Method for producing organic compound, and composition for culturing microorganism

Abstract

To provide a method for producing an organic compound that enables synthesis of various organic compounds using sugars derived from cellulose, and to provide a method for producing ethanol that enables efficient ethanol fermentation using sugars derived from cellulose while suppressing growth of acetic acid bacteria and acetic acid fermentation even if acetic acid bacteria are mixed in.SOLUTION: Provided is a method for producing an organic compound, preferably ethanol, including 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 having 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, preferably 0.01 to 0.2 times, the area value of peaks derived from glucose.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] In recent years, from the viewpoints of global environmental protection and effective utilization of waste, the synthesis and production of various organic compounds using biomass, which is a renewable resource, as a raw material have been studied. For example, in order to achieve carbon neutrality, bioethanol has been widely studied as a raw material for low-carbon footprint materials. In particular, the production of bioethanol from edible biomass and its olefination by dehydration reaction and its polymerization have been studied. Here, the conversion method from edible biomass to bioethanol has been technically established and is regarded as promising as a raw material production method for plastic products. However, there is a problem that edible biomass competes with food uses, and in recent years, the raw material conversion from edible biomass to inedible biomass has been progressing as a raw material for producing bioethanol. Cellulose, which is inedible biomass, can be decomposed into glucose by a hydrolysis reaction using an enzyme method or a sulfuric acid method, and can be further led to ethanol by a fermentation method using microorganisms from glucose. However, the decomposition reaction into glucose generally involves difficulty because cellulose has a chemically stable structure. For example, even if cellulose can be converted into glucose, the purity of the obtained glucose is not sufficient, and even if it is purified, ethanol cannot be obtained efficiently. Therefore, in a method for producing ethanol using cellulose, which is inedible biomass, as a starting material, a method for efficiently promoting alcohol fermentation by microorganisms or a method for obtaining a decomposition product of cellulose is required.

[0003] By the way, since the importance of environmental conservation due to climate change and carbon neutrality have been advocated, various methods for producing ethanol from biomass have been studied. 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 saccharifying enzyme reaction, and a solid acid catalyst reaction. Specifically, Patent Document 1 describes "a pressurized hot water reaction step of allowing pressurized hot water to act on biomass to selectively decompose hemicellulose contained in the biomass, a beating treatment step of beating the solid residue after the pressurized hot water reaction step, a primary saccharification step of allowing a saccharifying enzyme to act on the solid residue after the beating treatment step, and a secondary saccharification step of allowing a solid acid catalyst to act on the product obtained by the primary saccharification step, and a fermentation step of fermenting the monosaccharides obtained by the method for producing monosaccharides having these steps to produce ethanol." Further, Patent Document 2 describes "a bioethanol production method for producing ethanol from lignocellulose, wherein biomass is pulverized by a mill, and a biomass powder slurry is produced by mixing the pulverized biomass powder, a catalyst, and preheated steam in a preheater, and the biomass powder slurry supplied from the preheater is heated by heating steam in a hydrolysis tower."

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In recent years, attention has been focused on the production of organic compounds using biomass as a raw material. Among them, the production of bioethanol has advanced industrially and is used as a carbon-neutral fuel. In ethanol fermentation, it is known that not only the main reaction of converting glucose present in the hydrolysis product of cellulose into ethanol but also various side reactions occur. Therefore, by-products produced by side reactions reduce the yield and purity of ethanol. In particular, the amount of by-products produced tends to increase with the passage of fermentation time (reaction time) in ethanol fermentation.

