WO2025146776A1 - Method for producing organic compound, and composition for culturing microorganism - Google Patents

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

The present invention relates to a method for producing an organic compound and a composition for culturing microorganisms.

2. Description of the Related Art In recent years, from the viewpoint of protecting the global environment and making effective use of waste materials, the synthesis and production of various organic compounds using biomass, which is a renewable resource, as a raw material has been considered.
For example, bioethanol is being widely considered as a raw material for low carbon footprint materials to achieve carbon neutrality. In particular, the production of bioethanol from edible biomass, its dehydration reaction to olefination, and its polymerization are being considered. The conversion method from edible biomass to bioethanol is an established technology, and it is considered to be promising as a raw material production method for plastic products. However, there is a problem that edible biomass competes with food use, and in recent years, there has been a shift from edible biomass to non-edible biomass as a raw material for bioethanol production.
Cellulose, which is a non-edible biomass, can be decomposed into glucose by hydrolysis using an enzyme method or a sulfuric acid method, and then ethanol can be produced from the glucose by fermentation using microorganisms. However, the decomposition reaction into glucose is generally difficult because cellulose has a chemically stable structure. For example, even if cellulose can be converted into glucose, the purity of the glucose obtained is not sufficient, and even if it is refined, ethanol cannot be obtained efficiently. Therefore, in a method for producing ethanol using cellulose, which is a non-edible biomass, as a starting material, a method for efficiently proceeding with alcoholic fermentation by microorganisms or a method for obtaining a decomposition product of cellulose is required.

Incidentally, 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 considered. For example, Patent Document 1 describes a method for producing ethanol by fermenting monosaccharides obtained by hydrolyzing cellulose through three processes: a pressurized hot water reaction, a saccharification enzyme reaction, and a solid acid catalyst reaction. Specifically, Patent Document 1 describes "a method for producing ethanol having a fermentation process for fermenting monosaccharides obtained by a monosaccharide production process having a pressurized hot water reaction process in which pressurized hot water is applied to biomass to selectively decompose hemicellulose contained in the biomass, a beating process in which a solid residue after the pressurized hot 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 solid acid catalyst is applied to the product of the primary saccharification process." Patent Document 2 also describes a bioethanol production method for producing ethanol from lignocellulose, which is 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.

JP 2013-141415 A JP 2008-297229 A

In recent years, the production of organic compounds using biomass as a raw material has been attracting attention. In particular, the production of bioethanol has been industrialized and is used as a carbon-neutral fuel. In ethanol fermentation, in addition to the main reaction that converts the glucose present in the cellulose hydrolysate into ethanol, various side reactions are known to occur. 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) passes.

Acetic acid bacteria are bacteria that convert ethanol into acetic acid, and in the process of producing ethanol, they should be avoided from being mixed into the reaction system 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 suspended in the air, and are also present in the environment or reaction site where ethanol is produced by fermenting sugars and plant carbohydrates with yeast.
As described above, in order to solve the above problems caused by acetic acid bacteria, it is important to prevent the presence of acetic acid bacteria in the environment and reaction site during ethanol fermentation. However, it is not easy to remove acetic acid bacteria, which are normally present in the environment and reaction site. In particular, in ethanol production methods with an eye toward industrialization, the inclusion of acetic acid bacteria is unavoidable.
Therefore, in order to realize an efficient and/or industrial method for producing ethanol, it is necessary to suppress the proliferation of acetic acid bacteria and acetic acid fermentation even if acetic acid bacteria are contaminated.

As such, bioethanol is being widely considered and researched, but few organic compounds other than ethanol have been industrialized due to the high costs of collecting and transporting biomass and the need for advanced technology to convert it into the desired organic compound.

An object of the present invention is to provide a method for synthesizing an organic compound, which is capable of synthesizing various organic compounds while using sugars derived from cellulose.
An objective of one preferred embodiment of the present invention is to provide a method for producing ethanol that uses sugar derived from cellulose and that can efficiently perform ethanol fermentation while suppressing the growth of acetic acid bacteria and acetic acid fermentation even if acetic acid bacteria are contaminated.

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

The above production method <2> is synonymous with the production method [B1] below. Therefore, the production method [A1] of the organic compound of the present invention and the production method [B1] of ethanol of a preferred embodiment of the present invention can be distinguished and described as follows.
[A1] A method for producing an organic compound, comprising contacting a liquid containing glucose with a microorganism,
The method for producing an organic compound, wherein the glucose-containing liquid is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and 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 lower molecular weights than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose.
[A2] The manufacturing method according to [A1], wherein the cellulose comprises one of cellulose derived from board pulp and cellulose derived from pulp sludge, or a mixture thereof.
[A3] The production method according to [A1] or [A2], wherein the solid acid catalyst contains a 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 according to [A3] or [A4], wherein the carbon element is supported on the surface of the silica.
[A6] The method according to any one of [A3] to [A5], wherein the solid acid catalyst contains a titanium element.
[A7] The manufacturing method according to [A6], wherein the titanium element is contained in the silica.

[B1] A method for producing ethanol, comprising contacting a liquid containing glucose with a microorganism,
The method for producing ethanol, wherein the glucose-containing liquid is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and 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 lower molecular weights than glucose is 0.01 to 0.2 times the area value of the peak derived from glucose.
[B2] The production method according to [B1], wherein the solid acid catalyst contains carbon element and silica and has a sulfo group as a surface functional group.
[B3] The method according to [B2], wherein the silica has mesopores.
[B4] The method according to [B2] or [B3], wherein the carbon element is supported on the surface of the silica.
[B5] The method according to any one of [B2] to [B4], wherein the solid acid catalyst contains a titanium element.
[B6] The manufacturing method according to [B5], wherein the titanium element is contained in the silica.

The method for synthesizing an organic compound of the present invention can synthesize various organic compounds including ethanol, even though it uses sugar derived from cellulose (hydrolysate of cellulose). That is, it is possible to produce organic compounds from cellulose. In particular, the method for producing ethanol in a preferred embodiment of the present invention can efficiently ferment ethanol even though it uses sugar derived from cellulose, and can efficiently ferment ethanol while suppressing the growth of acetic acid bacteria and acetic acid fermentation even if acetic acid bacteria are contaminated. As a result, it is possible to efficiently produce ethanol from cellulose.
The above and other features and advantages of the present invention will become more apparent from the following description.

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

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, and 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 before and after "to" as the lower and upper limits.

[[Methods of producing organic compounds]]
Production method A of the present invention (hereinafter sometimes referred to as "production method A of the present invention") is a production method of an organic compound which comprises contacting a liquid containing glucose with a microorganism.
Furthermore, a preferred embodiment of the ethanol production method of the present invention (hereinafter, sometimes referred to as "preferable production method B of the present invention") is a method for producing ethanol which comprises contacting a liquid containing glucose with a microorganism.
In this specification, the manufacturing method A of the present invention and the preferred manufacturing method B of the present invention may be collectively referred to as the "manufacturing method of the present invention."

[Organic compounds 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 ability, and the like.
The organic compound produced by Production Method A of the present invention is not particularly limited, but 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 the preferred production method B of the present invention is ethanol.

The ingredients used in the manufacturing method of the present invention are described below.

[Liquid containing glucose]
In the production method of the present invention, a cellulose hydrolysate (sometimes referred to as a "cellulose hydrolysate" or "saccharified liquid") obtained by a method for producing a cellulose hydrolysate comprising contacting a liquid containing cellulose and water with a solid acid catalyst is used as the glucose-containing liquid.
As the glucose-containing liquid used in the production method A of the present invention, 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 with a differential refractive index detector is used. By contacting a cellulose hydrolysate having the above-mentioned origin and composition with a microorganism as a raw material mixture for the production method A of the present invention, various organic compounds can be produced, preferably in high amounts (high yields), while effectively utilizing cellulose, which is a non-edible biomass that can avoid competition with food applications. In addition, when producing the above-mentioned alcohol compounds as organic compounds, for example, by using a specific cellulose hydrolysate shown in Experimental Example 7 in the production method A of the present invention, the growth of acetic acid bacteria and acetic acid fermentation can be suppressed, 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.

As the glucose-containing liquid used in the preferred production method B of the present invention, among the above-mentioned cellulose hydrolysates, one in which the total area value of the peaks derived from components with a molecular weight lower than that of glucose is 0.01 to 0.2 times the area value of the peaks derived from glucose is used. In the preferred production method B of the present invention, by using such cellulose hydrolysates as a raw material mixture for ethanol fermentation, in addition to being able to produce ethanol, ethanol fermentation by microorganisms can be advanced or promoted while suppressing the occurrence and progress of side reactions, and ethanol can be produced efficiently (in high yields) even if the fermentation time is long. Moreover, even if acetic acid bacteria coexist during ethanol fermentation, the proliferation of acetic acid bacteria and acetic acid fermentation by acetic acid bacteria can be suppressed, and the ethanol converted from the cellulose hydrolysate can be suppressed from being converted into acetic acid. This is preferable, for example, when using the specific cellulose hydrolysate shown in Experimental Example 7. Therefore, the preferred production method B of the present invention can produce bioethanol, which is important for achieving carbon neutrality, in high yields (high yields) while effectively utilizing cellulose, which is a non-edible biomass that can avoid competition with food uses. In addition, because the growth of acetic acid bacteria and acetic acid fermentation can be inhibited, the excellent ethanol production efficiency is not compromised even under conditions or environments where exposure to or contact with acetic acid bacteria may occur during production, and industrialization is also within reach.

