanol production methods aimed at industrialization. Therefore, to realize efficient and / or industrial ethanol production methods, it is necessary to suppress the growth of acetic acid bacteria and acetic acid fermentation, even if they are present.

[0007] As such, bioethanol is being widely considered and researched, but few organic compounds other than ethanol have been industrialized due to reasons such as the high cost of collecting and transporting biomass and the need for advanced technology to convert them into the desired organic compounds.

[0008] An object of the present invention is to provide a method for synthesizing organic compounds that can synthesize various organic compounds using sugars derived from cellulose. In a preferred embodiment of the present invention, an object of the present invention is to provide a method for producing ethanol that can efficiently ferment ethanol using sugars derived from cellulose while suppressing the growth of acetic acid bacteria and acetic acid fermentation even if acetic acid bacteria are contaminated.

[0009] <1> A method for producing an organic compound, comprising contacting a liquid containing glucose with a microorganism, wherein the glucose-containing liquid is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and wherein, when the glucose-containing liquid is subjected to LC analysis using a differential refractive index detector, the total area value of peaks derived from components with molecular weights lower than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose. <2> The method according to <1>, wherein the organic compound is ethanol, and in the LC analysis, the total area value of peaks derived from components with molecular weights lower than glucose is 0.01 to 0.2 times the area value of the peak derived from glucose. <3> The method according to <1>, wherein the cellulose comprises one of cellulose derived from board pulp or cellulose derived from pulp sludge, or a mixture thereof. <4> The method according to <1> or <2>, wherein the solid acid catalyst contains carbon element and silica and has sulfo groups as surface functional groups. <5> The method according to <4>, wherein the silica has mesopores. <6> The manufacturing method according to <4> or <5>, wherein the carbon element is supported on the surface of the silica. <7> The manufacturing method according to any one of <4> to <6>, wherein the solid acid catalyst contains titanium element. <8> The manufacturing method according to <7>, wherein the titanium element is contained in the silica. <9> A composition for microbial culture comprising a liquid containing glucose, wherein the glucose-containing liquid is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and wherein, when the glucose-containing liquid is subjected to LC analysis using a differential refractive index detector, the total area value of peaks derived from components with lower molecular weights than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose.

[0010] The production method <2> above is synonymous with the production method [B1] below. Accordingly, the production method [A1] of an organic compound of the present invention and the production method [B1] of ethanol according to a preferred embodiment of the present invention can be distinguished and described as follows: [A1] A production method for an organic compound, comprising contacting a liquid containing glucose with a microorganism, wherein the glucose-containing liquid is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and wherein, when the glucose-containing liquid is subjected to LC analysis using a differential refractive index detector, the total area value of peaks derived from components having a lower molecular weight than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose. [A2] The production method according to [A1], wherein the cellulose comprises one of cellulose derived from board pulp or cellulose derived from pulp sludge, or a mixture thereof. [A3] The production method according to [A1] or [A2], wherein the solid acid catalyst comprises carbon element and silica and has a sulfo group as a surface functional group. [A4] The method according to [A3], wherein the silica has mesopores. [A5] The method for producing a catalyst according to [A3] or [A4], wherein the carbon element is supported on the surface of the silica. [A6] The method for producing a catalyst according to any one of [A3] to [A5], wherein the solid acid catalyst contains a titanium element. [A7] The method for producing a catalyst according to [A6], wherein the titanium element is contained in the silica.

[0011] [B1] A method for producing ethanol, comprising contacting a glucose-containing liquid with a microorganism, wherein the glucose-containing liquid is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and wherein, when the glucose-containing liquid is subjected to LC analysis using a differential refractive index detector, the total area value of peaks derived from components having a lower molecular weight than glucose is 0.01 to 0.2 times the area value of the peak derived from glucose. [B2] The method for producing ethanol according to [B1], wherein the solid acid catalyst contains carbon element and silica and has sulfo groups as surface functional groups. [B3] The method for producing ethanol according to [B2], wherein the silica has mesopores. [B4] The method for producing ethanol according to [B2] or [B3], wherein the carbon element is supported on the surface of the silica. [B5] The method for producing ethanol according to any one of [B2] to [B4], wherein the solid acid catalyst contains titanium element. [B6] The method for producing ethanol according to [B5], wherein the titanium element is contained in the silica.

[0012] The method for synthesizing an organic compound of the present invention can synthesize various organic compounds, including ethanol, using sugars derived from cellulose (cellulose hydrolysates). That is, organic compounds can be produced from cellulose. In particular, a preferred embodiment of the method for producing ethanol of the present invention can efficiently ferment ethanol using sugars derived from cellulose, and can efficiently ferment ethanol while suppressing the growth of acetic acid bacteria and acetic acid fermentation even when acetic acid bacteria are present. As a result, ethanol can be efficiently produced from cellulose. The above and other features and advantages of the present invention will become more apparent from the following description.

[0013] FIG. 1 is a chromatogram obtained by HPLC analysis of the cellulose degradation product prepared in Experimental Example 7(1). FIG. 2 is a graph showing the normal turbidity change of the culture solution in which yeast was cultured using the cellulose degradation product prepared in Experimental Example 7(1) in Experimental Examples 8-1 and 8-2. FIG. 3 is a graph showing the normal turbidity change of the culture solution in which acetic acid bacteria were cultured in Experimental Examples 8-1 and 8-2. FIG. 4 is a graph showing the normal turbidity change of the culture solution in which yeast was cultured using various carbon sources in Experimental Example 9. FIG. 5 is a graph showing the normal turbidity change of the culture solution in which acetic acid bacteria were cultured with the addition of levoglucosan at a predetermined concentration in Experimental Example 10. FIG. 6 is a graph showing the normal turbidity change of the culture solution in which acetic acid bacteria were cultured with the addition of HMF at a predetermined concentration in Experimental Example 10. FIG. 7 is a graph showing the normal turbidity change of the culture solution in which acetic acid bacteria were cultured with the addition of HMF at a predetermined concentration in Experimental Example 10. 1 is a graph showing the normal change in turbidity of a culture medium in which acetic acid bacteria were cultured with furfural added at a predetermined concentration.

[0014] In the present invention and this specification, "contamination with acetic acid bacteria" means contamination with acetic acid bacteria, i.e., coexistence of acetic acid bacteria. The cause of contamination or coexistence is not particularly limited, and includes, for example, contamination or coexistence as a result of exposure to or contact with acetic acid bacteria. In the present invention and this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" as the lower and upper limits.

[0015] [Method for Producing Organic Compounds] Production method A of the present invention (hereinafter sometimes referred to as "Production method A of the present invention") is a method for producing an organic compound, which comprises contacting a glucose-containing liquid with a microorganism. Furthermore, a preferred embodiment of the present invention for producing ethanol (hereinafter sometimes referred to as "preferred production method B of the present invention") is a method for producing ethanol, which comprises contacting a glucose-containing liquid with a microorganism. In this specification, Production method A of the present invention and preferred production method B of the present invention are collectively referred to as "production methods of the present invention."

[0016] [Organic Compound and Ethanol to be Produced] The organic compound produced by the production method of the present invention is not particularly limited and can be appropriately determined taking into consideration the composition of the glucose-containing liquid, the properties of the microorganism, the production capacity, etc. The organic compound produced by Production Method A of the present invention is not particularly limited, and examples thereof include alcohol compounds such as ethanol, propanol, and butanol; carboxylic acid compounds such as itaconic acid, succinic acid, adipic acid, and muconic acid; aromatic compounds such as shikimic acid, ferulic acid, protocatechuic acid, and phenol; amino acid compounds such as alanine, valine, methionine, and tryptophan; and diene compounds such as isoprene and butadiene. The organic compound produced by a preferred Production Method B of the present invention is ethanol.

[0017] The components used in the production method of the present invention will be described below.

[0018] [Glucose-Containing Liquid] In the production method of the present invention, the glucose-containing liquid used is a cellulose hydrolysate (sometimes referred to as a "cellulose hydrolysate" or "saccharified liquid") obtained by a method for producing a cellulose hydrolysate, which comprises contacting a liquid containing cellulose and water with a solid acid catalyst. The glucose-containing liquid used in Production Method A of the present invention is, among the above-mentioned cellulose hydrolysates, one in which the total area value of peaks derived from components with lower molecular weights than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose when subjected to LC analysis using a differential refractive index detector. By contacting a cellulose hydrolysate having the above-described origin and composition with a microorganism as a raw material mixture for Production Method A of the present invention, various organic compounds can be produced, preferably in high amounts (high yields), while effectively utilizing cellulose, a non-edible biomass that can avoid competition with food applications. Furthermore, when producing the above-mentioned alcohol compounds or the like as organic compounds, in Production Method A of the present invention, for example, Experimental Example 7. By using the specific cellulose hydrolysate shown in the above, the growth of acetic acid bacteria and acetic acid fermentation can be inhibited, so that even under conditions or environments where acetic acid bacteria may be mixed in during production, the excellent production efficiency of organic compounds is not impaired, and industrialization can be considered.

[0019] The glucose-containing liquid used in the preferred production method B of the present invention is a cellulose hydrolysate described above in which the total area value of peaks derived from components with molecular weights lower than glucose is 0.01 to 0.2 times the area value of the peak derived from glucose. In the preferred production method B of the present invention, by using such a cellulose hydrolysate as a raw material mixture for ethanol fermentation, not only can ethanol be produced, but microbial ethanol fermentation can be promoted while suppressing the occurrence and progression of side reactions, allowing for efficient (high yield) ethanol production even over long fermentation times. Furthermore, even if acetic acid bacteria coexist during ethanol fermentation, the growth of acetic acid bacteria and acetic acid fermentation by acetic acid bacteria can be suppressed, thereby preventing the ethanol converted from the cellulose hydrolysate from being converted into acetic acid. This is particularly advantageous when using the specific cellulose hydrolysate shown in Experimental Example 7. Therefore, the preferred production method B of the present invention effectively utilizes cellulose, a non-edible biomass that can avoid competition with food uses, while producing high amounts (high yields) of bioethanol, which is important for achieving carbon neutrality. Furthermore, because the growth of acetic acid bacteria and acetic acid fermentation can be inhibited, the excellent efficiency of ethanol production is not impaired even under conditions or environments where exposure to or contact with acetic acid bacteria may occur during production, and industrialization is also within reach.

[0020] As described above, the production method of the present invention uses the glucose-containing liquid described above. This liquid is the cellulose degradation product obtained by the degradation product production method described below, and has the composition (component area ratio) described below. The cellulose degradation product may have the composition described below, and may contain components other than those contained in the cellulose degradation product. The other components may be any components that do not inhibit the production of organic compounds or ethanol fermentation, such as water, hemicellulose, and lignocellulose.

[0021] The cellulose degradation product used in Production Method A of the present invention has a composition such that, when subjected to LC analysis using a differential refractive index detector, the total area value of peaks derived from components with molecular weights lower than glucose is 0.01 to 2.0 times the area value of the glucose-derived peak. When the area ratio of this total area value to the area value of the glucose-derived peak (hereinafter sometimes referred to as the "component area ratio") is 0.01 to 2.0, the composition and properties of the cellulose degradation product are improved, and various organic compounds can be synthesized by Production Method A of the present invention. When the component area ratio exceeds 2.0, the amount of glucose in the cellulose degradation product decreases, making it difficult to produce it at a high concentration and potentially reducing workability and productivity. In the present invention, the upper limit of the component area ratio is preferably 1.0 or less, and more preferably 0.2 or less, in terms of the yield of the organic compound, workability, productivity, etc. On the other hand, the lower limit of the component area ratio is not particularly limited, and is, for example, preferably 0.01 or more, more preferably 0.05 or more, and even more preferably 0.1 or more. In addition, in the production method A of the present invention, when an alcohol compound, particularly ethanol, is produced using a cellulose decomposition product having the above-mentioned component area ratio of 0.01 to 0.2 times, the same excellent effects as those of the preferred production method B of the present invention are achieved.