[0006] By the way, acetic acid bacteria are bacteria that convert ethanol into acetic acid. In the method for producing ethanol, they are bacteria that should be avoided from mixing into the reaction system in order to reduce the yield and purity of ethanol. However, acetic acid bacteria widely exist in nature as resident bacteria and are easily mixed. For example, acetic acid bacteria float in the air and also exist in an environment or reaction field where sugar or plant carbohydrates are fermented by yeast to produce ethanol in nature. As described above, in order to solve the above problems caused by acetic acid bacteria, it is important not to allow acetic acid bacteria to exist in the environment and reaction field in ethanol fermentation. However, it is not easy to remove acetic acid bacteria, which are resident bacteria, from the environment and reaction field. Especially in the method for producing ethanol with industrialization in view, acetic acid bacteria are bacteria whose mixing cannot be avoided. Therefore, in order to realize an efficient and / or industrial production method of ethanol, it is required to suppress the growth and acetic acid fermentation of acetic acid bacteria even if they are mixed in.

[0007] Thus, although extensive studies and research have been carried out on bioethanol, for organic compounds other than ethanol, few have been industrialized due to reasons such as the high cost of biomass collection and transportation and the need for advanced technology for conversion into the target organic compound.

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

Means for Solving the Problems

[0009] <1>A method for producing an organic compound, comprising contacting a liquid containing glucose with a microorganism, wherein the liquid containing glucose is a hydrolysis product of cellulose obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and when the liquid containing glucose is subjected to LC analysis using a refractive index detector, the total area value of peaks derived from components having a lower molecular weight than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose. A method for producing an organic compound. <2>The method according to <1>, wherein the organic compound is ethanol, and in the LC analysis, the total area value of peaks derived from components having a lower molecular weight than glucose is 0.01 to 0.2 times the area value of the peak derived from glucose. <3>The method according to <1>, wherein the cellulose contains any one of cellulose derived from board pulp or cellulose derived from pulp sludge, or a mixture thereof. <4>The 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 culturing microorganisms, which consists of a liquid containing glucose, wherein the liquid containing glucose is a hydrolyzate of cellulose obtained by bringing a liquid containing cellulose and water into contact with a solid acid catalyst, and when the liquid containing glucose is subjected to LC analysis using a 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. A composition for culturing microorganisms.

[0010] The production method described in <2> above is synonymous with the following production method [B1]. Therefore, when the production method [A1] of the organic compound of the present invention and the production method [B1] of ethanol, a preferred embodiment of the present invention, are described separately, they are as follows. 〔A1〕A method for producing an organic compound, which includes bringing a liquid containing glucose into contact with a microorganism, wherein the liquid containing glucose is a hydrolyzate of cellulose obtained by bringing a liquid containing cellulose and water into contact with a solid acid catalyst, and when the liquid containing glucose is subjected to LC analysis using a 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. A method for producing an organic compound. 〔A2〕The production method according to [A1], wherein the cellulose includes any one of cellulose derived from board pulp or cellulose derived from pulp sludge, or a mixture thereof. 〔A3〕The production method according to [A1] or [A2], wherein the solid acid catalyst 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 production method according to [A3] or [A4], wherein the carbon element is supported on the surface of the silica. 〔A6〕The production method according to any one of [A3] to [A5], wherein the solid acid catalyst contains a titanium element. 〔A7〕The production method according to [A6], wherein the titanium element is contained in the silica.

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

Advantages of the Invention

[0012] The method for synthesizing an organic compound of the present invention can synthesize various organic compounds including ethanol while using a sugar derived from cellulose (a hydrolysis product of cellulose). That is, an organic compound can be produced from cellulose. In particular, the method for producing ethanol in a preferred embodiment of the present invention can efficiently perform ethanol fermentation while using a sugar derived from cellulose, and can efficiently perform ethanol fermentation while suppressing the growth of acetic acid bacteria and acetic acid fermentation even if acetic acid bacteria are mixed in. As a result, ethanol can be efficiently produced from cellulose.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] In the present invention and this specification, "contamination with acetic acid bacteria" means contamination with acetic acid bacteria, that is, coexistence with acetic acid bacteria, and the factors for contamination or coexistence are not particularly limited. For example, it includes contamination or coexistence as a result of exposure to acetic acid bacteria or contact with acetic acid bacteria. In the present invention and this specification, the numerical range represented by "~" means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value.