As described above, the production method of the present invention uses the above-mentioned glucose-containing liquid. This liquid is the above-mentioned cellulose decomposition product obtained by the decomposition product production method described below, and has a composition (component area ratio) described below.
The cellulose decomposition product may have the following composition and may contain other components in addition to the components contained in the cellulose decomposition 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.

The cellulose decomposition product used in the production method A of the present invention has a composition in which the total area value of the peaks derived from components having a molecular weight lower than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose when subjected to LC analysis with a differential refractive index detector. When the area ratio of the total area value to the area value of the peak derived from glucose (hereinafter, sometimes referred to as the "component area ratio") is 0.01 to 2.0 times, the composition and characteristics of the cellulose decomposition product are improved, and various organic compounds can be synthesized by the production method A of the present invention. When the component area ratio exceeds 2.0 times, the amount of glucose in the cellulose decomposition product decreases, making it difficult to produce it at a high concentration, which may reduce workability and productivity. In the present invention, the upper limit of the component area ratio is preferably 1.0 times or less, and more preferably 0.2 times or less, in terms of the amount of organic compound obtained and workability and productivity. On the other hand, the lower limit of the component area ratio is not particularly limited, and is, for example, preferably 0.01 times or more, more preferably 0.05 times or more, and even more preferably 0.1 times 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.

On the other hand, the production method B of the present invention uses a cellulose decomposition product having a composition in which the component area ratio is 0.01 to 0.2 times when subjected to LC analysis with a differential refractive index detector. In the preferred production method B of the present invention, when a cellulose decomposition product having a component area ratio of 0.01 to 0.2 times is used, the composition and characteristics are improved, the occurrence or promotion of side reactions is suppressed, and ethanol fermentation of glucose can be preferentially promoted as a main reaction. Moreover, the growth of acetic bacteria and acetic fermentation, which could not be suppressed in the past when acetic bacteria were contaminated, can be highly suppressed, and the yield and selectivity (content) of ethanol by ethanol fermentation can be not only not reduced but also further increased. In terms of further increasing the efficiency of ethanol fermentation and further suppressing the growth of acetic bacteria and acetic fermentation, and producing ethanol with high yield and high selectivity, the component area ratio is preferably 0.01 to 0.2 times, more preferably 0.05 to 0.2 times, and even more preferably 0.1 to 0.2 times.
In the present invention, the component area ratio is a value measured by the method described in the examples below.

Generally, when cellulose is hydrolyzed, the resulting decomposition product (composition) contains glucose as the main component and other components. Examples of the other components include partial hydrolysis products of cellulose, glucose and other reactants, such as tetramer or higher oligosaccharides, sugars such as cellotriose, cellobiose, mannose, fructose, and levoglucosan, and further 5-hydroxymethylfurfural (HMF), furfural, etc. In the present invention, the main component refers to the component that is contained in the largest amount by mass among the components contained.
The cellulose decomposition product used in the present invention may contain the above-mentioned partial hydrolysis product of cellulose, reactants such as glucose, etc., but satisfies the above-mentioned component area ratio for components with lower molecular weights than glucose identified by LC analysis (sometimes referred to as "low molecular weight components"). 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, etc., but usually include components with lower molecular weights than glucose among the partial hydrolysis product of cellulose, reactants such as glucose, etc. Examples include levoglucosan, HMF, furfural, and even unidentifiable components. The number of types of low molecular weight components that the cellulose decomposition product can contain is not particularly limited, and may be single or multiple.

The content (also called concentration, the unit of content is mass%) of each low molecular weight component in the cellulose hydrolyzate can be appropriately determined taking into consideration the area ratio of the components. For example, the content of HMF in the cellulose hydrolyzate 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%.

Among the low molecular weight components mentioned above, the cellulose decomposition product preferably contains furfural from the viewpoint of inhibiting the proliferation of acetic acid bacteria. The concentration of furfural is not particularly limited as long as it is a concentration capable of inhibiting the proliferation of acetic acid bacteria, and is, for example, preferably 0.035 g/L (0.0035 mass%) or more, and more preferably 0.07 g/L (0.007 mass%) or more. Furthermore, from the viewpoint of having little effect on the proliferation of yeast and ethanol fermentation, it is preferably 5.0 g/L (0.5 mass%) or less, more preferably 2.0 g/L (0.2 mass%) or less, and even more preferably 1.0 g/L (0.1 mass%) or less.

The glucose content in the cellulose decomposition product cannot be determined unequivocally 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 at least a content that provides a component area ratio of 0.01 to 2.0 times the above, and is a content that is in the above preferred range, a more preferred range, or an even more preferred range. In terms of the mass ratio of glucose in the cellulose decomposition product to all components contained in the cellulose decomposition composition, the glucose content is preferably, for example, 50 to 99 mass%, and more preferably 83 to 99 mass%, from the viewpoint of efficient production of ethanol.

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

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

- Cellulose -
The cellulose used in the decomposition production method may be any carbohydrate (polysaccharide) represented by the molecular formula (C ₆ H ₁₀ O ₅ ) _n , and includes hemicellulose and lignocellulose in addition to cellulose. The cellulose used in the decomposition production method 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 a lignin removal step described below.

The cellulose may be a synthetic product or a commercially available product, may be derived from non-edible biomass, or may be a waste or recovered product 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 powder of plants with an alkali. Considering the possibility of replacing bioethanol derived from edible biomass, it is preferable to use cellulose derived from non-edible biomass for the cellulose used in the decomposition production method. Plants are representative of non-edible biomass, but it is preferable to use waste or recovered products thereof in terms of effective use of resources and cost reduction. Examples of non-edible biomass include wood flour and wood chips from trees, thinned wood, and wood, various pulps (unused products), and further, waste pulp such as board pulp and pulp sludge (paper sludge) discharged in the papermaking or pulp manufacturing process, and pulp recovered from waste diapers.
In the present invention, plate pulp refers to the dehydrated (squeezed) product of pulp discharged during the papermaking or pulp manufacturing process, and the shape of the product does not necessarily have to be plate-like. Pulp sludge is a dehydrated (squeezed) solid product obtained by adding additives such as aluminum sulfate, anionic coagulants, or cationic coagulants to a slurry solution of pulp discharged during the papermaking or pulp manufacturing process, and generally tends to contain more impurities than plate pulp.

In the production method of the present invention, among the above-mentioned celluloses, waste pulp such as board pulp, pulp sludge, and pulp recovered from waste diapers is particularly preferred because it can be recovered in large quantities and resources can be effectively utilized (costs can also be reduced), and it can also avoid poisoning of the solid acid catalyst by lignin without having to carry out the lignin removal step described below.
In the production method of the present invention, the cellulose used is preferably waste pulp, and more preferably plate pulp or pulp sludge, from the viewpoint of efficiently producing organic compounds, particularly ethanol, while effectively utilizing resources. In addition to the above waste pulp, it is also a preferred embodiment to use synthetic or commercially available products from the viewpoint of efficiently producing organic compounds, particularly ethanol.

Cellulose is usually crystalline, with two or more cellulose molecules bound together by hydrogen bonds. The decomposition product manufacturing method can use cellulose that exhibits crystallinity (sometimes called "crystalline cellulose"), or cellulose in which the crystallinity of crystalline cellulose has been reduced by a conventional method (sometimes called "low crystalline cellulose" or "microcrystalline cellulose"). Low crystalline cellulose may be crystalline cellulose in which the crystallinity has been partially reduced, or crystalline cellulose in which the crystallinity has been (almost) completely eliminated. There are no particular limitations on the treatment method for reducing crystallinity, and a treatment that can at least partially generate single-stranded cellulose molecules by breaking the hydrogen bonds is preferred. Cellulose that contains at least partially single-stranded cellulose molecules has a significantly higher hydrolysis reaction efficiency. Specific examples of treatment methods for reducing crystallinity include the various methods described in Patent Document 1, and among these, physical methods such as jet mills, hammer mills, ball mills, and bead mills are preferred.

The shape of the cellulose used in the decomposition product production method is not particularly limited, but in terms of hydrolysis reaction efficiency and glucose yield, it is preferable that it be 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 account the hydrolysis reaction efficiency and glucose yield.

- Water -
The water used in the decomposition product production method is not particularly limited, but industrial water, well water, city water, ion-exchanged water, purified water, (ultra)pure water, etc. can be used, and ion-exchanged water, purified water, and (ultra)pure water are preferred.