[0022] On the other hand, Production Method B of the present invention uses a cellulose hydrolyzate having a composition such that the component area ratio is 0.01 to 0.2 when subjected to LC analysis using a differential refractive index detector. In the preferred Production Method B of the present invention, using a cellulose hydrolyzate having a component area ratio of 0.01 to 0.2 improves the composition and properties, suppresses the occurrence or promotion of side reactions, and allows ethanol fermentation of glucose to proceed preferentially as the main reaction. Furthermore, the growth of acetic acid bacteria and acetic acid fermentation, which could not be suppressed in the past when acetic acid bacteria were present, can be highly suppressed, and the yield and selectivity (content) of ethanol produced by ethanol fermentation can be not only maintained but also increased. In terms of further increasing the efficiency of ethanol fermentation and further suppressing the growth of acetic acid bacteria and acetic acid fermentation, thereby producing ethanol with high yield and high selectivity, the component area ratio is preferably 0.01 to 0.2, more preferably 0.05 to 0.2, and even more preferably 0.1 to 0.2. In the present invention, the component area ratio is a value measured by the method described in the examples below.

[0023] Generally, when cellulose is hydrolyzed, the resulting degradation product (composition) contains glucose as the main component and other components. Examples of other components include partial hydrolysis products of cellulose, glucose and other reaction products, such as tetramer or higher oligosaccharides, sugars such as cellotriose, cellobiose, mannose, fructose, and levoglucosan, as well as 5-hydroxymethylfurfural (HMF) and furfural. In the present invention, the term "major component" refers to the component with the largest mass content among the components. The cellulose degradation product used in the present invention may contain the above-mentioned partial hydrolysis products of cellulose, glucose and other reaction products, but satisfies the above-mentioned component area ratio for components with molecular weights lower than glucose identified by LC analysis (sometimes referred to as "low molecular weight components"). While the low molecular weight components cannot be uniquely determined due to variations in the origin and composition of the cellulose used in the hydrolysis reaction, the contact conditions between glucose and the solid acid catalyst, and other factors, they typically include components with molecular weights lower than glucose among the partial hydrolysis products of cellulose, glucose and other reaction products. Examples include levoglucosan, HMF, furfural, and even unidentifiable components. The number of types of low-molecular-weight components that the cellulose degradation product may contain is not particularly limited, and may be one or more.

[0024] The content (also referred to as concentration, unit of content is mass%) of each low-molecular-weight component in the cellulose decomposition product can be appropriately determined taking into consideration the area ratio of the components. For example, the content of HMF in the cellulose decomposition product is preferably 1.5 mass% or less, more preferably 0.01 to 0.5 mass%, and even more preferably 0.02 to 0.01 mass%.

[0025] Among the low-molecular-weight components, the cellulose degradation product preferably contains furfural from the viewpoint of inhibiting the growth of acetic acid bacteria. The concentration of furfural is not particularly limited as long as it is a concentration that can inhibit the growth of acetic acid bacteria, and is, for example, preferably 0.035 g / L (0.0035% by mass) or more, more preferably 0.07 g / L (0.007% by mass) or more. Furthermore, from the viewpoint of minimizing the effect on yeast growth and ethanol fermentation, it is preferably 5.0 g / L (0.5% by mass) or less, more preferably 2.0 g / L (0.2% by mass) or less, and even more preferably 1.0 g / L (0.1% by mass) or less.

[0026] The glucose content in the cellulose decomposition product cannot be uniquely determined due to variations in the origin and composition of the cellulose used in the hydrolysis reaction, the contact conditions between the glucose and the solid acid catalyst, etc., but is a content such that the component area ratio is at least 0.01 to 2.0 times, and is a content within the above-mentioned preferred range, more preferred range, or even more preferred range. In terms of the mass ratio of glucose in the cellulose decomposition product, from the viewpoint of efficient ethanol production, the glucose content is preferably, for example, 50 to 99 mass%, and more preferably 83 to 99 mass%, based on the total mass of all components contained in the cellulose decomposition composition.

[0027] <Method for producing cellulose decomposition product> A cellulose decomposition product having the above composition can be prepared by mixing components such as glucose, but in the present invention, a cellulose decomposition product that satisfies the above composition is used that is obtained by the method for producing a cellulose decomposition product described below (sometimes referred to as the "decomposition product production method"). This decomposition product production method involves contacting a liquid containing cellulose and water with a solid acid catalyst. The decomposition product production method using a solid acid catalyst has high cellulose hydrolysis reaction efficiency and can suppress the excessive occurrence and promotion of various side reactions in the hydrolysis reaction to glucose, making it possible to obtain glucose with a selectivity that satisfies the above component area ratio and in a high yield.

[0028] (Liquid Containing Cellulose and Water) The liquid containing cellulose and water (sometimes referred to as the "reaction liquid") used in the hydrolysis product production method need only contain cellulose and water, and may also contain other components. Other components may be any that do not inhibit the cellulose hydrolysis reaction, such as hemicellulose, lignocellulose, fatty acids, polymeric surfactants, and aluminum sulfate. The cellulose content in the reaction liquid is not particularly limited, but is preferably 0.01 to 1 kg per 1 L of water, and more preferably 0.05 to 0.5 kg, in terms of the cellulose hydrolysis reaction efficiency (hereinafter sometimes simply referred to as "hydrolysis reaction efficiency") and the glucose yield (hereinafter sometimes referred to as "glucose yield"). In the present invention, the cellulose content in the reaction liquid refers to the substantial cellulose content (cellulose equivalent) when a mixture such as waste pulp is used as the cellulose source. The reaction liquid is typically obtained as a suspension or dispersion by mixing cellulose with water.

[0029] - Cellulose - The cellulose used in the decomposition production method has the molecular formula (C ６ H １０ O ５ ) ｎ The term "cellulose" may be used in any form, including carbohydrates (polysaccharides) represented by the formula (1), which includes not only cellulose but also hemicellulose and lignocellulose. The cellulose used in the method for producing the degradation product may be a mixture of cellulose, hemicellulose, and / or lignocellulose. However, when lignocellulose is used or when lignocellulose is contained, it is preferable to carry out the lignin removal step described below.

[0030] The cellulose may be synthetic or commercially available, may be derived from non-edible biomass, or may be waste or recovered materials thereof. Examples of cellulose derived from non-edible biomass include cellulose (lignocellulose) derived from plants such as trees, thinned wood, and wood, and cellulose obtained by appropriately treating chemical pulp obtained by bleaching defatted plant flour with alkali. Considering its potential as an alternative to bioethanol derived from edible biomass, it is preferable to use cellulose derived from non-edible biomass for the cellulose used in the degradation product production method. Plants are typical examples of non-edible biomass, but it is preferable to use waste or recovered materials from these biomass in terms of efficient resource utilization and cost reduction. Examples of non-edible biomass include wood flour and wood chips from trees, thinned wood, and wood, various pulps (unused), and waste pulp such as board pulp and pulp sludge (paper sludge) discharged during papermaking or pulp manufacturing processes, and pulp recovered from used diapers. In the present invention, plate pulp refers to the dehydrated (pressed) product of pulp discharged during the papermaking or pulp manufacturing process, and its shape does not necessarily have to be plate-like. Pulp sludge is the dehydrated (pressed) solid product of pulp slurry solution discharged during the papermaking or pulp manufacturing process to which additives such as aluminum sulfate, anionic coagulants, or cationic coagulants have been added, and generally tends to contain more impurities than plate pulp.

[0031] Among the above-mentioned celluloses, waste pulp, such as board pulp, pulp sludge, and pulp recovered from used diapers, is particularly preferred in that it is abundantly recovered, allows for effective utilization of resources (potentially reducing costs), and avoids the poisoning of solid acid catalysts by lignin without the need for the lignin removal step described below. In the production method of the present invention, waste pulp is preferred, and board pulp or pulp sludge is more preferred, in that it allows for efficient production of organic compounds, particularly ethanol, while making effective use of resources. Furthermore, from the viewpoint of efficient production of organic compounds, particularly ethanol, it is also a preferred embodiment to use synthetic or commercially available products in addition to the above-mentioned waste pulp.

[0032] Cellulose typically exhibits crystallinity due to the hydrogen bonding of two or more cellulose molecules. The hydrolysis product production method can use crystalline cellulose (sometimes referred to as "crystalline cellulose"), or cellulose obtained by reducing the crystallinity of crystalline cellulose using conventional methods (sometimes referred to as "low-crystalline cellulose" or "microcrystalline cellulose"). Low-crystalline cellulose may be crystalline cellulose in which the crystallinity has been partially reduced, or in which the crystallinity has been (almost) completely eliminated. The treatment method for reducing crystallinity is not particularly limited, but a treatment that can at least partially produce single-chain cellulose molecules by cleaving the hydrogen bonds is preferred. Cellulose containing at least partially single-chain cellulose molecules exhibits significantly higher hydrolysis reaction efficiency. Specific examples of treatment methods for reducing crystallinity include the various methods described in Patent Document 1, with physical methods such as jet mills, hammer mills, ball mills, and bead mills being preferred.

[0033] The shape of the cellulose used in the method for producing a degradation product is not particularly limited, but in terms of hydrolysis reaction efficiency and glucose yield, it is preferably in the form of particles, powder, small pieces, etc. The size of the cellulose is not particularly limited and can be appropriately determined taking into consideration the hydrolysis reaction efficiency and glucose yield.

[0034] - Water - The water used in the method for producing a hydrolyzed product is not particularly limited, but industrial water, well water, city water, ion-exchanged water, purified water, (ultra)pure water, etc. can be used, with ion-exchanged water, purified water, and (ultra)pure water being preferred.

[0035] (Solid Acid Catalyst) The solid acid catalyst used in the decomposition product production method is not particularly limited, and various solid acid catalysts can be used. Examples include inorganic solid acids such as zeolite, alumina, silica, silica alumina, zirconium sulfate, zirconium phosphate, heteropolyacid, and niobic acid, as well as inorganic solid acids, resins, or carbonaceous materials to which acidic groups such as sulfo groups have been introduced by acidification treatment. In the decomposition product production method, a solid acid catalyst containing carbon and silica and having sulfo groups as surface functional groups is preferably used as the solid acid catalyst, as it can hydrolyze cellulose with high efficiency and can produce a cellulose decomposition product that satisfies the above-mentioned component area ratio. It is more preferable to use a solid acid catalyst containing carbon, titanium, and silica and having sulfo groups as surface functional groups. Hereinafter, the above-mentioned preferred solid acid catalyst and more preferred solid acid catalyst will be collectively referred to as a "suitable solid acid catalyst." A suitable solid acid catalyst is a catalyst containing carbon and sulfo groups (-SO ) on a substrate (also referred to as a "silica substrate") primarily composed of silica (silicon dioxide). ３ H), or has carbon and titanium elements and a sulfo group. The shape of a suitable solid acid catalyst is not particularly limited, but in terms of hydrolysis reaction efficiency and glucose yield, particles, powder, small pieces, etc. are preferred. The size (average particle diameter) of a suitable solid acid catalyst is not particularly limited, but is preferably 0.1 to 10,000 μm, for example. The average particle diameter is a value measured by the method described in the Examples below. The substrate is preferably a porous substrate (porous body), more preferably a porous body having mesopores (mesoporous body), and even more preferably mesoporous silica, in terms of being able to increase hydrolysis reaction efficiency and glucose yield. In the present invention, mesopores refer to pores with an average pore diameter of 2 to 50 nm.