[0015] [[Method for Producing Organic Compounds]] The production method A of the present invention (hereinafter sometimes referred to as "the production method A of the present invention") is a method for producing an organic compound including contacting a liquid containing glucose with a microorganism. Further, a method for producing ethanol in a preferred embodiment of the present invention (hereinafter sometimes referred to as "preferred production method B of the present invention") is a method for producing ethanol including contacting a liquid containing glucose with a microorganism. In the present specification, the production method A of the present invention and the preferred production method B of the present invention may be collectively referred to as "the production method of the present invention".

[0016] [Organic compound and ethanol to be produced] The organic compound produced by the production method of the present invention is not particularly limited and can be appropriately determined in consideration of the composition of the liquid containing glucose, the properties of the microorganism, the production ability, and the like. The organic compound produced by the production method A of the present invention is not particularly limited. Examples 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.

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

[0018] [Liquid containing glucose] In the production method of the present invention, as the liquid containing glucose, a hydrolysate of cellulose (sometimes referred to as "cellulose degradation product" or "saccharified liquid") obtained by a method for producing a cellulose degradation product including contacting a liquid containing cellulose and water with a solid acid catalyst is used. As the liquid containing glucose used in Production Method A of the present invention, among the above-mentioned hydrolyzates of cellulose, those in which the total area value of the 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 when subjected to LC analysis using a refractive index detector are used. By bringing the hydrolyzate of cellulose having the origin and composition as described above into contact with microorganisms as the raw material mixture in Production Method A of the present invention, while effectively utilizing cellulose, which is a non-edible biomass that can avoid competition with food applications, various organic compounds can be produced, preferably in a high yield. Further, when producing the above alcohol compound or the like as an organic compound, in Production Method A of the present invention, for example, when using the hydrolyzate of a specific cellulose shown in Experimental Example 7, the growth of acetic acid bacteria and acetic acid fermentation can be suppressed, so that even under conditions or in an environment where acetic acid bacteria may be mixed during production, the excellent production efficiency of organic compounds is not impaired, and the realization of industrialization can also be considered.

[0019] As the liquid containing glucose used in the preferred production method B of the present invention, among the above-mentioned hydrolysis products of cellulose, those in which the total area value of the peaks derived from components having a lower molecular weight than the above-mentioned glucose is 0.01 to 0.2 times the area value of the peak derived from glucose are used. In the preferred production method B of the present invention, by using such a cellulose decomposition product as a raw material mixture for ethanol fermentation, in addition to being able to produce ethanol, the ethanol fermentation by microorganisms proceeds or is promoted while suppressing the occurrence and progress of side reactions. Even if the fermentation time is long, ethanol can be efficiently produced (in a high yield). Moreover, even if acetic acid bacteria coexist during ethanol fermentation, the growth of acetic acid bacteria and acetic acid fermentation by acetic acid bacteria can be suppressed, and the conversion of ethanol converted from the hydrolysis product of cellulose into acetic acid can be suppressed. This is preferably the case when using the hydrolysis product of a specific cellulose shown in, for example, Experimental Example 7. Therefore, the preferred production method B of the present invention can produce bioethanol, which is important for achieving carbon neutrality, in a high yield (high recovery rate) while effectively utilizing cellulose, a non-edible biomass that can avoid competition with food applications. In addition, since the growth of acetic acid bacteria and acetic acid fermentation can be suppressed, even under conditions or in an environment where exposure or contact with acetic acid bacteria may occur during production, the excellent production efficiency of ethanol is not impaired, and the realization of industrialization can also be taken into consideration.

[0020] As described above, the production method of the present invention uses the liquid containing glucose described above. This liquid is the above-mentioned cellulose decomposition product obtained by the decomposition product production method described later and has the composition (component area ratio) described later. The cellulose decomposition product only needs to have the following composition and may contain other components other than the components contained in the cellulose decomposition product. As the other components, those that do not inhibit the production of organic compounds and ethanol fermentation may be used, and examples include water, hemicellulose, lignocellulose, and the like.