(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. For example, inorganic solid acids such as zeolite, alumina, silica, silica alumina, zirconium sulfate, zirconium phosphate, heteropolyacid, niobic acid, inorganic solid acids to which acidic groups such as sulfo groups have been introduced by acidification treatment, resins, or carbonaceous materials can be mentioned. In the decomposition product production method, as the solid acid catalyst, it is preferable to use a solid acid catalyst containing carbon element and silica and having a sulfo group as a surface functional group, because it can hydrolyze cellulose with high efficiency and can obtain a cellulose decomposition product that satisfies the above component area ratio, and it is more preferable to use a solid acid catalyst containing carbon element, titanium element, and silica and having a sulfo group as a surface functional group. Hereinafter, the above-mentioned preferable solid acid catalyst and more preferable solid acid catalyst are collectively referred to as "suitable solid acid catalyst".
A suitable solid acid catalyst has a carbon element and a sulfo group ( _-SO3H ) on a substrate (also called a "silica substrate") mainly composed of silica (silicon dioxide), or has a carbon element, a titanium element and a sulfo group.
The shape of the suitable solid acid catalyst is not particularly limited, but is preferably particulate, powdery, or small piece shaped in terms of hydrolysis reaction efficiency and glucose yield. The size (average particle diameter) of the suitable solid acid catalyst is not particularly limited, but is preferably, for example, 0.1 to 10,000 μm. 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 that it can increase the hydrolysis reaction efficiency and glucose yield.
In the present invention, the mesopores refer to pores having an average pore diameter of 2 to 50 nm.

When the substrate is a porous substrate, its characteristics or physical properties, such as average pore diameter, specific surface area, total pore volume, etc., are not particularly limited and can be appropriately determined. For example, the average pore diameter is preferably 2 to 10 nm, more preferably 2 to 5 nm, in terms of hydrolysis reaction efficiency and glucose yield. The specific surface area is preferably 10 to 3000 ^{m 2} /g, more preferably 100 to 2000 ^{m 2} /g, in terms of hydrolysis reaction efficiency and glucose yield. The total pore volume is preferably 0.1 to 2 mL/g, more preferably 0.2 to 1 mL/g, in terms of hydrolysis reaction efficiency and glucose yield. The average pore diameter, specific surface area, and total pore volume of the porous substrate are values measured by the method described in the Examples below.

A suitable solid acid catalyst, usually a substrate, contains a carbon element. In the present invention, the substrate contains a carbon element, which means that the substrate has (exists) a carbon element on the surface and/or inside thereof, and examples thereof include a substrate having a carbon element supported or adsorbed on the substrate surface (a substrate supported with a carbon element), a substrate having a carbon element inside (a substrate formed of a composite of silica and a carbon element), and a substrate having a carbon element on the substrate surface and inside thereof. In the solid acid catalyst, the form (structure) containing a carbon element is preferably a substrate having a carbon element supported or adsorbed on the substrate surface (a substrate supported with a carbon element), in that the surface functional group described later can be bonded or fixed to the surface to increase the hydrolysis reaction efficiency and glucose yield.
In the present invention, the surface of a substrate generally refers to the outer surface, but when the substrate is porous, it includes the inner surfaces of the pores in addition to the outer surface, whereas the inside of the substrate refers to the portion not exposed to the surface.
The carbon element content in the suitable solid acid catalyst is not particularly limited, but is preferably 1 to 70 mass % and more preferably 5 to 50 mass % of the mass of the suitable solid acid catalyst (excluding the mass of sulfo groups) in terms of being able to increase the hydrolysis reaction efficiency and glucose yield. The carbon element content in the suitable solid acid catalyst is a value measured by the measurement method in the examples described below.

The above-mentioned more preferred solid acid catalyst, usually its substrate, contains titanium element. In the present invention, the substrate containing titanium element means that titanium element is present (present) on the surface and/or inside of the substrate, and examples thereof include a substrate in which titanium element is supported or adsorbed on the substrate surface (substrate supporting titanium element), a substrate in which titanium element is present (substrate formed of a composite of silica and titanium element), and a substrate in which titanium element is present on the substrate surface and inside. When the substrate has titanium element on its surface and/or inside, the hydrolysis reaction efficiency and glucose yield can be increased.
The content of titanium element contained in the more preferred solid acid catalyst is not particularly limited, but is preferably 0.1 to 10 mass %, 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 being able to increase the hydrolysis reaction efficiency and glucose yield. The more preferred carbon element content in the solid acid catalyst is a value measured by the measurement method in the examples described below.

A more preferred embodiment of the solid acid catalyst containing carbon and titanium elements is one in which the above-mentioned carbon element-containing embodiment and titanium element-containing embodiment can be combined as appropriate, but in terms of increasing the hydrolysis reaction efficiency and glucose yield, a combination of an embodiment in which titanium element is contained on the surface and/or inside of the substrate and an embodiment in which carbon element is supported or adsorbed on the surface of the substrate (a solid acid catalyst in this form is also called a carbon element-supported silica-titanium composite mesoporous body) is preferred.

A suitable solid acid catalyst has a sulfo group as a surface functional group. In the present invention, the term "a solid acid catalyst has 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 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, in terms of being able to increase the hydrolysis reaction efficiency and glucose yield. The content of sulfo groups in a suitable solid acid catalyst is a value measured by the measurement method in the Examples described below.

The solid acid catalyst may be a commercially available product, but since a suitable solid acid catalyst has a carbon element, preferably a titanium element, and a sulfo group, it is preferable to use an appropriately synthesized product.
The method of introducing carbon element into the substrate is not particularly limited, and various known methods can be applied. For example, a method of supporting or adsorbing carbon element on the surface of the substrate includes a method of 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, and usually, an organic compound is used, and for example, sugars such as sucrose, alcohols such as furfuryl alcohol, hydrocarbon compounds, and alkylene oxides are preferably used.
The method of introducing titanium element into the substrate is not particularly limited, and various known methods can be applied.For example, the method of supporting or adsorbing titanium element on the surface of the substrate can be the same as the method of supporting or adsorbing carbon element, except that a titanium-containing compound is used.On the other hand, the method of including titanium element inside or inside and on the surface of the substrate can be a method of forming the substrate using a mixture of a silica precursor compound described later and a titanium-containing compound described later, for example, the sol-gel method described later.
The method for introducing a sulfo group into the substrate is not particularly limited, and various known methods can be applied. For example, a method of treating a substrate, preferably a substrate having a carbon element supported or adsorbed on the surface, with sulfuric acid, specifically, the method described in Green Chem., 2010, 12, 1560-1563, can be mentioned.

Methods for producing porous silica as a substrate for a solid acid catalyst include the sol-gel method (also called the "molecular template method" or "template method"), which uses a silica source (silica precursor compound) and an amphiphilic compound as a template.
Hereinafter, a method for producing a carbon-supported mesoporous silica-titanium composite material having sulfo groups as surface functional groups by a sol-gel method will be described as an example of a more preferred solid acid catalyst.
A typical sol-gel method is, for example, to form an inorganic-organic nanocomposite by subjecting a silica precursor compound to a sol-gel reaction (hydrolysis reaction and condensation reaction) around self-assembled micelle particles formed with a cationic surfactant, and then calcining or treating the resulting compound with an acid (to remove the cationic surfactant).
A more preferred solid acid catalyst is a method for producing a mesoporous silica-titanium composite material containing titanium element on the surface and/or inside of a substrate by using the above-mentioned sol-gel method. At this time, the method can be carried out in the same manner as a general sol-gel method, except that a titanium precursor compound described later is used together with a silica precursor compound (coexistence of a silica precursor compound and a titanium precursor compound). Therefore, the type of each step in the sol-gel method applied when producing a mesoporous silica-titanium composite material, the operation and reaction conditions in each step, etc. can be appropriately determined with reference to a general sol-gel method. For example, the method described in Green Chem., 2010, 12, 1560-1563, and the series of steps in the synthesis method in the examples described later, the operation and reaction conditions in each step, etc. can be used as references.
In the above sol-gel method, like a general sol-gel method, the characteristics or physical properties of the porous body, such as the size (average pore diameter) and shape of the mesopores, and the skeletal shape of the porous body, can be adjusted or changed by changing the type of silica precursor compound, in particular the type of amphipathic compound, or preparation conditions such as the solvent composition, temperature, and time.

As the amphiphilic compound used as a template, compounds normally used in a general sol-gel method can be used without particular limitation, and usually, an amphiphilic surfactant is used, and cationic surfactants, block copolymers, etc. are preferably used. In the present invention, it is preferable to use a cationic surfactant as a template, in that the average pore diameter of the mesopores can be set relatively small, and the hydrolysis reaction efficiency and glucose yield can be further increased. As the cationic surfactant, surfactants normally used as templates in the sol-gel method can be used without particular limitation, and ammonium-based cationic surfactants and the like can be mentioned. As the ammonium-based cationic surfactant, alkyl ammonium salts are preferred, tetraalkyl ammonium salts are preferred, ammonium salts containing long-chain (6 or more carbon atoms) linear alkyl groups are more preferred, and mono-long-chain linear alkyl tri-short chain (4 or less carbon atoms) alkyl ammonium salts are particularly preferred. The anion forming the quaternary ammonium salt is not particularly limited, and inorganic anions such as hydroxide ions, halide ions, and perhalogen acid ions can be mentioned.
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.