[0036] When the substrate is a porous substrate, its characteristics or physical properties, such as the average pore diameter, specific surface area, and total pore volume, are not particularly limited and can be determined appropriately. For example, the average pore diameter is preferably 2 to 10 nm, more preferably 2 to 5 nm, from the viewpoints of hydrolysis reaction efficiency and glucose yield. The specific surface area is preferably 10 to 3,000 m, from the viewpoints of hydrolysis reaction efficiency and glucose yield. ２ / g, and 100 to 2000m ２ / g is more preferable. From the viewpoint of hydrolysis reaction efficiency and glucose yield, the total pore volume is preferably 0.1 to 2 mL / g, and more preferably 0.2 to 1 mL / g. The average pore diameter, specific surface area, and total pore volume of the porous substrate are values ​​measured by the methods described in the Examples below.

[0037] A suitable solid acid catalyst, typically a substrate, contains carbon. In the present invention, "a substrate containing carbon" refers to the presence (presence) of carbon on the surface and / or inside of the substrate. Examples include a substrate in which carbon is supported or adsorbed on the surface of the substrate (carbon-supported substrate), a substrate in which carbon is contained inside (a substrate formed of a composite of silica and carbon), and a substrate in which carbon is present on both the surface and inside. The carbon-containing form (structure) of a solid acid catalyst is preferably a substrate in which carbon is supported or adsorbed on the surface of the substrate (carbon-supported substrate), since this allows for the bonding or immobilization of surface functional groups (described below) to the surface, thereby improving the hydrolysis reaction efficiency and glucose yield. In the present invention, the "surface" of the substrate typically refers to the outer surface, but if the substrate is porous, it also includes the inner surfaces of the pores in addition to the outer surface. Meanwhile, the "interior" of the substrate refers to the portion not exposed to the surface. The carbon element content in a suitable solid acid catalyst is not particularly limited, but is preferably 1 to 70 mass %, more preferably 5 to 50 mass %, of the mass of the suitable solid acid catalyst (excluding the mass of sulfo groups) in order to increase the hydrolysis reaction efficiency and glucose yield. The carbon element content in a suitable solid acid catalyst is a value measured by the measurement method in the examples described below.

[0038] In the above-described more preferred solid acid catalyst, the substrate typically contains elemental titanium. In the present invention, "the substrate contains elemental titanium" refers to the presence (presence) of elemental titanium on the surface and / or inside of the substrate. Examples include an embodiment in which elemental titanium is supported or adsorbed on the surface of the substrate (elemental titanium-supported substrate), an embodiment in which elemental titanium is contained inside the substrate (an embodiment in which the substrate is formed of a composite of silica and elemental titanium), and an embodiment in which elemental titanium is contained on the surface and inside of the substrate. When elemental titanium is contained on the surface and / or inside of the substrate, the hydrolysis reaction efficiency and glucose yield can be improved. The content of elemental titanium contained in the more preferred solid acid catalyst is not particularly limited, but is preferably 0.1 to 10 mass% and more preferably 0.5 to 5 mass% of the mass of the more preferred solid acid catalyst (excluding the mass of carbon atoms and sulfo groups) in terms of improving the hydrolysis reaction efficiency and glucose yield. The content of elemental carbon in the more preferred solid acid catalyst is a value measured by the measurement method in the Examples described below.

[0039] A more preferred embodiment of the solid acid catalyst containing elemental carbon and elemental titanium is an embodiment in which the above-mentioned embodiment containing elemental carbon and embodiment containing elemental titanium are combined as appropriate, but from the viewpoint of improving the hydrolysis reaction efficiency and glucose yield, an embodiment in which elemental titanium is contained on the surface and / or inside of a substrate and an embodiment in which elemental carbon is supported or adsorbed on the surface of a substrate are combined (a solid acid catalyst of this embodiment is also referred to as a carbon-element-supported silica-titanium composite mesoporous material).

[0040] A suitable solid acid catalyst has a sulfo group as a surface functional group. In the present invention, a solid acid catalyst having a sulfo group as a surface functional group means that the sulfo group is chemically bonded to the surface of the substrate and / or carbon element. The sulfo group may be partially or entirely in the form of a salt, as long as it can promote the hydrolysis reaction of cellulose. The content of sulfo groups contained in a suitable solid acid catalyst is not particularly limited, but in terms of being able to increase the hydrolysis reaction efficiency and glucose yield, the amount present per 1 g of the suitable solid acid catalyst is preferably 0.01 to 2 mmol / g, and more preferably 0.05 to 1 mmol / g. The content of sulfo groups in a suitable solid acid catalyst is a value measured by the measurement method in the Examples described below.

[0041] Although commercially available solid acid catalysts may be used, suitable solid acid catalysts contain carbon, preferably titanium, and a sulfo group, and therefore, it is preferable to use an appropriately synthesized catalyst. The method for introducing carbon into the substrate is not particularly limited, and various known methods can be applied. For example, a method for supporting or adsorbing carbon onto the surface of the substrate includes mixing the substrate with a carbon source and then carbonizing the carbon source, specifically the method described in Green Chem., 2010, 12, 1560-1563. The carbon source is not particularly limited as long as it is a compound containing carbon atoms. Typically, organic compounds are used, and preferred examples include sugars such as sucrose, alcohols such as furfuryl alcohol, hydrocarbon compounds, and alkylene oxides. The method for introducing titanium into the substrate is not particularly limited, and various known methods can be applied. For example, a method for supporting or adsorbing titanium onto the surface of the substrate includes the same methods as those for supporting or adsorbing carbon, except that a titanium-containing compound is used. On the other hand, as a method for incorporating titanium element into the interior or the interior and surface of a substrate, a method for forming a substrate using a mixture of a silica precursor compound described below and a titanium-containing compound described below, such as the sol-gel method described below, can be used. The method for introducing sulfo groups into the substrate is not particularly limited, and various known methods can be applied. For example, a method for treating a substrate, preferably a substrate having carbon element supported or adsorbed on its surface, with sulfuric acid can be used, specifically the method described in Green Chem., 2010, 12, 1560-1563.

[0042] A method for producing porous silica as a substrate for a solid acid catalyst includes a sol-gel process (also known as a "molecular templating process" or "template process") using a silica source (silica precursor compound) and an amphiphilic compound as a template. Hereinafter, a more preferred solid acid catalyst will be described, taking as an example a method for producing a carbon-supported silica-titanium composite mesoporous material having sulfo groups as surface functional groups by the sol-gel process. A typical sol-gel process involves, for example, subjecting a silica precursor compound to a sol-gel reaction (hydrolysis and condensation reaction) around self-assembled micellar particles formed with a cationic surfactant to form an inorganic-organic nanocomposite, which is then calcined or acid-treated (to remove the cationic surfactant). A more preferred solid acid catalyst is a silica-titanium composite mesoporous material containing titanium on the surface and / or inside of a substrate, produced using the sol-gel process. This process can be carried out in the same manner as a typical sol-gel process, except that a titanium precursor compound (described below) is used together with the silica precursor compound (i.e., the silica precursor compound and the titanium precursor compound are allowed to coexist). Therefore, the type of each step in the sol-gel process used to produce a mesoporous silica-titanium composite, as well as the operations and reaction conditions for each step, can be appropriately determined with reference to conventional sol-gel processes. For example, the method described in Green Chem., 2010, 12, 1560-1563, and the series of steps in the synthesis method, the operations and reaction conditions for each step, in the Examples described below are useful references. The sol-gel process, like conventional sol-gel processes, allows for the adjustment or modification of the characteristics or physical properties of the porous body, such as the size (average pore diameter) and shape of the mesopores, as well as the skeletal shape of the porous body, by changing the type of silica precursor compound, particularly the type of amphiphilic compound, or preparation conditions such as the solvent composition, temperature, and time.

[0043] The amphiphilic compound used as the template can be any compound commonly used in a typical sol-gel process, without any particular limitations. Typically, an amphiphilic surfactant is used, with cationic surfactants, block copolymers, and the like being preferred. In the present invention, the use of a cationic surfactant as the template is preferred because it allows the average pore diameter of the mesopores to be set relatively small, further increasing the hydrolysis reaction efficiency and glucose yield. The cationic surfactant can be any surfactant commonly used as a template in the sol-gel process, without any particular limitations, and includes ammonium-based cationic surfactants. As the ammonium-based cationic surfactant, alkylammonium salts are preferred, with tetraalkylammonium salts being preferred. Ammonium salts containing long-chain (6 or more carbon atoms) linear alkyl groups are more preferred, and mono-long-chain linear alkyltrishort-chain (4 or less carbon atoms) alkylammonium salts are particularly preferred. The anion forming the quaternary ammonium salt is not particularly limited, but examples include inorganic anions such as hydroxide ions, halide ions, and perhalogen acid ions. A representative example of a mesoporous silica material prepared by the sol-gel method using a cationic surfactant is MCM-41, and a representative example of a porous silica material prepared by the sol-gel method using a block copolymer is SBA-15.

[0044] The silica precursor compound can be any compound commonly used in a general sol-gel process, without any particular limitations. Examples include silicon halides, hydroxides, and alkoxides, with silicon alkoxides and / or alkyl alkoxides being preferred. The number of carbon atoms in the alkyl group forming the alkyl alkoxide is not particularly limited, but is preferably 1 to 6, and more preferably 1 to 3. In the sol-gel process, a titanium-containing compound is used together with the silica precursor compound. Examples of the titanium-containing compound include titanium precursor compounds that are converted to elemental titanium together with the silica precursor compound by the sol-gel process. Examples of the titanium precursor compound include titanium halides, hydroxides, and alkoxides, with titanium alkoxides and / or titanium alkyl alkoxides (alkoxytitanium) being more preferred. The number of carbon atoms in the alkyl group forming the alkyl alkoxide is not particularly limited, but is preferably 1 to 6, and more preferably 1 to 3.

[0045] Next, carbon element is supported on or adsorbed onto the surface of the obtained silica-titanium composite mesoporous material. The method for supporting or adsorbing carbon element is not particularly limited and is as described above. Sulfo groups are introduced as surface functional groups onto the carbon element-supported silica-titanium composite mesoporous material thus obtained. The method for introducing sulfo groups is not particularly limited and is as described above. The silica-titanium composite mesoporous material, the carbon element-supported silica-titanium composite mesoporous material, and / or the element-supported silica-titanium composite mesoporous material into which sulfo groups have been introduced can also be crushed or disintegrated as appropriate.

[0046] As a preferred solid acid catalyst, a method for producing carbon-supported mesoporous silica having sulfo groups as surface functional groups by a sol-gel method can be mentioned, which is similar to the above-mentioned more preferred method for producing the solid acid catalyst, except that a silica precursor compound is used instead of a titanium precursor compound in the sol-gel method.

[0047] (Step of contacting a liquid containing cellulose and water with a solid acid catalyst) The method for producing a decomposition product includes contacting a liquid containing cellulose and water (reaction liquid) with a solid acid catalyst, and by this contact, cellulose is hydrolyzed in the presence of the solid acid catalyst to convert it to glucose, thereby obtaining a cellulose decomposition product. Each component used in this contacting step may be one type or two or more types.

[0048] The cellulose hydrolysis reaction that occurs in the contacting step is generally thought to involve hydrolysis of cellulose to oligosaccharides and hydrolysis of oligosaccharides to glucose. Therefore, the contacting step results in the hydrolysis of cellulose (saccharification treatment) to obtain a sugar-containing liquid containing glucose (also called a "saccharified liquid"). This sugar-containing liquid usually has the above-mentioned composition (satisfies the component area ratio).

[0049] In the method for producing a degradation product, the use of a suitable solid acid catalyst allows for efficient hydrolysis of cellulose to produce a cellulose degradation product containing glucose as the main component. The cellulose conversion rate and glucose selectivity (mass ratio of glucose content to other components) cannot be determined unequivocally because they vary depending on the type of cellulose, the type and amount of solid acid catalyst used, and the hydrolysis reaction conditions, but at least a glucose yield ((amount of glucose obtained) / (amount of cellulose charged)×100(%)) of 45% or more can be achieved.