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

[0022] On the one hand, the production method B of the present invention uses a cellulose decomposition product having a composition in which the above-mentioned component area ratio is 0.01 to 0.2 times when subjected to LC analysis using an indication refractometer detector. In the preferred production method B of the present invention, when using a cellulose decomposition product with a component area ratio of 0.01 to 0.2 times, its composition and properties are improved, the occurrence or promotion of side reactions is suppressed, and the ethanol fermentation of glucose can be preferentially advanced as the main reaction. Moreover, the growth of acetic acid bacteria and acetic acid fermentation, which could not be suppressed when acetic acid bacteria were conventionally mixed, can be highly suppressed, not only without reducing the yield and selectivity (content rate) of ethanol by ethanol fermentation, but also further increasing them. In terms of further enhancing the efficiency of ethanol fermentation and further suppressing the growth of acetic acid bacteria and acetic acid fermentation to produce ethanol with a high yield and high selectivity, the above-mentioned 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 above-mentioned component area ratio is a value measured by the method described in the examples described later.

[0023] Generally, when cellulose is subjected to a hydrolysis reaction, the obtained decomposition product (composition) contains glucose as the main component and other components. Examples of other components include partial hydrolysis reaction products of cellulose, reaction products such as glucose, for example, oligosaccharides with a degree of polymerization of tetramer or higher, cellotriose, cellobiose, mannose, fructose, saccharides such as levoglucosan, and further, 5-hydroxymethylfurfural (HMF), furfural, and the like. In the present invention, the main component refers to the component with the largest contained mass among the contained components. The cellulose degradation product used in the present invention may contain the above-described partial hydrolysis reaction product of cellulose, reaction products such as glucose, etc., but satisfies the above component area ratio for components having a lower molecular weight than glucose identified by LC analysis (sometimes referred to as "low molecular weight components"). Although the low molecular weight components cannot be uniquely determined by variations such as the origin and composition of the cellulose used in the hydrolysis reaction and the contact conditions between glucose and the solid acid catalyst, typically, among the partial hydrolysis reaction products of cellulose, reaction products such as glucose, etc., components having a lower molecular weight than glucose are included. For example, levoglucosan, HMF, furfural, etc., and further unidentified components can be mentioned. The number of types of low molecular weight components that the cellulose degradation product may contain is not particularly limited and may be singular or plural.

[0024] The content (also referred to as concentration, and the unit of content is mass%) of each low molecular weight component in the cellulose degradation product can be appropriately determined in consideration of the above component area ratio. For example, the content of HMF in the cellulose degradation product is preferably 1.5 mass% or less, more preferably 0.01 - 0.5 mass%, and even more preferably 0.02 - 0.1 mass%.

[0025] Among the above-described low molecular weight components, the cellulose degradation product preferably contains furfural from the viewpoint of suppressing the growth of acetic acid bacteria. The concentration of furfural is not particularly limited as long as it can suppress the growth of acetic acid bacteria. For example, it is preferably 0.035 g / L (0.0035 mass%) or more, and more preferably 0.07 g / L (0.007 mass%) or more. Also, from the viewpoint of having little influence on the growth 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.

[0026] The content of glucose in the cellulose degradation product cannot be uniquely determined by variations such as the origin and composition of the cellulose used in the hydrolysis reaction, and the contact conditions between glucose and the solid acid catalyst. However, it is at least the content such that the above component area ratio is 0.01 to 2.0 times, and is the content falling within the above preferred range, the above more preferred range, or the above even more preferred range. The content of glucose in the cellulose degradation product, in terms of the mass ratio in all components contained in the cellulose degradation composition, is preferably, for example, 50 to 99% by mass, and more preferably 83 to 99% by mass, in terms of being able to efficiently produce ethanol.