The silica precursor compound may be any compound commonly used in a general sol-gel method without any particular limitation, and examples thereof include silicon halides, hydroxides, and alkoxides, and silicon alkoxides and/or alkyl alkoxides are 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 method, a titanium-containing compound is used together with a silica precursor compound. Examples of the titanium-containing compound include titanium precursor compounds that are converted into elemental titanium together with the silica precursor compound by the sol-gel method. As the titanium precursor compound, titanium halides, hydroxides, alkoxides, etc. are preferred, and titanium alkoxides and/or titanium alkyl alkoxides (alkoxytitanium) are 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.

Next, carbon element is supported or adsorbed on 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 into the thus obtained carbon-supported silica-titanium composite mesoporous material. 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 sulfo-group-introduced element-supported silica-titanium composite mesoporous material can also be crushed or disintegrated as appropriate.

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 that is similar to the more preferred method for producing the solid acid catalyst described above, except that in the sol-gel method, a silica precursor compound is used instead of a titanium precursor compound.

(Step of contacting a liquid containing cellulose and water with a solid acid catalyst)
The method for producing the decomposition product includes contacting a liquid (reaction liquid) containing cellulose and water with a solid acid catalyst. By this contact, cellulose is subjected to a hydrolysis reaction in the presence of the solid acid catalyst to convert it into glucose, thereby obtaining a cellulose decomposition product.
The components used in this contacting step may be one type or two or more types.

The cellulose hydrolysis reaction that occurs in the contacting step is generally considered to involve hydrolysis of cellulose to oligosaccharides and hydrolysis of oligosaccharides to glucose. Therefore, in the contacting step, a sugar-containing liquid (also called a "saccharification liquid") that contains glucose is obtained as a result of the cellulose hydrolysis treatment (saccharification treatment). This sugar-containing liquid usually has the above composition (satisfies the component area ratio).

In the method for producing decomposition products, if a suitable solid acid catalyst is used, cellulose can be efficiently hydrolyzed to obtain a cellulose decomposition product containing glucose as the main component. At this time, the conversion rate of cellulose and the selectivity of glucose (mass ratio of glucose content to other component contents) 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) x 100 (%)) of 45% or more can be achieved.

In the method for producing the decomposition product, a liquid containing cellulose and water (reaction liquid) is contacted with a solid acid catalyst under appropriate reaction conditions, usually under heating.
The contacting method may be any method capable of contacting the three components of cellulose, water and solid acid catalyst, and examples thereof include a method of contacting a previously prepared reaction liquid with a solid acid catalyst, a method of contacting cellulose, water and a solid acid catalyst without previously preparing a reaction liquid (a method of contacting cellulose with a solid acid catalyst in the presence of water), etc. In the contacting step, when the reaction liquid is contacted with or introduced into a solid acid catalyst, this includes an embodiment in which cellulose and water are contacted with or introduced into a solid acid catalyst separately instead of the reaction liquid.
In the contacting step, for example, the reaction liquid and the solid acid catalyst are put into a sealed container and then heated. 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 that it can increase the efficiency of the hydrolysis reaction of cellulose and suppress the by-production of other components to increase the yield of glucose. The heating time (reaction time) is appropriately determined depending on the heating temperature, the hydrolysis reaction efficiency (conversion rate) of cellulose, the glucose yield, and the like, and can be, for example, 1 to 48 hours. The inside of the sealed container under heating may be at normal pressure, but usually, the partial pressure of water vapor is in a pressurized state of, for example, more than 0.1 MPa. In the present invention, the hydrolysis reaction can also be performed in an environment that is actively pressurized. The pressure at this time is not particularly limited, and can be, for example, more than 0.1 MPa and 20 MPa or less, and is preferably 0.1 to 10 MPa. The reaction environment (atmosphere) is not particularly limited and may be an inert gas atmosphere, but may also be an air atmosphere or a water vapor atmosphere. In consideration of industrial production, the reaction environment is preferably an air atmosphere and/or a water vapor atmosphere.

The amounts of the reaction solution and the solid acid catalyst used are not particularly limited, but at least the amount of water (amount of water present) in the reaction system is set to be equal to or more than the amount required for the hydrolysis reaction of cellulose. The amount of water in the reaction system is preferably 0.1 to 1000 times by mass, more preferably 1 to 100 times by mass, relative to the amount of cellulose present, in terms of the mixability (stirrability) and handleability 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, and the amount of water in the reaction system refers to the amount of water derived from the reaction solution, but when water is added, it is the total amount of water derived from the reaction solution and the added water.
The amount of the solid acid catalyst used can be appropriately determined taking into consideration the reaction conditions, the hydrolysis reaction efficiency, the glucose yield, and the like. For example, the amount is preferably 0.01 to 10 times by mass, and more preferably 0.1 to 5 times by mass, relative to the amount of cellulose present.

The contacting step can be carried out batchwise using a sealed container 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 in terms of hydrolysis reaction efficiency and glucose yield to mix the reaction liquid and the solid acid catalyst by appropriate means such as stirring or shaking. An example of a continuous process is a method in which the reaction liquid is passed continuously or intermittently through a heated reaction tube.

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

[Post-processing process]
In the method for producing the decomposition product, the reaction mixture after the contacting step can be post-treated to obtain a cellulose decomposition product. Examples of the post-treatment of the reaction mixture include a step of cooling the reaction mixture and a step of subjecting the reaction mixture to solid-liquid separation.
The reaction mixture can be separated into solid and liquid without cooling, but it is preferable to cool the mixture in order to suppress further reaction of glucose or in consideration of workability, safety, and the like. The reaction mixture may be cooled naturally or using various refrigerants. The cooling temperature of the reaction mixture is not particularly limited, and is usually 100°C or less. In terms of suppressing further reaction of glucose and maintaining a high yield of glucose, it is preferably 80°C or less, and in consideration of workability, safety, and the like, it is preferably near room temperature (for example, 15 to 40°C). The lower limit of the cooling temperature can be, for example, 0°C or more in terms of workability, and is preferably 15°C or more. The cooling rate at this time can be appropriately determined, and it is preferable to reduce the temperature by 0.1 to 10°C per minute. The cooling time can be appropriately determined depending on the heating temperature and the cooling rate.
The reaction mixture that has been appropriately cooled is subjected to solid-liquid separation to separate a sugar-containing liquid (cellulose decomposition product) mainly composed of glucose as a liquid phase and at least a solid acid catalyst and unreacted cellulose as a solid phase. The method of solid-liquid separation is not particularly limited, and examples thereof include filtration, centrifugation, and precipitation.

[Other steps]
In the method for producing the decomposition product, steps other than the contact step and the post-treatment step can also be carried out. For example, the steps include purifying the separated and recovered sugar-containing liquid so as to satisfy the above-mentioned component area ratio, adjusting the composition of the separated and recovered sugar-containing liquid, isolating the solid acid catalyst and unreacted cellulose from the separated and recovered solid phase, washing and regenerating the isolated solid acid catalyst, removing impurities and contaminants from the waste when the above-mentioned waste is used as a cellulose source, and removing lignin from cellulose.
The isolation and purification method of the sugar-containing liquid can be applied to various known isolation and purification methods without any 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 range of the above-mentioned component area ratio 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 isolation step, washing step and regeneration step of the solid acid catalyst. However, since the catalytic activity of the solid acid catalyst usually gradually decreases with increasing number of uses, the solid acid catalyst can also be washed and/or regenerated in consideration of the decrease in catalytic activity, hydrolysis reaction efficiency or glucose yield. The washing and regeneration method is not particularly limited.

The decomposition product production method can decompose cellulose into glucose by a simple and safe process of contacting a reaction liquid with a solid acid catalyst, and can preferably obtain a cellulose decomposition product that satisfies the above-mentioned composition. In particular, the decomposition product production method using a suitable solid acid catalyst can obtain glucose from cellulose in a relatively short time with a high yield by a simple and safe process. In addition, since the occurrence and promotion of each side reaction during the cellulose hydrolysis reaction can be suppressed, glucose can be obtained with high selectivity (high purity), and a cellulose decomposition product that satisfies the above-mentioned composition can be obtained. In this respect, the decomposition product production method can be said to be a method for producing glucose. As a result, the decomposition product production method is suitable as a method for obtaining a raw material for producing bioethanol using non-edible biomass as a raw material. Moreover, when waste pulp is used as a cellulose source, the efficiency of the cellulose hydrolysis reaction and the glucose yield can be further increased, and non-edible biomass resources can be effectively utilized. Therefore, the decomposition product production method can efficiently produce glucose from cellulose at low cost, and is also suitable for industrialization of the cellulose hydrolysis reaction and the bioethanol production method.