[0050] In the method for producing a hydrolysis product, a liquid (reaction liquid) containing cellulose and water is contacted with a solid acid catalyst under appropriate reaction conditions, usually under heating. The contacting method may be any method that allows the three components of cellulose, water, and the solid acid catalyst to come into contact, including a method in which a pre-prepared reaction liquid is contacted with the solid acid catalyst, and a method in which cellulose, water, and the solid acid catalyst are contacted without pre-preparing a reaction liquid (a method in which cellulose and the solid acid catalyst are contacted in the presence of water). In the contacting step, when the reaction liquid is contacted with or introduced into the solid acid catalyst, this includes embodiments in which cellulose and water are contacted with or introduced into the solid acid catalyst separately, instead of the reaction liquid. For example, the contacting step involves heating the reaction liquid and the solid acid catalyst after introducing them into a sealed container. The heating temperature (reaction temperature) of the hydrolysis reaction is not particularly limited, but can be, for example, 110 to 200°C. The heating temperature is preferably 120 to 180°C, more preferably 120 to 150°C, in order to increase the efficiency of the cellulose hydrolysis reaction while suppressing the by-production of other components and thereby increasing the glucose yield. The heating time (reaction time) is determined appropriately depending on the heating temperature, the cellulose hydrolysis reaction efficiency (conversion rate), the glucose yield, and the like, and can be, for example, 1 to 48 hours. The pressure inside the sealed container under heating may be normal pressure, but typically the pressure is increased to, for example, a water vapor partial pressure exceeding 0.1 MPa. In the present invention, the hydrolysis reaction can also be carried out in an actively pressurized environment. The pressure at this time is not particularly limited and can be, for example, greater than 0.1 MPa and 20 MPa or less, with 0.1 to 10 MPa being preferred. The reaction environment (atmosphere) is not particularly limited and can be an inert gas atmosphere, but can also be an air atmosphere or a water vapor atmosphere. Considering industrial production, the reaction environment is preferably an air atmosphere and / or a water vapor atmosphere.

[0051] The amounts of the reaction solution and solid acid catalyst used are not particularly limited, but the amount of water in the reaction system (amount of water present) should be at least the amount necessary for the cellulose hydrolysis reaction. The amount of water in the reaction system is preferably 0.1 to 1,000 times by mass, more preferably 1 to 100 times by mass, relative to the amount of cellulose present, in terms of the mixability (stirability) and ease of handling of the reaction system, as well as the hydrolysis reaction efficiency and glucose yield. In the contacting step, water can be added separately from the reaction solution. The amount of water in the reaction system refers to the amount of water derived from the reaction solution. When water is added, the amount refers to the total amount of water derived from the reaction solution and the added water. The amount of solid acid catalyst used can be determined appropriately taking into account the reaction conditions, hydrolysis reaction efficiency, glucose yield, and the like. For example, it is preferably 0.01 to 10 times by mass, more preferably 0.1 to 5 times by mass, relative to the amount of cellulose present.

[0052] The contacting step can be carried out batchwise using a sealed vessel such as an autoclave, or can be carried out continuously using a reaction tube filled with a solid acid catalyst. In the batchwise process, it is preferable to mix the reaction solution and the solid acid catalyst by appropriate means such as stirring or shaking, in terms of hydrolysis reaction efficiency and glucose yield. An example of a continuous process is a method in which the reaction solution is continuously or intermittently passed through a heated reaction tube.

[0053] As described above, cellulose can be subjected to a hydrolysis reaction in the presence of a solid acid catalyst to obtain a mixture (also referred to as a reaction mixture) of a sugar-containing liquid containing glucose as the main component and the solid acid catalyst.

[0054] [Post-treatment Step] In the decomposition product production method, the reaction mixture can be post-treated after the contacting step to obtain a cellulose decomposition product. Post-treatment of the reaction mixture includes a step of cooling the reaction mixture and a step of solid-liquid separation of the reaction mixture. While the reaction mixture can be solid-liquid separated without cooling, cooling is preferred to suppress further reaction of glucose or to take into account workability, safety, and the like. The reaction mixture can be cooled naturally or using various refrigerants. The cooling temperature of the reaction mixture is not particularly limited and can usually be 100°C or lower. From the viewpoint of suppressing further reaction of glucose and maintaining a high glucose yield, 80°C or lower is preferred. From the viewpoint of workability, safety, and the like, a temperature near room temperature (e.g., 15 to 40°C) is preferred. The lower limit of the cooling temperature can be, for example, 0°C or higher, and preferably 15°C or higher, from the viewpoint of workability. The cooling rate can be determined appropriately, and a decrease of 0.1 to 10°C per minute is preferred. The cooling time can be determined appropriately depending on the heating temperature, cooling rate, and the like. The reaction mixture, which has been appropriately cooled, is subjected to solid-liquid separation to separate a sugar-containing liquid (cellulose decomposition product) containing glucose as a main component as a liquid phase from at least the solid acid catalyst and unreacted cellulose as a solid phase. The method for solid-liquid separation is not particularly limited, and examples thereof include filtration, centrifugation, and precipitation.

[0055] [Other Steps] In the decomposition product production method, steps other than the contacting step and post-treatment step can also be performed. For example, a step of purifying the separated and recovered sugar-containing liquid so as to satisfy the above-mentioned component area ratio, a step of adjusting the composition of the separated and recovered sugar-containing liquid, a step of isolating the solid acid catalyst and unreacted cellulose from the separated and recovered solid phase, a step of washing and regenerating the isolated solid acid catalyst, and, when the above-mentioned waste is used as a cellulose source, a step of removing impurities and contaminants from the waste, a step of removing lignin from the cellulose, etc., can be mentioned. Various known isolation and purification methods can be applied as the isolation and purification method for the sugar-containing liquid without particular limitation. In addition, in the step of adjusting the composition of the separated and recovered sugar-containing liquid, the amount of components can be adjusted within the above-mentioned component area ratio range by adding glucose to the separated and recovered sugar-containing liquid. In addition, the solid acid catalyst used in the decomposition product production method can be reused in the separated and recovered state (for example, as a mixture with unreacted cellulose), and there is no need to perform the solid acid catalyst isolation step, washing step, and regeneration step. However, since the catalytic activity of a solid acid catalyst usually gradually decreases with increasing number of uses, the solid acid catalyst may be washed and / or regenerated in consideration of the decrease in catalytic activity, hydrolysis reaction efficiency, glucose yield, etc. The washing and regeneration methods are not particularly limited.

[0056] The decomposition product production method decomposes cellulose into glucose through a simple and safe process of contacting a reaction solution with a solid acid catalyst, thereby producing a cellulose decomposition product that preferably satisfies the above-mentioned composition. In particular, the decomposition product production method using a suitable solid acid catalyst can produce glucose from cellulose in a high yield in a relatively short time through a simple and safe process. Furthermore, since the occurrence and promotion of various side reactions during the cellulose hydrolysis reaction can be suppressed, glucose can be obtained with high selectivity (high purity), resulting in a cellulose decomposition product that satisfies the above-mentioned composition. In this respect, the decomposition product production method can be considered a method for producing glucose. As a result, the decomposition product production method is suitable as a method for obtaining raw materials for bioethanol production using non-edible biomass as a raw material. Moreover, when waste pulp is used as a cellulose source, the cellulose hydrolysis reaction efficiency and glucose yield can be further increased, enabling the effective use of non-edible biomass resources. Therefore, the decomposition product production method not only enables efficient production of glucose from cellulose at low cost, but is also suitable for industrialization of cellulose hydrolysis and bioethanol production.

[0057] [Step of Contacting a Glucose-Containing Liquid with Microorganisms (Fermentation Step)] The production method of the present invention involves contacting a glucose-containing liquid having the above-described composition (hereinafter sometimes referred to as glucose liquid) with microorganisms. In this contacting step, the water content and pH are appropriately adjusted to create conditions suitable for various organic compound synthesis methods, such as ethanol fermentation. Typically, microorganisms are inoculated into the glucose liquid, and the glucose is converted into organic compounds such as ethanol by the action of the microorganisms. For convenience, the liquid obtained by mixing the glucose liquid with microorganisms and culturing for a certain period of time, in which some or all of the glucose has been converted into organic compounds such as ethanol, is referred to as the "fermentation liquid."

[0058] <Microorganisms> The microorganisms used in the production method of the present invention may have any properties or characteristics to synthesize and produce various organic compounds, and the microorganisms used in the preferred production method B of the present invention may have any properties or characteristics to produce ethanol. Such microorganisms are not particularly limited, and examples thereof include yeast, eubacteria, and archaea.

[0059] Examples of the microorganisms used in the production method A of the present invention include those of the genus Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, and the like. genus Rhodococcus, genus Pseudomonas, genus Bacillus, genus Lactobacillus, genus Lactococcus, genus Enterococcus, genus Alcaligenes, Klebsiella lla), Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Issatchenkia, Kluyvero Examples of suitable microorganisms include the genera Kluyveromyces, Yarrowia, Pichia, Candida, and Hansenula, as well as the genera Saccharomyces, Acetobacterium, and Eubacterium. Furthermore, when the organic compound to be produced in Production Method A of the present invention is an alcohol, particularly ethanol, the microorganisms described below that are used in the preferred Production Method B of the present invention can also be used. Microorganisms to be used in Production Method A of the present invention can be appropriately selected from the above-mentioned microorganisms and used, for example, a microorganism capable of producing the organic compound to be produced.

[0060] In the preferred production method B of the present invention, the yeast used is preferably one that can ferment sugars (hexose, pentose). Specifically, the yeast may be a yeast of the genus Saccharomyces such as Saccharomyces cerevisiae, a yeast of the genus Pichia such as Pichia stipitis, a yeast of the genus Candida such as Candida shihatae, a yeast of the genus Pachysolen such as Pachysolen tannophilus, a yeast of the genus Issatchenkia such as Issatchenkia orientalis, or a yeast of the genus Kluyveromyces marxianus. Examples of suitable yeasts include yeasts of the genus Kluyveromyces, such as Saccharomyces or Isachenkia, preferably Saccharomyces cerevisiae or Isachenkia orientalis. Genetically modified yeasts produced using genetic engineering techniques can also be used. Genetically modified yeasts can be used without particular limitations as long as they can ferment sugars (hexoses and pentoses), and preferably yeasts that can simultaneously ferment hexoses and pentoses.

[0061] In the preferred production method B of the present invention, examples of the eubacteria or archaea used include eubacteria such as Zymomonas, Escherichia coli, Corynebacterium, Clostridium, and Halomonas, and archaea such as Halobacteria. From the viewpoint of ethanol productivity, eubacteria of the genera Zymomonas, Escherichia coli, Corynebacterium, Clostridium, and Halomonas are preferred, with Escherichia coli and Clostridium being more preferred. More specific examples of eubacteria include Zymomonas mobilis, genetically modified Escherichia coli (strain KO11), Clostridium ljungdahlii, Clostridium autoethanogenum, and Halomonas (Halomonas sp. strain KM-1), which are preferably used.

[0062] In addition to the above, the microorganisms used in the production method of the present invention may also be those described in paragraph

[0043] of Patent Document 1, the contents of which are incorporated herein by reference. In the production method of the present invention, one or more types of microorganisms may be used, and it is preferable to use them as a culture medium. The culture medium for the microorganism can be prepared by conventional methods and conditions.