[0027] <Method for producing cellulose degradation product> The cellulose degradation product having the above composition can also be prepared by mixing each component such as glucose. However, in the present invention, among the cellulose degradation products obtained by the method for producing a cellulose degradation product (sometimes referred to as a "degradation product production method") described below, those satisfying the above composition are used. This degradation product production method is a production method including bringing a liquid containing cellulose and water into contact with a solid acid catalyst. The above degradation product production method using a solid acid catalyst has high hydrolysis reaction efficiency of cellulose and can suppress excessive occurrence and promotion of each side reaction with respect to the hydrolysis reaction to glucose. Therefore, glucose can be obtained at a selectivity satisfying the above component area ratio and at a high yield.

[0028] (Liquid containing cellulose and water) The liquid containing cellulose and water (sometimes referred to as a "reaction liquid") used in the degradation product production method only needs to contain cellulose and water, and may contain other components. As other components, those that do not inhibit the hydrolysis reaction of cellulose may be used, and examples thereof include hemicellulose, lignocellulose, fatty acids, polymer surfactants, and sulfate bands. The content of cellulose in the reaction solution is not particularly limited, but in terms of the hydrolysis reaction efficiency of cellulose (hereinafter sometimes simply referred to as "hydrolysis reaction efficiency") and the yield of glucose (hereinafter sometimes referred to as "glucose yield"), it is preferably 0.01 to 1 kg, more preferably 0.05 to 0.5 kg, per 1 L of water. In the present invention, when a mixture such as waste pulp is used as the cellulose source, the content of cellulose in the reaction solution refers to the substantial cellulose content (cellulose equivalent amount). The reaction solution is usually obtained as a suspension or dispersion by mixing cellulose and water.

[0029] - Cellulose - The cellulose used in the decomposition product production method may be a carbohydrate (polysaccharide) represented by the molecular formula (C6H 10 O5) n and includes hemicellulose and lignocellulose in addition to cellulose. The cellulose used in the decomposition product production method may be a mixture of cellulose and hemicellulose and / or lignocellulose. However, when lignocellulose is used or included, it is preferable to perform the lignin removal step described later.

[0030] The cellulose may be a synthetic product or a commercially available product, or may be derived from inedible biomass, or may be these wastes or recovered materials. Examples of cellulose derived from inedible biomass include cellulose (lignocellulose) derived from plants such as trees, thinned wood, and lumber, and cellulose obtained by appropriately subjecting chemical pulp obtained by bleaching defatted powder of plants and the like to alkali treatment. Considering the substitutability such as bioethanol derived from edible biomass, it is preferable to use cellulose derived from inedible biomass as the cellulose used in the decomposition product production method. As inedible biomass, plants are typical, but in terms of effective utilization of resources and cost reduction, it is preferable to use their wastes or recovered materials. Examples of inedible biomass include wood powder and wood chips such as trees, thinned wood, and lumber, various pulps (unused products), and further, board pulp and pulp sludge (paper sludge) discharged in the manufacturing process of paper or pulp, and waste pulp such as pulp recovered from waste diapers. In the present invention, the board pulp means a dehydrated product (squeezed product) of pulp discharged during the manufacturing process of paper or pulp, and its shape does not necessarily have to be plate-like. The pulp sludge is a dehydrated product (squeezed product) of solids obtained by adding additives such as sulfuric acid band, anionic flocculant, and cationic flocculant to a slurry-like solution of pulp discharged during the manufacturing process of paper or pulp, and usually has a tendency to have a larger amount of impurities than the board pulp.

[0031] In the production method of the present invention, among the above-mentioned celluloses, waste pulps such as board pulp, pulp sludge, and pulp recovered from waste diapers are particularly preferable in that the recovery amount is abundant and resources can be effectively utilized (cost reduction is also possible), and poisoning of the solid acid catalyst by lignin can be avoided without performing the lignin removal step described later. In the production method of the present invention, as the cellulose to be used, waste pulp is preferable in terms of efficiently producing organic compounds, particularly ethanol, while effectively utilizing resources, and it is more preferable that it is board pulp or pulp sludge. Further, from the viewpoint of efficiently producing organic compounds, particularly ethanol, in addition to the above waste pulp, using synthetic products or commercially available products is also one of the preferable embodiments.