[Step of contacting a glucose-containing liquid with a microorganism (fermentation step)]
In the production method of the present invention, a liquid containing glucose having the above-mentioned composition (hereinafter sometimes referred to as glucose liquid) is contacted with a microorganism. In this contacting step, the water content and pH are appropriately adjusted to provide conditions suitable for various organic compound synthesis methods, such as ethanol fermentation, and usually, the glucose liquid is inoculated with a microorganism to convert the glucose into an organic compound such as ethanol by the action of the microorganism. The liquid in which the glucose liquid and the microorganism are mixed and cultured for a certain period of time and part or all of the glucose is converted into an organic compound such as ethanol is referred to as a "fermentation liquid" for convenience.

<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.

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. la), Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Lactococcus, Enterococcus, Alcaligenes, Klebsiella The genus Paenibacillus, the genus Arthrobacter, the genus Corynebacterium, the genus Brevibacterium, the genus Schizosaccharomyces, the genus Issatchenkia, the genus Kluyvero Examples of suitable microorganisms include the genus Kluyveromyces, the genus Yarrowia, the genus Pichia, the genus Candida, the genus Hansenula, or the genus Saccharomyces, the genus Acetobacterium, the genus Eubacterium, etc. In addition, when the organic compound to be produced in the production method A of the present invention is alcohol, particularly ethanol, the microorganisms described below for use in the preferred production method B of the present invention can also be used.
As the microorganism used in the production method A of the present invention, for example, a microorganism capable of producing the desired organic compound can be appropriately selected from the above-mentioned microorganisms and used.

In the preferred production method B of the present invention, the yeast used is preferably one capable of fermenting 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, or a yeast of the genus Pachysolen such as Pachysolen tannophilus. Examples of yeasts include yeasts of the genus Saccharomyces, such as Issatchenkia orientalis, and yeasts of the genus Kluyveromyces, such as Kluyveromyces marxianus. Preferred are yeasts belonging to the genera Saccharomyces and Issatchenkia, and more preferably Saccharomyces cerevisiae and Issatchenkia orientalis. Genetically modified yeasts produced using recombinant technology can also be used. As the genetically modified yeast, any yeast that can ferment sugars (hexose and pentose) can be used without particular limitation, and preferably yeast that can ferment hexose and pentose simultaneously can be used.

In the preferred production method B of the present invention, examples of the eubacteria or archaea used include eubacteria of the genus Zymomonas, Escherichia coli, Corynebacterium, Clostridium, Halomonas, and other species, and archaea of the class Halobacteria. From the viewpoint of ethanol productivity, eubacteria of the genus 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 (KO11 strain), Clostridium ljungdahlii, Clostridium autoethanogenum, and Halomonas sp. KM-1 strain, which are preferably used.

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 their entirety.
In the production method of the present invention, one or more kinds of microorganisms can be used, and it is preferable to use them as a culture medium. The culture medium for the microorganism can be prepared by a conventional method and under conventional conditions.

The glucose solution contains cellulose decomposition products. In addition to the cellulose decomposition products, the glucose solution may contain a nutrient source necessary for the proliferation and activity of microorganisms. The nutrient source is not particularly limited, and examples thereof include yeast extract and polypeptone. In addition, the nutrient source described in paragraph [0043] of Patent Document 1 can be used, and the contents thereof are incorporated as a part of the description of this specification.
The glucose solution may also contain various components that are commonly used depending on the microorganism used, such as a pH adjuster, a buffering agent, a chelating agent, an antibiotic, an expression inducer, and an antifoaming agent.
In addition, since the influence of acetic acid bacteria can be suppressed in the fermentation process, acetic acid bacteria may be mixed into the glucose liquid. The acetic acid bacteria is not particularly limited, and various known acetic acid bacteria such as Acetobacter and Gluconacetobacter can be mentioned. However, it is preferable that the amount of acetic acid bacteria that may be mixed into the glucose liquid is about 0.2 mass% or less as an inoculation amount of the acetic acid bacteria culture liquid (OD660 = 2).

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

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.

The glucose liquid usually contains glucose and water derived from the glucose liquid. The glucose content (concentration) in the glucose liquid usually coincides with the above-mentioned glucose content in the cellulose decomposition product. However, when the concentration in the glucose liquid is adjusted by adding water appropriately, the glucose concentration in the glucose liquid becomes lower than the glucose concentration in the cellulose decomposition product. The glucose content in the glucose liquid at this time can be, for example, 0.1 to 10 mass%, and is preferably 1 to 5 mass% in terms of efficient ethanol production. The water content in the glucose liquid can be, for example, 90 to 99.9 mass%, and is preferably 95 to 99 mass% in terms of efficient ethanol production.

The pH of the fermentation liquid 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 liquid in the fermentation step is not particularly limited as long as it is within the optimum temperature range for the microorganism, and is preferably, for example, 20 to 40° C., and more preferably 30 to 40° C.
The amount of microorganisms to be inoculated into the glucose solution is not particularly limited, and may be any amount that allows the inoculated microorganisms to grow. For example, the amount of microorganism culture solution (OD660=6) to be inoculated may be 0.01 to 10% by mass, and preferably 0.1 to 1% by mass in terms of favorable growth of the microorganisms.
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 liquid is in contact with the microorganisms, usually 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).

The amount of ethanol produced in the fermentation broth is not particularly limited and is determined appropriately depending on the ethanol production ability of the microorganism and the culture method. For example, it can be 1 to 120 g/L, or it can also be 3 to 50 g/L. Since ethanol has bactericidal properties, the amount of ethanol produced in the fermentation broth cannot be so high that it affects the survival and activity of the microorganisms, and it is preferable to set the upper limit at 150 g/L, for example.

The atmosphere in the fermentation process can be appropriately determined depending on the microorganisms used, and can be an aerobic atmosphere during contact, but can also be an anaerobic atmosphere during fermentation.

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 process can be carried out in a batch or continuous manner using a fermenter. The fermenter can be of any shape, such as a stirring type, airlift type, bubble column type, loop type, open bond type, or photobio type.

In the manner described above, a fermentation mixture containing microorganisms and organic compounds such as ethanol can be obtained.
When focusing on the production process, the production method of the present invention can also be said to be a method for producing organic compounds such as ethanol from cellulose, which includes a step of contacting with the above-mentioned decomposition product production method.

[Post-processing process]
In the production method of the present invention, the reaction mixture (fermentation mixture) obtained after the above-mentioned contacting step can be post-treated to obtain organic compounds such as ethanol. Examples of post-treatment of the fermentation mixture include a step of separating the fermentation mixture from solid and liquid, and a step of isolating ethanol.
The fermentation mixture may be subjected directly to a step of isolating organic compounds such as ethanol, but is usually subjected to solid-liquid separation to separate a liquid containing organic compounds such as ethanol as a liquid phase from a solid content containing microorganisms as a solid phase. The method of solid-liquid separation is not particularly limited, and examples thereof include filtration, centrifugation, and precipitation.
In the step of isolating an organic compound such as ethanol, the liquid containing the organic compound such as ethanol after solid-liquid separation can usually be distilled to isolate the organic compound. As the distillation method and conditions, a conventional distillation method can be applied, and examples of the distillation include atmospheric distillation and reduced pressure distillation.

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

The production method A of the present invention can produce a target organic compound, preferably in high yield, from a cellulose decomposition product that satisfies the above component area ratio, using a microorganism.
In addition, the preferred production method B of the present invention can efficiently (highly) produce ethanol from a cellulose decomposition product that satisfies the above component area ratio by suppressing the occurrence and progress of side reactions while promoting ethanol fermentation by microorganisms, even if the fermentation time is long. In addition, a cellulose decomposition product prepared using a suitable solid acid catalyst satisfies the above component area ratio, and can further efficiently proceed with ethanol fermentation. Moreover, the 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 if acetic acid bacteria coexist during ethanol fermentation, and can suppress the conversion of ethanol converted from a cellulose hydrolysate to acetic acid. As a result, 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. Moreover, when a cellulose decomposition product prepared using waste pulp is used as a cellulose source, a cellulose decomposition product with an improved glucose content can be obtained while satisfying the above component area ratio, and effective use of non-edible biomass resources is also possible. Therefore, the preferred production method B of the present invention can efficiently produce ethanol from cellulose at low cost, and is also suitable for industrialization of bioethanol production.

[[Composition for microbial culture]]
The composition for microbial culture of the present invention comprises a liquid containing glucose. The liquid containing glucose is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and is a cellulose hydrolysate in which, 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. The cellulose hydrolysate constituting the composition for microbial culture of the present invention is the same as the cellulose hydrolysate 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 that are normally used depending on the microorganism to be used, components that are normally used in culture (culture medium), etc. Examples of such other components include a nutrient source necessary for the proliferation and activity of the microorganism, a pH adjuster, a buffer, a chelating agent, an antibiotic, an expression inducer, and an antifoaming agent. Each component is not particularly limited and is as described above. In addition, the composition for microbial culture may contain the above-mentioned acetic acid bacteria since the effect of the acetic acid bacteria can be suppressed.