[0063] The glucose solution contains cellulose degradation products. In addition to the cellulose degradation products, it may also contain nutrients necessary for microbial growth and activity. Nutrient sources are not particularly limited and include, for example, yeast extract and polypeptone. Nutrient sources such as those described in paragraph

[0043] of Patent Document 1 can also be used, the contents of which are incorporated herein by reference. The glucose solution may also contain components commonly used depending on the microorganism used, such as a pH adjuster, a buffer, a chelating agent, an antibiotic, an expression inducer, and an antifoaming agent. Acetobacter may be mixed into the glucose solution to suppress the effects of acetic acid bacteria during the fermentation process. Acetobacter species are not particularly limited and include various known acetic acid bacteria such as those of the Acetobacter and Gluconacetobacter genera. However, it is preferable that the amount of acetic acid bacteria that may be mixed into the glucose solution be approximately 0.2% by mass or less as an inoculation amount of an acetic acid bacteria culture solution (OD660 = 2).

[0064] In the fermentation step, the method and conditions (e.g., fermentation method and fermentation conditions) for contacting the glucose solution with the microorganism are not particularly limited, and the atmosphere, temperature, pH, time, etc. can be appropriately selected and set depending on the microorganism to be used, etc. In Production Method A of the present invention, the method and conditions for contacting the glucose solution with the microorganism can be, for example, the following method and conditions in Production Method B of the present invention.

[0065] For example, the contacting method and conditions in the preferred production method B of the present invention (including the embodiment in which ethanol is produced in the production method A of the present invention) can be appropriately selected and determined with reference to known ethanol fermentation methods and conditions, specifically, the method and conditions described in Patent Document 1. An example is described below.

[0066] The glucose solution typically contains glucose and water derived from the glucose solution. The glucose content (concentration) in the glucose solution typically corresponds to the glucose content in the cellulose degradation product. However, when adjusting the concentration in the glucose solution by adding water appropriately, the glucose concentration in the glucose solution will be lower than the glucose concentration in the cellulose degradation product. The glucose content in the glucose solution in this case can be, for example, 0.1 to 10% by mass, with 1 to 5% by mass being preferred in terms of efficient ethanol production. The water content in the glucose solution can be, for example, 90 to 99.9% by mass, with 95 to 99% by mass being preferred in terms of efficient ethanol production.

[0067] The pH of the fermentation broth in the fermentation step is not particularly limited, but is preferably maintained in the range of 3 to 10, and more preferably in the range of 4 to 8. The temperature of the fermentation broth in the fermentation step is not particularly limited as long as it is within the optimal temperature range for the microorganism, and is, for example, preferably 20 to 40°C, and more preferably 30 to 40°C. The amount of microorganism to be inoculated into the glucose solution is not particularly limited, as long as it is an amount that allows the inoculated microorganism to grow. For example, the inoculation amount of a microbial culture solution (OD660 = 6) can be 0.01 to 10% by mass, and 0.1 to 1% by mass is preferred in terms of favorable microbial growth. The fermentation time cannot be uniquely determined depending on the glucose content, fermentation temperature, pH, the presence or absence of a nutrient source, etc., but can be, for example, 12 to 240 hours, and is preferably 24 to 120 hours. When the fermentation step is carried out in a continuous manner as described below, the fermentation time refers to the time during which the glucose solution is in contact with the microorganisms, and usually refers to the average residence time from when it is supplied into the reaction tank until it is transferred out of the reaction tank (average residence time).

[0068] The content of ethanol produced in the fermentation broth is not particularly limited and is determined appropriately depending on the ethanol-producing ability of the microorganism, the culture method, etc. For example, it can be 1 to 120 g / L, or can also be 3 to 50 g / L. Note that, because ethanol has bactericidal properties, the content in the fermentation broth cannot be so high as to affect the survival and activity of the microorganisms, and it is preferable, for example, to set the upper limit to 150 g / L or less.

[0069] The atmosphere for the fermentation step can be appropriately determined depending on the microorganisms used. An aerobic atmosphere can be used during contact, but an anaerobic atmosphere can also be used during fermentation.

[0070] The fermentation step may be carried out in one step or in multiple steps. When the fermentation step is carried out in multiple steps, the fermentation conditions in each fermentation step may be the same or different. The fermentation step may be carried out in a batch or continuous manner using a fermenter. Any shape of fermenter may be used, and for example, a stirring type, airlift type, bubble column type, loop type, open bond type, or photobio type fermenter may be used.

[0071] As described above, a fermentation mixture containing a microorganism and an organic compound such as ethanol can be obtained. Focusing on the production process, the production method of the present invention can also be said to be a method for producing an organic compound such as ethanol from cellulose, which includes a step of contacting with the above-mentioned method for producing a degradation product.

[0072] [Post-treatment step] In the production method of the present invention, the reaction mixture (fermentation mixture) obtained after the contacting step can be post-treated to obtain organic compounds such as ethanol. Post-treatment of the fermentation mixture includes a step of solid-liquid separation of the fermentation mixture and a step of isolating ethanol. The fermentation mixture may be subjected to the step of isolating organic compounds such as ethanol as is, but typically, solid-liquid separation is performed to separate a liquid containing organic compounds such as ethanol as a liquid phase from a solid fraction containing microorganisms as a solid phase. The method of solid-liquid separation is not particularly limited, and examples include filtration, centrifugation, and precipitation. In the step of isolating organic compounds such as ethanol, the liquid containing organic compounds such as ethanol after solid-liquid separation can typically be distilled to isolate the organic compounds. Conventional distillation methods and conditions can be used for the distillation method and conditions, and examples include atmospheric distillation and reduced-pressure distillation.

[0073] [Other Steps] In the production method of the present invention, steps other than the fermentation step and post-treatment step can also be performed. For example, a step of purifying an organic compound such as distilled ethanol can be included. The method for purifying the organic compound can be any known purification method without any particular limitation, and examples thereof include a concentration purification method using a zeolite membrane.

[0074] Production method A of the present invention can produce target organic compounds using microorganisms, preferably in high yields, from cellulose degradation products that satisfy the above-mentioned component area ratios. Furthermore, preferred production method B of the present invention can promote or accelerate microbial ethanol fermentation from cellulose degradation products that satisfy the above-mentioned component area ratios while suppressing the occurrence and progression of side reactions, thereby producing ethanol efficiently (in high yields) even over long fermentation times. Furthermore, cellulose degradation products prepared using a suitable solid acid catalyst satisfy the above-mentioned component area ratios, allowing ethanol fermentation to proceed more efficiently. Furthermore, preferred production method B of the present invention can suppress the growth of acetic acid bacteria and acetic acid fermentation by acetic acid bacteria, even in the presence of acetic acid bacteria during ethanol fermentation, thereby preventing the conversion of ethanol converted from cellulose hydrolysates to acetic acid. As a result, preferred production method B of the present invention is suitable as a method for producing bioethanol using nonedible biomass as a raw material. Furthermore, when using cellulose degradation products prepared using waste pulp as a cellulose source, cellulose degradation products that satisfy the above-mentioned component area ratios and have an even higher glucose content can be obtained, enabling the effective use of nonedible biomass resources. Therefore, the preferred production method B of the present invention is capable of producing ethanol from cellulose efficiently at low cost, and is also suitable for industrialization of bioethanol production methods.

[0075] [Composition for Microbial Culture] The composition for microbial culture of the present invention comprises a liquid containing glucose. The glucose-containing liquid is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst. When the glucose-containing liquid is subjected to LC analysis using a differential refractive index detector, the total area value of peaks derived from components with lower molecular weights than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose. The cellulose hydrolyzate constituting the composition for microbial culture of the present invention is the same as the cellulose hydrolyzate used in the production method of the present invention, for example, Production Method A of the present invention. The composition for microbial culture of the present invention may contain components typically used depending on the microorganism used, such as components typically used in culture media. Examples of such other components include nutrients necessary for microbial growth and activity, pH adjusters, buffers, chelating agents, antibiotics, expression inducers, and antifoaming agents. Each component is not particularly limited and is as described above. The composition for microbial culture may also contain the aforementioned acetic acid bacteria to suppress the effects of acetic acid bacteria.

[0076] The total content of cellulose degradation products in the composition for microbial culture is not particularly limited and can be determined appropriately taking into consideration factors such as the production efficiency of organic compounds, and can be, for example, 0.1% by mass or more. In particular, the content of glucose in the composition for microbial culture can be set to 0.1 to 10% by mass in terms of factors such as the production efficiency of organic compounds. The total content of other components in the composition for microbial culture is not particularly limited and can be determined appropriately taking into consideration factors such as the production efficiency of organic compounds, and the content of acetic acid bacteria in the composition for microbial culture is preferably about 0.2% by mass or less as the inoculation amount of acetic acid bacteria culture solution (OD660 = 2).

[0077] The composition for microbial cultivation of the present invention contains a cellulose decomposition product having the above-mentioned specific component area ratio and can produce various organic compounds, and is therefore suitable for use as a medium in the production method of the present invention.

[0078] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0079] [Preparation of Solid Acid Catalyst] Experimental Example 1: Preparation of Solid Acid Catalyst for Cellulose Decomposition (1) Preparation of Porous Silica (First Step) 625.5 g of a 16% by mass aqueous solution of hexadecyltrimethylammonium hydroxide was stirred, and a mixed solution of 9.25 g of tetraisopropyl titanate and 50.0 g of 2-propanol was added dropwise to the stirred solution at room temperature (25°C). After stirring for 30 minutes, 190.5 g of tetramethyl orthosilicate was added dropwise. After adding 5.0 g of 2-propanol, stirring was continued for 3 hours, resulting in the formation of a precipitate.

[0080] (Second Step) The resulting precipitate was filtered and washed with 5 L of ion-exchanged water, and the washed solid was dried under reduced pressure at 100° C. for 5 hours.

[0081] (Third Step) 20 g of the dried solid obtained in the second step was placed in a flask, and a mixed solution of 200 mL of methanol and 10 g of concentrated hydrochloric acid (content 36% by mass) was added (acid treatment). Heating was continued at reflux temperature for 1 hour with stirring, and after cooling, the liquid phase was removed by filtration. The same procedure was repeated once more using a mixed solution of 200 mL of methanol and 5 g of concentrated hydrochloric acid. Finally, after refluxing with 200 mL of methanol for 1 hour, the solid finally filtered out was dried under reduced pressure at 120 °C and 10 mmHg for 1.5 hours.

[0082] (Fourth Step) The solid obtained in the third step was heat-treated (calcined) at 600° C. for 3 hours in an air stream.

[0083] (Fifth Step) The fired solid obtained in the fourth step was pulverized eight times with a hammer mill to obtain a powdered porous silica (silica-titanium composite mesoporous material) having an average particle size of 10 μm. The average particle size was measured using a laser diffraction / scattering particle size distribution analyzer (LA-950 manufactured by Horiba, Ltd.) using ion-exchanged water as a dispersion medium, and calculated on a volume basis.

[0084] (2) Preparation of Porous Silica-Carbon Composite 3 g of porous silica and 30 g of propylene oxide (reagent, Fujifilm Wako Pure Chemical Industries, Ltd.) serving as a carbon source were added to an autoclave equipped with a stirrer. The autoclave was pressurized with nitrogen to 1 MPa-G and then depressurized to normal pressure, a process repeated three times to replace the atmosphere inside the autoclave with nitrogen. The autoclave was immersed in an oil bath, and the stirrer was set to a rotation speed of 500 rpm to stir the contents inside the autoclave. The oil bath temperature was adjusted so that the internal temperature of the autoclave reached 100°C. After the internal temperature of the autoclave reached 100°C, the mixture was stirred for 6 hours. The solid obtained by filtration was air-dried and then heated in an electric furnace under a nitrogen atmosphere at a rate of 6°C / min to 500°C, followed by heat treatment at 500°C for 2 hours to prepare 3.2 g of porous silica-carbon composite as a carbon-loaded silica-titanium composite mesoporous material.