[0032] Cellulose usually shows crystallinity when two or more cellulose molecules are bonded by hydrogen bonds. The decomposition product production method can also use cellulose showing crystallinity (sometimes referred to as "crystalline cellulose"), and can also use cellulose with reduced crystallinity of crystalline cellulose by a conventional method (sometimes referred to as "low-crystalline cellulose" or "microcrystalline cellulose"). As the low-crystalline cellulose, those with partially reduced crystallinity of crystalline cellulose may be used, or those with (substantially) completely disappeared crystallinity may be used. The treatment method for reducing crystallinity is not particularly limited, and it is preferably a treatment that can cut the above hydrogen bond to generate single-stranded cellulose molecules at least partially. Cellulose containing at least partially single-stranded cellulose molecules has a significantly higher hydrolysis reaction efficiency. Specific examples of the treatment method for reducing crystallinity include various methods described in Patent Document 1. Among them, physical methods such as jet mills, hammer mills, ball mills, and bead mill methods are preferable.

[0033] 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 preferably in the form of particles, powder, small flakes, etc. The size of the cellulose is not particularly limited and can be appropriately determined in consideration of hydrolysis reaction efficiency and glucose yield.

[0034] - Water - The water used in the decomposition product production method is not particularly limited, and industrial water, well water, city water, ion-exchanged water, purified water, (ultra) pure water, etc. can be used, and ion-exchanged water, purified water, (ultra) pure water are preferable.

[0035] (Solid acid catalyst) The solid acid catalyst used in the decomposition product manufacturing 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, and niobic acid, inorganic solid acids into which acidic groups such as sulfonic groups are introduced by acidification treatment, resins, or carbonaceous materials, etc. can be mentioned. In the decomposition product manufacturing method, as the solid acid catalyst, a solid acid catalyst containing carbon element and silica and having a sulfonic group as a surface functional group is preferably used in that it can hydrolyze cellulose with high efficiency and obtain a cellulose decomposition product satisfying 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 sulfonic group as a surface functional group. Hereinafter, the above preferable solid acid catalyst and more preferable solid acid catalyst are collectively referred to as "suitable solid acid catalyst". The suitable solid acid catalyst has a carbon element and a sulfonic group (-SO3H), or a carbon element, a titanium element, and a sulfonic group on a substrate mainly composed of silica (also referred to as "silica substrate"). The shape of the suitable solid acid catalyst is not particularly limited, but in terms of hydrolysis reaction efficiency and glucose yield, it is preferably in the form of particles, powder, flakes, etc. The size (average particle diameter) of the suitable solid acid catalyst is not particularly limited, but for example, it is preferably 0.1 to 10000 μm. The average particle diameter is the value measured by the method described in the examples below. The substrate is preferably a porous substrate (porous body) in that it can enhance the hydrolysis reaction efficiency and glucose yield, more preferably a porous body having mesopores (mesoporous body), and even more preferably mesoporous silica. In the present invention, mesopores refer to pores having an average pore diameter of 2 to 50 nm.

[0036] When the substrate is a porous substrate, its properties 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, from the viewpoints 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, from the viewpoints 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, from the viewpoints of hydrolysis reaction efficiency and glucose yield. The average pore diameter, specific surface area, and total pore volume of the porous substrate are the values measured by the method described in the examples below.