The total content of the cellulose decomposition products in the composition for microbial culture is not particularly limited and can be appropriately determined in consideration of the production efficiency of the organic compound, 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 0.1 to 10% by mass in terms of the production efficiency of the organic compound, etc.
The total content of other components in the composition for microbial culture is not particularly limited and can be appropriately determined taking into consideration the production efficiency of the organic compound, etc., and it is preferable that the content of acetic acid bacteria in the composition for microbial culture is about 0.2 mass% or less as the inoculation amount of acetic acid bacteria culture solution (OD660 = 2).

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

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

[Preparation of solid acid catalyst]
Experimental Example 1: Preparation of solid acid catalyst for decomposing cellulose
(1) Preparation of porous silica (first step)
625.5 g of a 16% by mass hexadecyltrimethylammonium hydroxide aqueous solution 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.

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

(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 for 1 hour at reflux temperature while stirring, and after cooling, the liquid phase was removed by filtration. The same operation 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.

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

(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 type particle size distribution measuring device (LA-950 manufactured by Horiba, Ltd.) using ion-exchanged water as a dispersion medium, and calculated on a volume basis.

(2) Preparation of porous silica-carbon composite 3 g of porous silica and 30 g of propylene oxide (a reagent manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a carbon source were added to an autoclave equipped with a stirrer. The inside of the autoclave was pressurized with nitrogen to 1 MPa-G and depressurized to normal pressure, and this operation was repeated three times to replace the inside of the autoclave with nitrogen. The autoclave was immersed in an oil bath, and the stirrer was set to rotate at 500 rpm to stir the inside of the autoclave, while adjusting the oil bath temperature so that the inside temperature of the autoclave was 100°C. After stirring for 6 hours after the inside temperature of the autoclave reached 100°C, the solid obtained by filtration was air-dried, and further heated to 500°C at 6°C/min in an electric furnace under a nitrogen atmosphere, and heat-treated at 500°C for 2 hours to prepare 3.2 g of porous silica-carbon composite as a carbon element-supported silica-titanium composite mesoporous body.

(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 mixture was washed with water at 50° C. until the pH of the washing liquid 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.

Experimental Example 2 Pore Analysis of Porous Silica Material
The powdered porous silica (silica-titanium composite mesoporous material) prepared as described above had a specific surface area of 1160 ^{m 2} /g, a total pore volume of 0.60 mL/g, and an average pore diameter of 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 Microtrack-Bell) after vacuum degassing the porous silica at 120°C for 2 hours, and then using the BET method and the nitrogen adsorption amount near the relative pressure (P/P0 = 0.96). The average pore diameter was calculated from the total pore volume and specific surface area using the following formula (1):

(Formula 1):
Average pore diameter (nm) = 4 x total pore volume (mL/g) / specific surface area (m ² /g) x 1000

In addition, the Ti concentration in the powdered porous silica was analyzed by XRF and found to be 1.5% by mass.

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.

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 a potentiometric automatic titrator (Kyoto Electronics). 50 mg of the solid acid catalyst was weighed out in a screw tube bottle, 30 mL of a 0.05N NaCl aqueous solution was added using a whole pipette, and the mixture was stirred with a stirrer for 15 hours. After stirring, the solution in the screw tube bottle was filtered, and 10 mL of the filtrate was collected using a whole 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 0.005N NaOH aqueous solution was dropped in 0.1 mL at a time for titration. The sulfo group content was calculated from the equivalent point of the titration result to determine the Na ⁺ exchange amount. The sulfo group content of the solid acid catalyst thus obtained was 0.09 mmol/g.

<Experimental Example 5. Methods for obtaining and preparing raw materials (board pulp, pulp sludge)>
The raw material, board pulp, was obtained by dehydrating the raw material used in the papermaking process for household paper.
The raw material, 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 during the papermaking process, solidifying it, and then dewatering it.

<Experimental Example 6. Method for pre-treating raw materials>
The plate pulp obtained in the above Experimental Example 5 was dehydrated, dried at 60° C. for 12 hours, and then vacuum dried at 120° C. for 12 hours. After vacuum drying, the plate pulp was finely chopped with scissors to obtain a pretreated plate pulp.
The pulp sludge obtained in the above Experimental Example 5 was dehydrated, dried at 60° C. for 12 hours, and then vacuum dried at 120° C. for 12 hours. After vacuum drying, the pulp sludge was finely chopped with scissors to obtain a pretreated pulp sludge.

Experimental Example 7: Preparation of cellulose decomposition product
(1) Hydrolysis Reaction of Cellulose The cellulose used as the reaction substrate was treated as follows.
1 kg of zirconia balls with a diameter of 1 cm 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 rotating platform and subjected to ball mill treatment at 200 rpm for 120 hours. This operation was carried out in two batches, yielding 9 g of ball milled cellulose.
Next, the ball milled cellulose, solid acid catalyst, and purified water were added to an autoclave equipped with a stirrer, and the mixture was heated from room temperature to 150° C. in about 30 minutes while stirring at 300 rpm, and then hydrolysis reaction of cellulose 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. Then, the reaction liquid was filtered to separate it into liquid and solid, and the two batches were mixed to obtain a cellulose decomposition product and a solid residue.
In each experimental example, the term "cellulose hydrolysate" refers to the cellulose hydrolysate prepared in Experiment 7.(1).

(2) Hydrolysis Reaction of Plate Pulp The plate pulp used as the reaction substrate was treated as follows.
1 kg of zirconia balls with a diameter of 1 cm and 5 g of the pretreated (finely chopped) plate pulp obtained in Experimental Example 6 above were placed in a ceramic pot mill. The plate pulp was set on the rotating table of the tabletop pot mill and treated with a ball mill at 200 rpm for 120 hours. This operation was carried out in multiple batches, and the plate pulp attached to the wall of the ceramic bottle was collected as a sample to obtain 9 g of ball milled plate pulp.
Next, the plate pulp treated with a ball mill, the solid acid catalyst, and purified water were added to an autoclave equipped with a stirring device, and the temperature was raised from room temperature to 150°C in about 30 minutes while stirring at 300 rpm, and then the plate pulp hydrolysis reaction was carried out for three batches at 150°C for 24 hours (internal pressure 0.5 MPa). In the first batch of ball mill treatment, the amounts of plate pulp, solid acid catalyst, and purified water added were 3g, 3g, and 37.5g, respectively. After the reaction was completed, the autoclave was cooled to room temperature. Then, the reaction liquid was filtered to separate into liquid and solid. The solid obtained by filtration and separation was vacuum dried at 120°C for 12 hours, and 3g of newly ball milled plate pulp and 37.5g of purified water were added to carry out the plate pulp hydrolysis reaction, thereby carrying out the plate pulp hydrolysis reaction for the second batch. The same operation as in the second batch was repeated to carry out the plate pulp hydrolysis reaction for 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 1/3 of its volume, to obtain a “plate pulp decomposition product” as a cellulose decomposition product.

(3) Hydrolysis Reaction of Pulp Sludge The pulp sludge used as the reaction substrate was treated as follows.
1 kg of zirconia balls with a diameter of 1 cm and 5 g of the pretreated (finely chopped) pulp sludge obtained in Experimental Example 6 above were placed in a ceramic pot mill. The mixture was set on a tabletop pot mill rotating platform and treated with a ball mill at 200 rpm for 120 hours. This operation was carried out in multiple batches, and the plate pulp attached to the wall of the ceramic bottle was collected as a sample to obtain 21 g of ball milled plate pulp.
Next, the ball milled pulp sludge, solid acid catalyst, and purified water were added to an autoclave equipped with a stirrer, and the temperature was raised from room temperature to 150° C. in about 30 minutes while stirring at 300 rpm, and then 7 batches of hydrolysis reaction of the pulp sludge were carried out at 150° C. for 24 hours (internal pressure 0.5 MPa). In the ball mill treatment of the first batch, 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. Then, the reaction liquid was filtered to separate into 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 of the second batch. The same operation as in the second batch was repeated to carry out the hydrolysis reaction of the pulp sludge of the third, fourth, fifth, sixth, and seventh batches. The seven batches of liquid were mixed to obtain a pulp sludge decomposition liquid. The pulp sludge decomposition liquid was concentrated at 50° C. to a volume ratio of 1/7 to obtain a “pulp sludge decomposition product” as a cellulose decomposition product.