[0085] (3) Preparation of solid acid catalyst 1 g of porous silica-carbon composite and 20 mL of 95% by mass sulfuric acid were added to an autoclave and treated at 150°C for 15 hours. After the treatment, the composite was washed with water at 50°C until the pH of the washings was the same as that of purified water. After washing with water, the solid separated by filtration was dried under reduced pressure at 120°C for 12 hours to obtain 1 g of a solid acid catalyst as a carbon-supported silica-titanium composite mesoporous material having sulfo groups as surface functional groups.

[0086] Experimental Example 2: Pore analysis of porous silica material The powdered porous silica material (silica-titanium composite mesoporous material) prepared as described above had a specific surface area of ​​1,160 m ２ / g, total pore volume 0.60 mL / g, average pore diameter 2.1 nm. The specific surface area and total pore volume were determined by measuring the nitrogen adsorption isotherm at liquid nitrogen temperature using a BELSORP MINI X (manufactured by Microtrac-Bell) after vacuum degassing the silica porous body at 120°C for 2 hours, and then using the BET method and the nitrogen adsorption amount near a relative pressure (P / P0 = 0.96). The average pore diameter was calculated from the total pore volume and specific surface area values ​​using the following (Equation 1): (Equation 1): average pore diameter (nm) = 4 × total pore volume (mL / g) / specific surface area (m ２ / g) × 1000

[0087] Furthermore, the Ti concentration in the powdered porous silica was analyzed by XRF and found to be 1.5 mass %.

[0088] Experimental Example 3: Measurement of carbon content in porous silica-carbon composite The carbon content of the porous silica-carbon composite was determined from the weight loss rate by thermogravimetric analysis. The thermogravimetric analysis was performed in an air atmosphere by increasing the furnace temperature to 1000°C at a rate of 5°C / min. The carbon content of the obtained porous silica-carbon composite was 12% by mass.

[0089] Experimental Example 4: Measurement of sulfo group content of solid acid catalyst The sulfo group content of the solid acid catalyst was measured by titration using an automatic potentiometric titrator (Kyoto Electronics). 50 mg of the solid acid catalyst was weighed into a screw cap bottle, and 30 mL of a 0.05 N aqueous NaCl solution was added using a volumetric pipette, followed by stirring for 15 hours with a stirrer. After stirring, the solution in the screw cap bottle was filtered, and 10 mL of the filtrate was sampled using a volumetric pipette and transferred to a 50 mL beaker. Two drops of phenolphthalein solution were added to the 50 mL beaker using a Pasteur pipette, and titration was performed by adding 0.1 mL of 0.005 N aqueous NaOH solution dropwise. The Na content was calculated from the equivalence point of the titration result. ＋ The amount of exchange was calculated to determine the sulfo group content. The sulfo group content of the solid acid catalyst thus obtained was 0.09 mmol / g.

[0090] Experimental Example 5. Methods for Obtaining and Preparing Raw Materials (Pulp Boards and Pulp Sludge) The pulp boards were obtained by dehydrating raw materials used in a papermaking machine for household paper in the papermaking process. The pulp sludge was obtained by adding aluminum sulfate, anionic coagulants, and cationic coagulants to a slurry solution of pulp discharged from a papermaking machine for household paper in the papermaking process, solidifying the solution, and then dehydrating the solidified solution.

[0091] <Experimental Example 6. Method of Pretreating Raw Materials> The plate pulp obtained in Experimental Example 5 above was dehydrated, then dried at 60°C for 12 hours, then vacuum dried at 120°C for 12 hours, and after vacuum drying, was finely chopped with scissors to obtain pretreated plate pulp. The pulp sludge obtained in Experimental Example 5 above was dehydrated, then dried at 60°C for 12 hours, then vacuum dried at 120°C for 12 hours, and after vacuum drying, was finely chopped with scissors to obtain pretreated pulp sludge.

[0092] Experimental Example 7. Preparation of Cellulose Decomposition Product (1) Cellulose Hydrolysis Reaction The cellulose used as the reaction substrate was treated as follows. 1 kg of 1 cm diameter zirconia balls and 5 g of microcrystalline cellulose (Avicel PH-101, a reagent manufactured by Sigma-Aldrich) were placed in a ceramic pot mill. The mixture was placed on a tabletop pot mill turntable and ball milled at 200 rpm for 120 hours. This procedure was performed in two batches, yielding 9 g of ball mill-treated cellulose. Next, the ball mill-treated cellulose, solid acid catalyst, and purified water were added to an autoclave equipped with a stirrer. The mixture was heated from room temperature to 150°C over approximately 30 minutes while stirring at 300 rpm, and then the cellulose hydrolysis reaction was carried out in two batches at 150°C for 24 hours (internal pressure 0.5 MPa). In the first batch of ball mill treatment, the amounts of cellulose, solid acid catalyst, and purified water added were 2 g, 2 g, and 25 g, respectively. Similarly, in the second batch of ball mill treatment, the amounts of cellulose, solid acid catalyst, and purified water added were 3 g, 3 g, and 38 g, respectively. After the reaction was completed, the autoclave was cooled to room temperature. The reaction solution was then filtered to separate the liquid and solid, and the two batches were mixed to obtain a cellulose decomposition product and a solid residue. In each experimental example, when simply referring to a "cellulose decomposition product," it refers to the cellulose decomposition product prepared in Experimental Example 7 (1).

[0093] (2) Hydrolysis Reaction of Plate Pulp The plate pulp used as the reaction substrate was treated as follows. 1 kg of 1 cm diameter zirconia balls and 5 g of the pretreated (finely chopped) plate pulp obtained in Experimental Example 6 above were placed in a ceramic pot mill. The pot mill was set on a rotating table and ball milled for 120 hours at 200 rpm. This procedure was repeated multiple times, and the plate pulp adhering to the ceramic bottle wall was collected as a sample, yielding 9 g of ball milled plate pulp. Next, the ball milled plate pulp, solid acid catalyst, and purified water were added to an autoclave equipped with a stirrer. The mixture was heated from room temperature to 150°C over approximately 30 minutes while stirring at 300 rpm. After that, three batches of plate pulp hydrolysis reactions were carried out at 150°C for 24 hours (internal pressure: 0.5 MPa). In the first batch of ball milling, the amounts of plate pulp, solid acid catalyst, and purified water added were 3 g, 3 g, and 37.5 g, respectively. After the reaction was completed, the autoclave was cooled to room temperature. Thereafter, the reaction solution was filtered to separate the liquid and solid. The solid obtained by filtration was vacuum dried at 120 ° C for 12 hours, and 3 g of newly ball-milled plate pulp and 37.5 g of purified water were added to carry out the hydrolysis reaction of the plate pulp, thereby carrying out the hydrolysis reaction of the plate pulp of the second batch. The same operation as in the second batch was repeated to carry out the hydrolysis reaction of the plate pulp of the third batch. The liquids for the three batches were mixed to obtain a plate pulp decomposition liquid. The plate pulp decomposition liquid was concentrated at 50 ° C to a volume ratio of 1 / 3, and a "plate pulp decomposition product" was obtained as a cellulose decomposition product.

[0094] (3) Hydrolysis Reaction of Pulp Sludge The pulp sludge used as the reaction substrate was treated as follows. 1 kg of 1 cm diameter zirconia balls and 5 g of the pretreated (finely chopped) pulp sludge obtained in Experimental Example 6 above were placed in a ceramic pot mill. The pot mill was set on a rotating table and ball milled for 120 hours at 200 rpm. This operation was performed in multiple batches, and the pulp plate adhering to the wall of the ceramic bottle was collected as a sample, yielding 21 g of ball milled pulp plate. Next, the ball milled pulp sludge, solid acid catalyst, and purified water were placed in an autoclave equipped with a stirrer. While stirring at 300 rpm, the temperature was raised from room temperature to 150°C over approximately 30 minutes, and then the pulp sludge was hydrolyzed at 150°C for 24 hours (internal pressure: 0.5 MPa) for 7 batches. In the first batch of ball mill treatment, the amounts of pulp sludge, solid acid catalyst, and purified water added were 3 g, 3 g, and 37.5 g, respectively. After the reaction was completed, the autoclave was cooled to room temperature. The reaction solution was then filtered to separate the liquid and solid. The solid obtained by filtration and separation was vacuum-dried at 120°C for 12 hours, and 3 g of newly ball-milled pulp sludge and 37.5 g of purified water were added to carry out the hydrolysis reaction of the pulp sludge, thereby carrying out the hydrolysis reaction of the pulp sludge in the second batch. The same operation as in the second batch was repeated to carry out the hydrolysis reaction of the pulp sludge in the third, fourth, fifth, sixth, and seventh batches. The liquids from the seven batches were mixed to obtain a pulp sludge decomposition solution. The pulp sludge decomposition solution was concentrated at 50°C to a volume ratio of 1 / 7, obtaining a "pulp sludge decomposition product" as a cellulose decomposition product.

[0095] (4) High-Performance Liquid Chromatography (HPLC) Analysis The cellulose hydrolysate, plate pulp hydrolysate, and pulp sludge hydrolysate obtained in (1), (2), and (3) above were subjected to HPLC analysis, and the HPLC analysis results of the cellulose hydrolysate, plate pulp hydrolysate, and pulp sludge hydrolysate were obtained. Among the chromatograms obtained by HPLC analysis, the chromatogram of the cellulose hydrolysate obtained in (1) above is shown in Figure 1. The HPLC analysis was performed using an LC-20A (manufactured by Shimadzu Corporation) with a Shodex SP0810 column (manufactured by Resonac, inner diameter 8 mm, length 300 mm), purified water as the mobile phase (0.5 mL / min), and a differential refractive index detector (RID-20A) as the detector, at a column temperature of 70 ° C., a sample injection volume of 20 μL, an analysis time of 70 minutes, and a sampling rate of 100 ms. The resulting chromatograms were used to identify the types of components in the cellulose hydrolyzate, plate pulp hydrolyzate, and pulp sludge hydrolyzate, and their contents were quantified. Specifically, each component detected in the chromatogram was identified by comparing it with the retention time of each compound reagent on, for example, the Shodex SP0810 column. Next, the concentrations (mass%) of glucose, cellobiose, fructose, levoglucosan, HMF (5-hydroxymethylfurfural), and furfural in the cellulose hydrolyzate were quantified by an absolute calibration curve method using the reagents for each component. The quantitative results for the cellulose hydrolyzate, plate pulp hydrolyzate, and pulp sludge hydrolyzate obtained in (1), (2), and (3) above are shown in Tables 1 to 3, respectively.

[0096]

[0097]

[0098]

[0099] The peak area values ​​of the peaks derived from glucose and components lower in molecular weight than glucose that elute after the retention time of glucose in each chromatogram obtained from HPLC analysis of cellulose hydrolyzate, plate pulp hydrolyzate, and pulp sludge hydrolyzate using a differential refractive index detector are shown in Tables 4 to 6 below. "Other 1" to "Other 10" shown in Tables 4 to 6 are not cellobiose or fructose, but components lower in molecular weight than glucose. Note that "Other n" in each table (where n is 1 or 2 in Table 4, an integer from 1 to 6 in Table 5, and an integer from 1 to 10 in Table 6) indicates peaks derived from unidentifiable substances in each chromatogram in the order of detection using n. The "Other n" peaks with the same n value do not refer to peaks derived from substances common to all chromatograms (Tables 4 to 6). The waveform processing parameters used for peak detection were a slope of 200 μV / min, a width of 5 sec, and a minimum area of ​​1000 counts as the minimum area / height, and area values ​​were calculated under these conditions.