[0037] In a preferred solid acid catalyst, usually, the substrate contains a carbon element. In the present invention, that the substrate contains a carbon element means that the carbon element is present on the surface and / or inside of the substrate. For example, the mode in which the carbon element is supported or adsorbed on the surface of the substrate (carbon element-supported substrate), the mode in which the carbon element is contained inside the substrate (the mode in which the substrate is formed of a composite of silica and a carbon element), the mode in which the carbon element is present on the surface and inside of the substrate, etc. can be mentioned. The form (structure) containing a carbon element in the solid acid catalyst is preferably the mode in which the carbon element is supported or adsorbed on the surface of the substrate (carbon element-supported substrate) in terms of being able to increase the hydrolysis reaction efficiency and glucose yield by bonding or fixing the surface functional groups described below to the surface. In the present invention, the surface of the substrate usually refers to the outer surface. However, when the substrate is a porous body, it includes the inner surface of the pores in addition to the outer surface. On the other hand, the inside of the substrate refers to the portion not exposed on the surface. The carbon element content in the suitable solid acid catalyst is not particularly limited, but in terms of enhancing the hydrolysis reaction efficiency and glucose yield, it is preferably 1 to 70% by mass, more preferably 5 to 50% by mass, based on the mass of the suitable solid acid catalyst (excluding the mass of the sulfo group). The carbon element content in the suitable solid acid catalyst shall be the value measured by the measurement method described in the examples below.

[0038] In the above-mentioned more preferable solid acid catalyst, usually, its substrate contains titanium element. In the present invention, that the substrate contains titanium element means that the substrate has (exists) titanium element on the surface and / or inside of the substrate. For example, the mode of supporting or adsorbing titanium element on the surface of the substrate (titanium element-supported substrate), the mode in which the substrate contains titanium element inside (the mode in which the substrate is formed of a composite of silica and titanium element), the mode in which the substrate has titanium element on the surface and inside, etc. can be mentioned. When the substrate has titanium element on its surface and / or inside, the hydrolysis reaction efficiency and glucose yield can be enhanced. The titanium element content in the more preferable solid acid catalyst is not particularly limited, but in terms of enhancing the hydrolysis reaction efficiency and glucose yield, it is preferably 0.1 to 10% by mass, more preferably 0.5 to 5% by mass, based on the mass of the more preferable solid acid catalyst (excluding the mass of carbon atoms and sulfo groups). The carbon element content in the more preferable solid acid catalyst shall be the value measured by the measurement method described in the examples below.

[0039] The mode in which the more preferable solid acid catalyst contains carbon element and titanium element can be appropriately combined with the above-mentioned mode containing carbon element and the mode containing titanium element. However, in terms of enhancing the hydrolysis reaction efficiency and glucose yield, the mode in which titanium element is contained on the surface and / or inside of the substrate and the form in which carbon element is supported or adsorbed on the surface of the substrate are combined (this form of solid acid catalyst is also referred to as a carbon element-supported silica-titanium composite mesoporous body).

[0040] A suitable solid acid catalyst has a sulfo group as a surface functional group. In the present invention, that the 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 the carbon element. As long as this sulfo group can promote the hydrolysis reaction of cellulose, part or all of it may be in the form of a salt. The content of the sulfo group contained in the suitable solid acid catalyst is not particularly limited, but in terms of enhancing the hydrolysis reaction efficiency and glucose yield, it is preferably 0.01 to 2 mmol / g, more preferably 0.05 to 1 mmol / g, as the amount present per 1 g of the suitable solid acid catalyst. The content of the sulfo group in the suitable solid acid catalyst is the value measured by the measurement method in the examples described later.