(4) High-performance liquid chromatography (HPLC) analysis The cellulose decomposition product, plate pulp decomposition product, and pulp sludge decomposition product obtained in (1), (2), and (3) above were subjected to HPLC analysis to obtain HPLC analysis results for the cellulose decomposition product, plate pulp decomposition product, and pulp sludge decomposition product. Among the chromatograms obtained by HPLC analysis, the chromatogram of the cellulose decomposition product obtained in (1) above is shown in FIG. 1. The HPLC analysis was performed using an LC-20A (manufactured by Shimadzu Corporation) with a column of Shodex SP0810 (manufactured by Resonaq, inner diameter 8 mm, length 300 mm), 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., a sample injection amount of 20 μL, an analysis time of 70 minutes, and a sampling rate of 100 ms.
The obtained chromatogram was used to identify the type of each component of the cellulose decomposition product, the plate pulp decomposition product, and the pulp sludge decomposition product, and to quantify the content thereof. Specifically, each component detected in this chromatogram was identified, for example, by comparing with the retention time of each compound reagent in the above column: Shodex SP0810. Next, the concentrations (mass%) of glucose, cellobiose, fructose, levoglucosan, HMF (5-hydroxymethylfurfural), and furfural in the cellulose decomposition product were quantified by an absolute calibration curve method using the reagents of each component. The quantitative results of the cellulose decomposition product, the plate pulp decomposition product, and the pulp sludge decomposition product obtained in (1), (2), and (3) above are shown in Tables 1 to 3, respectively.

As a result of HPLC analysis of cellulose hydrolyzate, plate pulp hydrolyzate, and pulp sludge hydrolyzate using a differential refractive index detector, the peak area values of each chromatogram derived from glucose and components with lower molecular weight than glucose that elute after the retention time of glucose are shown in Tables 4 to 6 below. "Other 1" to "Other 10" shown in Tables 4 to 6 are neither cellobiose nor fructose, but components with lower molecular weight than glucose. Note that "Other n" in each table (n is 1 or 2 in Table 4, an integer of 1 to 6 in Table 5, and an integer of 1 to 10 in Table 6) indicates peaks derived from unidentifiable substances in each chromatogram in the order of detection using n, and "Other n" in which n has the same value does not mean 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 the area value was calculated.

From the area values shown in Tables 4 to 6, the area ratios (component area ratios) were calculated.
Specifically, in Table 4, the area value of glucose was 10750862, and the total area value of the peaks derived from "components having a lower molecular weight than glucose" (other 1 + other 2 + levoglucosan + HMF + furfural) was 783106. Thus, in Table 4 (the cellulose decomposition product obtained in (1) above), the area ratio of the total area value of "components having a lower molecular weight than glucose" to the area value of "glucose" was 0.072 times.
Similarly, the area ratio was also determined 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 10159454 and 10901449, respectively, and the total area values of the peaks derived from "components with lower molecular weight 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 8387038 and 8357042, respectively. Therefore, in Tables 5 and 6, the area ratio of the total area value of "components with lower molecular weight than glucose" to the area value of "glucose" was 0.826 times and 0.767 times, respectively.

<Experimental Example 8-1. Cultivation of microorganisms using cellulose decomposition products as a carbon source: ethanol fermentation>
The materials used in this experiment are as follows:

(Culture medium)
- Yeast medium (carbon source: cellulose decomposition product obtained in Experimental Example 7.(1)) -
The components were mixed in ultrapure water to give the following concentrations, the pH was adjusted to 5.6, and the medium was sterilized with high-pressure steam.
Cellulose hydrolyzate in an amount equivalent to 20 g/L of glucose, 20 g/L of polypeptone (manufactured by Nippon Seiyaku Co., Ltd.), and 10 g/L of yeast extract (manufactured by Thermo Fisher Scientific Co., Ltd.)

- Acetic acid bacteria medium (carbon source: cellulose decomposition product obtained in Experimental Example 7. (1)) -
The components were mixed in ultrapure water to give the following concentrations, the pH was adjusted to 7.0, and the medium was sterilized by high-pressure steam.
Cellulose hydrolyzate in an amount equivalent to 20 g/L of glucose, 5 g/L polypeptone, 3 g/L yeast extract, 3 g/L meat extract (manufactured by BD), 2 g/L ammonium sulfate (manufactured by Nacalai Tesque), 1 g/L monopotassium dihydrogen phosphate (manufactured by Nacalai Tesque), 0.5 g/L magnesium sulfate heptahydrate (manufactured by Nacalai Tesque)

(microorganisms)
- Ethanol-producing bacteria -
Yeast (Saccharomyces cerevisiae S288C NBRC1136)
- Ethanol decomposition bacteria -
Acetic acid bacteria (Acetobacter aceti NBRC14818)

(Microbial cultivation method: Ethanol fermentation)
From the glycerol stock (microorganism frozen storage solution obtained by adding glycerol to a microorganism culture solution to a final concentration of 30% by mass and then freezing), a microorganism corresponding to the medium was inoculated into a 5.0 mL medium/test tube (5.0 mL of each medium was placed in one test tube), and cultured overnight at 30° C. (hereinafter referred to as "preculture"). 0.1 mL of each of the obtained precultures was added to a 5.0 mL medium/test tube (a test tube containing the same medium as the preculture), and cultured at 30° C. and 200 rpm for 48 hours (hereinafter referred to as "main culture").

(Measurement of turbidity of the main culture solution)
During the main culture process, samples were taken from each main culture over time (0 hours, 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 hours, 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.

(Measurement of glucose concentration in the main culture solution)
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 obtained filtrate was subjected to HPLC analysis, and the glucose concentration (mass%) in the filtrate was quantified by an absolute calibration curve method using a glucose reagent. HPLC analysis was performed using an LC-20A (Shimadzu Corporation) with a Shodex SP0810 (Resonac Corporation) column, 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.

The residual glucose rate was calculated from the HPLC analysis results according to the following formula (2).
The obtained residual glucose rate was used to determine the "glucose assimilation rate" described below.

(Formula 2):
Glucose residual rate (%)=(glucose concentration in the main culture after 48 hours of culture)/(glucose concentration in the main culture at 0 hours of culture)×100

(Measurement of ethanol concentration in the main culture solution)
Using a headspace gas chromatography apparatus, the filtrate was heated at 80° C. for 30 minutes, and the gas phase portion 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)

<Experimental Example 8-2. Cultivation of microorganisms using glucose as a carbon source: ethanol fermentation>
The turbidity, glucose concentration, and ethanol concentration of the main culture solution 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 after 0 hours, 24 hours, and 48 hours from 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, in which commercially available glucose was used as the glucose source.

(Culture medium)
- Yeast medium (carbon source: glucose) -
A medium in which glucose (reagent manufactured by Nacalai Tesque) was used at 20 g/L in place of the cellulose decomposition product in the “yeast medium (carbon source: cellulose decomposition product obtained in Experimental Example 7 (1))” in Experimental Example 8-1.
- Acetic acid bacteria medium (carbon source: glucose) -
A medium in which glucose (a reagent manufactured by Nacalai Tesque) was used at 20 g/L in place of the cellulose decomposition product in the “medium for acetic acid bacteria (carbon source: cellulose decomposition product obtained in Experimental Example 7 (1))” in Experimental Example 8-1.

<Experimental Example 8-3. Evaluation of ethanol fermentation>
(Determination of Microbial Growth)
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 (%) in each main culture (cultured for 48 hours) was calculated and evaluated according to the following criteria. The results are shown in Table 7 together with the relative bacterial cell concentration values.

- judgement -
A: Relative bacterial cell concentration ≧75%
B: 75% > relative bacterial cell concentration ≧18%
C: 18% > relative bacterial cell concentration

From the results in Table 7 and Figures 2 and 3, it was found 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 became constant and yeast growth stopped after about 24 hours. When acetic acid bacteria were used (Figure 3), the turbidity remained constant until 24 hours and no growth of acetic acid bacteria was observed, but after 24 hours, the turbidity increased, indicating that acetic acid bacteria were growing.
In contrast, when cellulose decomposition products were used as the carbon source, an increase in yeast cell concentration was observed, but no increase in acetic acid bacteria cell concentration was observed. Moreover, when cellulose decomposition products were used, the relative cell concentration was significantly improved compared to when glucose was used. Specifically, when yeast was used (Figure 2), under the conditions of the above Experimental Example 8-1, the turbidity increased even after 24 hours, indicating that yeast proliferation continued. When acetic acid bacteria were used (Figure 3), the turbidity remained almost unchanged, indicating that acetic acid bacteria proliferation and acetic acid fermentation (ethanol decomposition) were suppressed.

(Determination of glucose utilization rate)
The glucose residual rate (%) calculated by the above formula 2 was taken as the glucose assimilation rate and was evaluated according to the following criteria. The results are shown in Table 8 together with the glucose residual rate.

- judgement -
A: Glucose residual rate ≦25%
B: 25% < glucose residual rate ≦ 75%
C: 75% or less residual glucose rate

The results in Table 8 show that when glucose was used as a carbon source, both yeast and acetic acid bacteria utilized the glucose. In contrast, when cellulose decomposition products were used as a carbon source, yeast utilized the glucose contained in the cellulose decomposition products, but acetic acid bacteria did not utilize the glucose contained in the cellulose decomposition products.

(Determination of ethanol productivity)
The ethanol concentration was calculated from the GC-MS analysis results and judged according to the following criteria.

- judgement -
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

The results in Table 9 show that the same amount of ethanol was detected when cellulose decomposition products and glucose were used as the carbon source. Furthermore, when cellulose decomposition products were used, the ethanol concentration (fermentation efficiency) was almost the same as when glucose was used alone. This shows that the use of cellulose decomposition products makes it possible to efficiently produce ethanol using yeast.