[0100]

[0101]

[0102]

[0103] Area ratios (component area ratios) were calculated from the area values ​​shown in Tables 4 to 6. Specifically, in Table 4, the area value of glucose was 10,750,862, and the total area value of the peaks derived from "components with lower molecular weights than glucose" (Other 1 + Other 2 + Levoglucosan + HMF + Furfural) was 783,106. Therefore, in Table 4 (the cellulose hydrolyzate obtained in (1) above), the area ratio of the total area value of "components with lower molecular weights than glucose" to the area value of "glucose" was 0.072 times. Similarly, the area ratios were also calculated for the plate pulp hydrolyzate and pulp sludge hydrolyzate obtained in (2) and (3) above. That is, in Tables 5 and 6, the area values ​​of glucose were 10,159,454 and 10,901,449, respectively, and the total area values ​​of the peaks derived from "components with lower molecular weights than glucose" (Table 5: Other 1 + Other 2 + Other 3 + Other 4 + Other 5 + Other 6 + Levoglucosan + HMF; Table 6: Other 1 + Other 2 + Other 3 + Other 4 + Other 5 + Other 6 + Other 7 + Other 8 + Other 9 + Other 10 + Levoglucosan + HMF + Furfural) were 8,387,038 and 8,357,042, respectively. Thus, in Tables 5 and 6, the area ratios of the total area value of "components with lower molecular weights than glucose" to the area value of "glucose" were 0.826 times and 0.767 times, respectively.

[0104] Experimental Example 8-1. Cultivation of Microorganisms Using Cellulose Decomposition Product as a Carbon Source: Ethanol Fermentation The following materials were used in this Experimental Example. (Culture Medium) - Yeast medium (carbon source: cellulose decomposition product obtained in Experimental Example 7 (1)) - A medium prepared by mixing the components in ultrapure water to the following concentrations, adjusting the pH to 5.6, and then sterilizing with high-pressure steam: Cellulose decomposition product in an amount equivalent to 20 g / L of glucose, 20 g / L polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), and 10 g / L yeast extract (manufactured by Thermo Fisher Scientific) - Acetic acid bacteria medium (carbon source: cellulose decomposition product obtained in Experimental Example 7 (1)) - A medium prepared by mixing the components in ultrapure water to the following concentrations, adjusting the pH to 7.0, and then sterilizing with high-pressure steam. Cellulose hydrolysate in an amount equivalent to 20 g / L of glucose, 5 g / L of polypeptone, 3 g / L of yeast extract, 3 g / L of meat extract (manufactured by BD), 2 g / L of ammonium sulfate (manufactured by Nacalai Tesque), 1 g / L of monopotassium dihydrogen phosphate (manufactured by Nacalai Tesque), and 0.5 g / L of magnesium sulfate heptahydrate (manufactured by Nacalai Tesque).

[0105] (Microorganisms) - Ethanol-producing bacteria - Yeast (Saccharomyces cerevisiae S288C NBRC1136) - Ethanol-decomposing bacteria - Acetic acid bacteria (Acetobacter aceti NBRC14818)

[0106] (Microbial Culture Method: Ethanol Fermentation) Microorganisms corresponding to the culture medium were inoculated from glycerol stocks (microorganism frozen cultures prepared by adding glycerol to a final concentration of 30% by mass and then freezing) into 5.0 mL culture medium / test tubes (5.0 mL of each culture medium was placed in one test tube) and cultured overnight at 30°C (hereinafter referred to as "preculture"). 0.1 mL of each resulting preculture was added to 5.0 mL culture medium / test tubes (test tubes containing the same medium as the precultures), and cultured at 30°C and 200 rpm for 48 hours (hereinafter referred to as "main culture").

[0107] (Measurement of Turbidity of Main Culture Solution) During the main culture process, samples were taken from each main culture solution over time (0 hour, 24 hours, and 48 hours after the main culture), and the turbidity (OD660) was measured using a spectrophotometer (Shimadzu Corporation, UV1800). The changes in turbidity after 0 hour, 24 hours, and 48 hours after the main culture are shown in Figures 2 and 3. In Figures 2 and 3, "■" indicates the results of Experimental Example 8-1, in which a cellulose decomposition product was used as the glucose source.

[0108] (Measurement of glucose concentration in main culture) After 48 hours of culture, 1 mL of each main culture was centrifuged, and the supernatant was sterilized by filtration through a 0.22 μm filter. The resulting filtrate was subjected to HPLC analysis, and the glucose concentration (mass%) in the filtrate was quantified by the absolute calibration curve method using a glucose reagent. HPLC analysis was performed using an LC-20A (Shimadzu Corporation) column with Shodex SP0810 (Resonac Inc.), purified water (0.5 mL / min) as the mobile phase, and a differential refractive index detector (RID-20A) as the detector, at a column temperature of 70°C.

[0109] The residual glucose rate was calculated from the HPLC analysis results using the following formula (2). The obtained residual glucose rate was used to determine the "glucose assimilation rate" described below. (Formula 2): residual glucose rate (%) = (glucose concentration in the main culture after 48 hours of culture) / (glucose concentration in the main culture at 0 hours of culture) × 100

[0110] (Measurement of ethanol concentration in the main culture solution) Using a headspace gas chromatography device, the filtrate was heated at 80°C for 30 minutes, and the gas phase was measured with a gas chromatograph-mass spectrometer (GC-MS) under the following MS measurement conditions. The obtained GC-MS analysis results were used to determine the "ethanol productivity" described below. - MS measurement conditions - Measurement mode: Selected ion monitoring (SIM) mode Monitor ion: Quantitative ion m / z 31 (ethanol)

[0111] Experimental Example 8-2. Cultivation of Microorganisms Using Glucose as a Carbon Source: Ethanol Fermentation The turbidity of the main culture medium, the glucose concentration in the main culture medium, and the ethanol concentration in the main culture medium were measured in the same manner as in Experimental Example 8-1, except that the following materials were used for the yeast medium and the acetic acid bacteria medium in Experimental Example 8-1. The changes in turbidity 0 hours, 24 hours, and 48 hours after the start of main culture are shown in Figures 2 and 3. In Figures 2 and 3, "●" indicates the results of Experimental Example 8-2, which used commercially available glucose as the glucose source.

[0112] (Culture media) - Yeast medium (carbon source: glucose) - A medium in which glucose (reagent manufactured by Nacalai Tesque) was used at 20 g / L instead of the cellulose degradation product in the "Yeast medium (carbon source: cellulose degradation product obtained in Experimental Example 7 (1))" of Experimental Example 8-1. - Acetic acid bacteria medium (carbon source: glucose) - A medium in which glucose (reagent manufactured by Nacalai Tesque) was used at 20 g / L instead of the cellulose degradation product in the "Acetic acid bacteria medium (carbon source: cellulose degradation product obtained in Experimental Example 7 (1))" of Experimental Example 8-1.

[0113] Experimental Example 8-3. Evaluation of ethanol fermentation (Determination of microbial growth rate) In the measurement of the turbidity of the main culture in Experimental Example 8-2, the turbidity value of the experiment in which the culture was carried out for 48 hours using glucose as a carbon source was taken as 100% bacterial cell concentration, and the relative bacterial cell concentration (%) of each main culture (cultured for 48 hours) was calculated and determined according to the following criteria. The results are shown in Table 7 together with the relative bacterial cell concentration values. - Determination - A: Relative bacterial cell concentration ≧ 75% B: 75% > relative bacterial cell concentration ≧ 18% C: 18% > relative bacterial cell concentration

[0114]

[0115] The results in Table 7 and Figures 2 and 3 show that when glucose was used as a carbon source, the cell concentrations of both yeast and acetic acid bacteria increased. Specifically, when yeast was used (Figure 2), under the conditions of the above experimental example, the turbidity leveled off and yeast growth stopped after about 24 hours. When acetic acid bacteria were used (Figure 3), the turbidity leveled off and no growth of acetic acid bacteria was observed until 24 hours. However, after 24 hours, the turbidity increased, indicating the growth of acetic acid bacteria. In contrast, when cellulose degradation products were used as a carbon source, an increase in yeast cell concentration was observed, but no increase in acetic acid bacteria cell concentration was observed. Moreover, when cellulose degradation products were used, the relative cell concentration was significantly higher than when glucose was used. Specifically, when yeast was used (Figure 2), under the conditions of Experimental Example 8-1, the turbidity increased even after 24 hours, indicating that yeast growth continued. When acetic acid bacteria were used (Figure 3), the turbidity remained almost unchanged, indicating that acetic acid bacteria growth and acetic acid fermentation (ethanol decomposition) were suppressed.

[0116] (Determination of glucose utilization rate) The glucose utilization rate (%) calculated by the above (Equation 2) was determined as the glucose utilization rate and was determined according to the following criteria. The results are shown in Table 8 together with the glucose utilization rate values. -Determination- A: Glucose utilization rate ≦ 25% B: 25% < glucose utilization rate ≦ 75% C: 75% < glucose utilization rate

[0117]

[0118] The results in Table 8 show that when glucose was used as a carbon source, both yeast and acetic acid bacteria assimilated the glucose. In contrast, when a cellulose degradation product was used as a carbon source, yeast assimilated the glucose contained in the cellulose degradation product, but acetic acid bacteria did not assimilate the glucose contained in the cellulose degradation product.

[0119] (Determination of Ethanol Productivity) The ethanol concentration was calculated from the GC-MS analysis results and determined according to the following criteria: Determination A: Ethanol concentration ≥ 3.0 g / L B: 3.0 g / L > ethanol concentration ≥ 0.1 g / L C: 0.1 g / L > ethanol concentration

[0120]

[0121] The results in Table 9 show that the same amount of ethanol was detected when cellulose degradation products and glucose were used as carbon sources. Furthermore, the ethanol concentration (fermentation efficiency) was almost the same when cellulose degradation products were used as compared to when glucose was used alone. This demonstrates that the use of cellulose degradation products enables efficient ethanol production by yeast.

[0122] Experimental Example 9: Cultivation of yeast using cellulose decomposition product, board pulp decomposition product, and pulp sludge decomposition product as carbon sources: ethanol fermentation The following materials were used in this experimental example: (Culture medium) The components were mixed in ultrapure water to give the following concentrations, the pH was adjusted to 5.6, and the medium was sterilized by filtration using a 0.22 μm filter. Yeast medium (carbon source: none) 6.7 g / L Yeast Nitrogen Base (YNB), w / Ammonium Sulfate (manufactured by MP Biomedicals), 790 mg / L Complete Supplement Mixture (CSM), Powder (manufactured by MP Biomedicals) Yeast medium (carbon source: glucose) 20 g / L glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 6.7 g / L YNB, w / Ammonium Sulfate, 790 mg / L CSM, Powder Yeast medium (carbon source: cellulose decomposition product obtained in Experimental Example 7 (1)) Cellulose hydrolysate in an amount equivalent to 20 g / L in terms of glucose, 6.7 g / L YNB, with ammonium sulfate, 790 mg / L CSM, powder - yeast medium (carbon source: board pulp hydrolysate obtained in Experimental Example 7 (2)) - board pulp hydrolysate in an amount equivalent to 20 g / L in terms of glucose, 6.7 g / L YNB, with ammonium sulfate, 790 mg / L CSM, powder - yeast medium (carbon source: pulp sludge hydrolysate obtained in Experimental Example 7 (3)) - pulp sludge hydrolysate in an amount equivalent to 20 g / L in terms of glucose, 6.7 g / L YNB, with ammonium sulfate, 790 mg / L CSM, powder

[0123] (Microorganism) Yeast (Saccharomyces cerevisiae S288C NBRC1136)

[0124] (Yeast Culturing Method) 4.0 mL of yeast medium (carbon source: glucose) was placed in a test tube, and yeast was inoculated from a glycerol stock and cultured overnight at 30°C and 225 rpm (hereinafter referred to as "pre-preculture"). 0.5 mL of the resulting pre-preculture was placed in a 250 mL baffled Erlenmeyer flask containing 50 mL of yeast medium (carbon source: glucose), and cultured overnight at 30°C and 100 rpm (hereinafter referred to as "preculture"). The resulting preculture was collected, centrifuged to remove the supernatant, and then washed with yeast medium (carbon source: none), and the supernatant was removed to obtain yeast cells. The resulting cells were suspended in each yeast medium (carbon source: none, glucose, cellulose hydrolyzate, board pulp hydrolyzate, or pulp sludge hydrolyzate) to an inoculum concentration of OD600 = 0.7. 20 mL of each yeast culture solution was placed in a 125 mL baffled Erlenmeyer flask and cultured at 30°C and 100 rpm for 48 hours (hereinafter referred to as "main culture").