<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 materials used in this experiment are as follows:

(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 decomposition product in an amount equivalent to 20 g/L of glucose, 6.7 g/L YNB, w/ ammonium sulfate, 790 mg/L CSM, powder

- Yeast medium (carbon source: decomposition product of plate pulp obtained in Experimental Example 7. (2)) -
Amount of glucose equivalent to 20 g/L of board pulp hydrolyzate, 6.7 g/L YNB, w/ ammonium sulfate, 790 mg/L CSM, powder

- Yeast medium (carbon source: pulp sludge decomposition product obtained in Experimental Example 7.(3)) -
Pulp sludge decomposition product in an amount equivalent to 20 g/L of glucose, 6.7 g/L YNB, w/ ammonium sulfate, 790 mg/L CSM, powder

(microorganisms)
Yeast (Saccharomyces cerevisiae S288C NBRC1136)

(Method of culturing yeast)
A test tube was charged with 4.0 mL of yeast medium (carbon source: glucose), and yeast was inoculated from a glycerol stock and cultured overnight at 30°C and 225 rpm (hereinafter referred to as "pre-preculture"). A 250 mL baffled Erlenmeyer flask was charged with 50 mL of yeast medium (carbon source: glucose), and 0.5 mL of the obtained pre-preculture was charged and cultured overnight at 30°C and 100 rpm (hereinafter referred to as "preculture"). The obtained preculture was collected, the supernatant was removed by centrifugation, and the yeast was washed with yeast medium (carbon source: none) to remove the supernatant, and yeast cells were obtained. The obtained cells were suspended in each yeast medium (carbon source: none, glucose, cellulose decomposition product, plate pulp decomposition product or pulp sludge decomposition product) so that the inoculum concentration was 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”).

(Measurement of turbidity of the main culture solution)
During the main culture, samples were taken from each main culture over time (0 hours, 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 FIG. 4.

(Determination of yeast growth)
In measuring the turbidity of the main culture, the turbidity value of the experimental example in which the culture was performed for 48 hours using glucose as a carbon source was taken as 100% bacterial cell concentration, and the relative bacterial cell concentration (%) in each main culture (cultured for 48 hours) was calculated and evaluated according to the following criteria. The results are shown in Table 10 together with the relative bacterial cell concentration values.

- judgement -
A: Relative bacterial cell concentration ≧75%
B: 75% > relative bacterial cell concentration ≧18%
C: 18% > relative bacterial cell concentration

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 decomposition products, sheet pulp decomposition products, and pulp sludge decomposition products were used as carbon sources. When glucose was used as the carbon source, the turbidity became constant and yeast growth stopped after about 10 hours. In contrast, when cellulose decomposition products or sheet pulp decomposition products were used as the carbon source, yeast growth continued even after 24 hours, and the relative cell concentration was higher than that of glucose after 48 hours.

(Measurement of ethanol concentration in the main culture solution)
During the main culture, samples were taken from each main culture broth at 0 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 Co., Ltd.) Note that the results of an experiment without a carbon source are omitted.

(Determination of ethanol productivity)
Based on the ethanol concentration in the main culture medium, the evaluation was made according to the following criteria.

- judgement -
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

The results in Table 11 show that ethanol was detected in all cases where glucose, cellulose hydrolysate, plate pulp 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 plate pulp hydrolysate was used as the carbon source, the ethanol concentration (fermentation efficiency) was significantly higher than when glucose was used as the carbon source.

<Experimental Example 10. Identification of growth inhibitors of acetic acid bacteria>
The materials used in this experiment are as follows:

(Culture medium)
The components were mixed in ultrapure water to give the following concentrations, the pH was adjusted to 7.0, and the medium was sterilized by high-pressure steam.
- Acetic acid bacteria medium (carbon source: glucose) -
20 g/L glucose (Fujifilm Wako Pure Chemical Industries, Ltd.), 5 g/L polypeptone (Shioya 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, we assumed that the substances that have the growth inhibitory effect on acetic acid bacteria would be levoglucosan, HMF, or furfural. We therefore selected these substances as candidates for growth inhibitors and added them to the acetic acid bacteria medium (carbon source: glucose) at the following concentrations.

Levoglucosan (manufactured by 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): 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

(microorganisms)
Acetic acid bacteria (Acetobacter aceti NBRC14818)

(Method of culturing acetic acid bacteria)
A glycerol stock of acetic acid bacteria was inoculated into a 125 mL baffled Erlenmeyer flask containing 20 mL of acetic acid bacteria medium (carbon source: glucose) 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) containing each growth inhibitor candidate so that the inoculum concentration was OD600 = 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").

(Measurement of turbidity of the main culture solution)
During the main culture, samples were taken from each main culture over time (0 hours, 8 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, 8 hours, 24 hours, and 48 hours after the main culture are shown in Figures 5 to 7.

(Determination of the growth rate of acetic acid bacteria)
In measuring the turbidity of the main culture, the turbidity value of the experimental example in which the culture was performed for 48 hours without adding a growth inhibitor candidate was taken as 100% bacterial cell concentration, and the relative bacterial cell concentration (%) in each main culture (cultured for 48 hours) was calculated and evaluated according to the following criteria. The results are shown in Tables 12 to 14 together with the relative bacterial cell concentration values.

- judgement -
A: Relative bacterial cell concentration ≧75%
B: 75% > relative bacterial cell concentration ≧20%
C: 20% > relative bacterial cell concentration

The results in Tables 12 to 14 and Figures 5 to 7 show that levoglucosan and HMF had no effect on the growth of acetic acid bacteria, but furfural clearly inhibited the growth of acetic acid bacteria. Specifically, when 0.035 g/L furfural was added to the acetic acid bacteria medium, the relative bacterial cell concentration was 53% of the experimental example without furfural addition (0 g/L), and when 0.070 g/L to 0.560 g/L furfural was added to the acetic acid bacteria medium, the relative bacterial cell concentration was 6 to 7% of the experimental example without furfural addition (0 g/L). Under the conditions in the above experimental examples, it was found that 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 more.

From the above results, it was shown that cellulose decomposition products, plate pulp decomposition products, and pulp sludge decomposition products that satisfy the above component area ratios can be used as carbon sources for ethanol production by yeast. In particular, it was shown that cellulose decomposition products and plate pulp decomposition products show fermentation efficiency equal to or higher than that when glucose is used alone. It was also shown that the cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst can highly inhibit the growth of acetic acid bacteria by containing 0.035 g/L or more, preferably 0.070 g/L or more of furfural.
In addition, the cellulose decomposition product obtained in Experimental Example 7. (1) showed a fermentation efficiency almost equal to that when glucose was used alone, but surprisingly, it was also shown to have an effect of suppressing the growth of acetic acid bacteria (suppression of acetic acid fermentation). Therefore, by using a cellulose decomposition product satisfying the above component area ratio, even if acetic acid bacteria are mixed in, it is possible to prevent the growth of acetic acid bacteria and ethanol decomposition by acetic acid fermentation, and even if the fermentation time is extended to 48 hours, ethanol can be efficiently produced by yeast. Thus, the preferred production method B of the present invention can produce ethanol from a cellulose decomposition product satisfying the above component area ratio using a microorganism 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 if acetic acid bacteria are mixed in. 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.

Although the present invention has been described in conjunction with its embodiments, we do not intend to limit our invention to any of the details of the description unless otherwise specified, and believe that the appended claims should be interpreted broadly without departing from the spirit and scope of the invention as set forth in the appended claims.

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 as part of the present specification.

Claims (14)

A method for producing an organic compound, comprising contacting a liquid containing glucose with a microorganism,
The method for producing an organic compound, wherein the glucose-containing liquid is a cellulose hydrolysate obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and 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 lower molecular weights than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose. The method of claim 1, wherein the organic compound is ethanol, and the total area value of peaks derived from components with lower molecular weights than glucose in the LC analysis is 0.01 to 0.2 times the area value of the peak derived from glucose. The method of claim 1, wherein the cellulose comprises one of cellulose derived from board pulp and cellulose derived from pulp sludge, or a mixture thereof. The method according to claim 1, wherein the solid acid catalyst contains carbon and silica and has a sulfo group as a surface functional group. The method according to claim 2, wherein the solid acid catalyst contains carbon and silica and has a sulfo group as a surface functional group. The method of claim 4, wherein the silica has mesopores. The method of claim 5, wherein the silica has mesopores. The method of claim 4, wherein the carbon element is supported on the surface of the silica. The method of claim 5, wherein the carbon element is supported on the surface of the silica. The method of claim 6, wherein the carbon element is supported on the surface of the silica. The method of claim 7, wherein the carbon element is supported on the surface of the silica. The method according to any one of claims 4 to 11, wherein the solid acid catalyst contains titanium element. The manufacturing method according to claim 12, wherein the titanium element is contained in the silica. A composition for microbial culture comprising 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, and 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 lower molecular weights than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose.

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