[0125] (Measurement of Turbidity of Main Culture) During the main culture, samples were taken from each main culture over time (0 hour, 2 hours 30 minutes, 5 hours, 7 hours 30 minutes, 10 hours, 24 hours, and 48 hours after the main culture), and the turbidity (OD600) was measured using a spectrophotometer (Multiskan Sky, manufactured by Thermo Fisher). The changes in turbidity after 0 hours, 2 hours 30 minutes, 5 hours, 7 hours 30 minutes, 10 hours, 24 hours, and 48 hours after the main culture are shown in Figure 4.

[0126] (Determination of yeast growth rate) In measuring the turbidity of the main culture, the turbidity value of the experimental example in which the culture was carried out for 48 hours using glucose as a carbon source was taken as 100% bacterial cell concentration, and the relative bacterial cell concentration (%) of each main culture (cultured for 48 hours) was calculated and determined according to the following criteria. The results are shown in Table 10 together with the relative bacterial cell concentration values. - Determination - A: Relative bacterial cell concentration ≧ 75% B: 75% > relative bacterial cell concentration ≧ 18% C: 18% > relative bacterial cell concentration

[0127]

[0128] The results in Table 10 and Figure 4 show that yeast did not grow at all without a carbon source, but an increase in yeast cell concentration was observed when glucose, cellulose hydrolysate, board pulp hydrolysate, and pulp sludge hydrolysate were used as carbon sources. When glucose was used as the carbon source, the turbidity leveled off and yeast growth stopped after about 10 hours. In contrast, when cellulose hydrolysate or board pulp hydrolysate was used as the carbon source, yeast growth continued even after 24 hours, and the relative cell concentration after 48 hours was higher than that of glucose.

[0129] (Measurement of ethanol concentration in main culture solution) During the main culture process, samples were taken from each main culture solution at 0 hours and 24 hours after the start of the main culture, and the ethanol concentration was measured using E-kit Liquid Ethanol (manufactured by J.K. International). Note that the results of an experiment without a carbon source are omitted.

[0130] (Assessment of ethanol productivity) Based on the ethanol concentration in the main culture medium, the productivity was judged according to the following criteria: - Judgment - S: > ethanol concentration ≥ 9.0 g / L A: 9.0 g / L > ethanol concentration ≥ 3.0 g / L B: 3.0 g / L > ethanol concentration ≥ 0.2 g / L C: 0.2 g / L > ethanol concentration

[0131]

[0132] The results in Table 11 show that ethanol was detected in all cases where glucose, cellulose hydrolysate, pulp board hydrolysate, and pulp sludge hydrolysate were used as the carbon source. When cellulose hydrolysate was used as the carbon source, the ethanol concentration (fermentation efficiency) was almost the same as when glucose was used as the carbon source. When pulp board hydrolysate was used as the carbon source, the ethanol concentration (fermentation efficiency) was significantly higher than when glucose was used as the carbon source.

[0133] Experimental Example 10: Identification of a Growth Inhibitor of Acetic Acid Bacteria The following materials were used in this experiment. (Culture Medium) The components were mixed in ultrapure water to the following concentrations, adjusted to pH 7.0, and then autoclaved. Acetic acid bacteria culture medium (carbon source: glucose) - 20 g / L glucose (Fujifilm Wako Pure Chemical Industries, Ltd.), 5 g / L polypeptone (Shiotani MS Co., Ltd.), 3 g / L yeast extract (Biokar diagnostics), 3 g / L meat extract (Gibco), 2 g / L ammonium sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 g / L monopotassium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.5 g / L magnesium sulfate heptahydrate (Fujifilm Wako Pure Chemical Industries, Ltd.) (Candidate for Growth Inhibitor) In Table 1, it was assumed that the substance with a growth inhibitory effect on acetic acid bacteria would be levoglucosan, HMF, or furfural. These substances were then selected as growth inhibitor candidates and added to the acetic acid bacteria medium (carbon source: glucose) at the following concentrations: Levoglucosan (Fujifilm Wako Pure Chemical Industries, Ltd.): 0 g / L, 0.049 g / L, 0.098 g / L, 0.195 g / L, 0.390 g / L 5-hydroxymethylfurfural (HMF) (Nacalai Tesque, Inc.): 0 g / L, 0.066 g / L, 0.133 g / L, 0.265 g / L, 0.530 g / L Furfural (Fujifilm Wako Pure Chemical Industries, Ltd.): 0 g / L, 0.035 g / L, 0.070 g / L, 0.140 g / L, 0.280 g / L, 0.560 g / L

[0134] (Microorganism) Acetobacter aceti (NBRC14818)

[0135] (Acetate Bacterial Cultivation Method) 20 mL of acetic acid bacteria medium (carbon source: glucose) was placed in a 125 mL baffled Erlenmeyer flask, and the acetic acid bacteria were inoculated from a glycerol stock and cultured at 30°C and 225 rpm for 3 days (hereinafter referred to as "preculture"). The resulting preculture was collected, and the supernatant was removed by centrifugation to obtain acetic acid bacteria cells. The resulting cells were suspended in acetic acid bacteria medium (carbon source: glucose) supplemented with each growth inhibitor candidate so that the inoculum concentration reached an OD600 of 0.084. 4 mL of each acetic acid bacteria culture was placed in a test tube, and cultured at 30°C and 225 rpm for 48 hours (hereinafter referred to as "main culture").

[0136] (Measurement of Turbidity of Main Culture) During the main culture, samples were taken from each main culture over time (0, 8, 24, and 48 hours after the main culture), and the turbidity (OD600) was measured using a spectrophotometer (Multiskan Sky, manufactured by Thermo Fisher). The changes in turbidity after 0, 8, 24, and 48 hours after the main culture are shown in Figures 5 to 7.

[0137] (Determination of growth of acetic acid bacteria) In measuring the turbidity of the main culture, the turbidity value of the experimental example in which the culture was carried out for 48 hours without adding a candidate growth inhibitor was taken as 100% bacterial cell concentration, and the relative bacterial cell concentration (%) in each main culture (cultured for 48 hours) was calculated and determined according to the following criteria. The results are shown in Tables 12 to 14 together with the relative bacterial cell concentration values. - Determination - A: Relative bacterial cell concentration ≧ 75% B: 75% > relative bacterial cell concentration ≧ 20% C: 20% > relative bacterial cell concentration

[0138]

[0139]

[0140]

[0141] The results in Tables 12 to 14 and Figures 5 to 7 indicate that levoglucosan and HMF had no effect on the growth of acetic acid bacteria, but furfural clearly inhibited their growth. Specifically, when 0.035 g / L of furfural was added to the acetic acid bacteria medium, the relative bacterial cell concentration was 53% of that in the experiment without furfural (0 g / L). When 0.070 g / L to 0.560 g / L of furfural was added to the acetic acid bacteria medium, the relative bacterial cell concentration was 6 to 7% of that in the experiment without furfural (0 g / L). Under the conditions of the above experiments, furfural partially inhibited the growth of acetic acid bacteria at 0.035 g / L and completely inhibited the growth of acetic acid bacteria at 0.070 g / L or higher.

[0142] These results demonstrate that cellulose hydrolyzates, pulp board hydrolyzates, and pulp sludge hydrolyzates satisfying the above-mentioned component area ratios can be used as carbon sources for yeast-mediated ethanol production. In particular, cellulose hydrolyzates and pulp board hydrolyzates exhibited fermentation efficiencies equivalent to or greater than those obtained when glucose was used alone. Furthermore, it was also demonstrated that cellulose hydrolyzates obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, containing 0.035 g / L or more, preferably 0.070 g / L or more, of furfural, can highly inhibit the growth of acetic acid bacteria. Furthermore, it was surprisingly demonstrated that the cellulose hydrolyzate obtained in Experimental Example 7 (1) exhibited fermentation efficiencies similar to those obtained when glucose was used alone, while also suppressing the growth of acetic acid bacteria (inhibition of acetic acid fermentation). Therefore, by using cellulose hydrolyzates satisfying the above-mentioned component area ratios, even when acetic acid bacteria are present, the growth of acetic acid bacteria and ethanol decomposition by acetic acid fermentation can be prevented. Furthermore, ethanol can be efficiently produced by yeast even when the fermentation time is extended to 48 hours. Thus, the preferred production method B of the present invention can use microorganisms to produce ethanol from cellulose hydrolyzate that satisfies the above-mentioned component area ratios with sufficient efficiency for industrial production and at low cost. Furthermore, the preferred production method B of the present invention can produce ethanol without reducing the ethanol fermentation efficiency even when acetic acid bacteria are present. Therefore, it can be seen that the preferred production method B of the present invention is suitable as a method for producing bioethanol using non-edible biomass as a raw material.

[0143] While the present invention has been described in connection with embodiments thereof, we do not intend to limit our invention to any of the details of the description unless otherwise specified, and believe that the claims should be construed broadly without departing from the spirit and scope of the invention as set forth in the appended claims.

[0144] This application claims priority based on Japanese Patent Application No. 2024-000267 filed in Japan on January 4, 2024, and Japanese Patent Application No. 2024-209793 filed in Japan on December 2, 2024, the contents of which are incorporated herein by reference.

Claims

1. A method for producing an organic compound comprising contacting a liquid containing glucose with a microorganism, wherein the liquid containing glucose is a hydrolyzate of cellulose obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and when the liquid containing glucose is subjected to LC analysis using a refractive index detector, the total area value of peaks derived from components having a lower molecular weight than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose.

2. The production method according to claim 1, wherein the organic compound is ethanol, and in the LC analysis, the total area value of peaks derived from components having a lower molecular weight than glucose is 0.01 to 0.2 times the area value of the peak derived from glucose.

3. The production method according to claim 1, wherein the cellulose contains any one of cellulose derived from board pulp or cellulose derived from pulp sludge, or a mixture thereof.

4. The production method according to claim 1, wherein the solid acid catalyst contains a carbon element and silica and has a sulfo group as a surface functional group.

5. The production method according to claim 2, wherein the solid acid catalyst contains a carbon element and silica and has a sulfo group as a surface functional group.

6. The method according to claim 4, wherein the silica has mesopores.

7. The method according to claim 5, wherein the silica has mesopores.

8. The production method according to claim 4, wherein the carbon element is supported on the surface of the silica.

9. The production method according to claim 5, wherein the carbon element is supported on the surface of the silica.

10. The production method according to claim 6, wherein the carbon element is supported on the surface of the silica.

11. The production method according to claim 7, wherein the carbon element is supported on the surface of the silica.

12. The production method according to any one of claims 4 to 11, wherein the solid acid catalyst contains a titanium element.

13. The production method according to claim 12, wherein the titanium element is contained in the silica.

14. A composition for microorganism culture comprising a liquid containing glucose, wherein the liquid containing glucose is a hydrolyzate of cellulose obtained by bringing a liquid containing cellulose and water into contact with a solid acid catalyst, and when the liquid containing glucose is subjected to LC analysis using a refractive index detector, the total area value of peaks derived from components having a lower molecular weight than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose.

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