[0041] Commercially available products may be used as the solid acid catalyst, but since a suitable solid acid catalyst has a carbon element, preferably a titanium element, and a sulfo group, it is preferable to use a suitably synthesized one. The method for introducing a carbon element into the substrate is not particularly limited, and various known methods can be applied. For example, as a method for supporting or adsorbing a carbon element on the surface of the substrate, there is a method of carbonizing the carbon source after mixing the substrate and 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 a carbon atom, and usually an organic compound is used. For example, saccharides such as sucrose, alcohols such as furfuryl alcohol, hydrocarbon compounds, and alkylene oxides are preferably used. The method for introducing a titanium element into the substrate is not particularly limited, and various known methods can be applied. For example, as a method for supporting or adsorbing a titanium element on the surface of the substrate, the same method as the method for supporting or adsorbing the above carbon element can be mentioned except for using a titanium-containing compound. On the other hand, as a method for incorporating a titanium element into the inside or inside and surface of the substrate, there is a method of forming a 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 the substrate, preferably a substrate having carbon element supported or adsorbed on its surface, with sulfuric acid can be mentioned. Specifically, the method described in Green Chem., 2010, 12, 1560-1563 can be mentioned.

[0042] As a method for producing a silica porous body as the substrate of the solid acid catalyst, a sol-gel method (also referred to as "molecular templating method" or "template method") using a silica source (silica precursor compound) and an amphiphilic compound as a template can be mentioned. Hereinafter, as a more preferable solid acid catalyst, a method for producing a carbon element-supported silica-titanium composite mesoporous body having a sulfo group as a surface functional group by a sol-gel method will be described by way of example. A general sol-gel method is, for example, a method in which a silica precursor compound is subjected to a sol-gel reaction (hydrolysis reaction and condensation reaction) around self-organized micelle particles formed by a cationic surfactant to form an inorganic-organic nanocomposite, and then this is calcined or acid-treated (to remove the cationic surfactant). A more preferable solid acid catalyst is first produced by using the above sol-gel method to produce a silica-titanium composite mesoporous body in which titanium element is contained in the surface and / or inside of the substrate. At this time, it can be carried out in the same manner as the general sol-gel method except for using a titanium precursor compound described later together with the silica precursor compound (coexisting the silica precursor compound and the titanium precursor compound). Therefore, the types of each step, the operations and reaction conditions in each step in the sol-gel method applied when producing the silica-titanium composite mesoporous body can be appropriately determined with reference to the general sol-gel method. For example, the method described in Green Chem., 2010, 12, 1560-1563, and a series of steps, the operations and reaction conditions in the synthesis method in the examples described later can be used as a reference. In the above sol-gel method, by changing the type of silica precursor compound, particularly the type of amphiphilic compound, or the preparation conditions such as solvent composition, temperature, and time, the characteristics or physical properties of the porous material such as the size of the mesopores (average pore diameter) and the shape of the mesopores, and the skeletal shape of the porous material can be adjusted or changed, which is the same as the general sol-gel method.

[0043] As the amphiphilic compound used as a template, the compounds usually used in the general sol-gel method can be used without particular limitation. Usually, amphiphilic surfactants are 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, the surfactants usually used as templates in the sol-gel method can be used without particular limitation, and examples include ammonium-based cationic surfactants. As the ammonium-based cationic surfactant, alkylammonium salts are preferred, tetraalkylammonium salts are preferred, ammonium salts containing a long-chain (6 or more carbon atoms) straight-chain alkyl group are more preferred, and mono-long-chain straight-chain alkyltri-short-chain (4 or less carbon atoms) alkylammonium salts are particularly preferred. The anion forming the quaternary ammonium salt is not particularly limited, and examples include inorganic anions such as hydroxide ions, halide ions, and perhalate ions. In addition, examples of the representative of the silica mesoporous material prepared by the sol-gel method using a cationic surfactant include MCM-41, and examples of the representative of the silica porous material prepared by the sol-gel method using a block copolymer include SBA-15.

[0044] As the silica precursor compound, compounds commonly used in the general sol-gel method can be used without particular limitation. Examples include silicon halides, hydroxides, alkoxides, etc., and silicon alkoxides and / or alkyl alkoxides are preferred. The carbon number of 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 above sol-gel method, a titanium-containing compound is used together with the silica precursor compound. Examples of the titanium-containing compound include titanium precursor compounds that are converted into titanium elements together with the silica precursor compound by the sol-gel method. Preferred examples of the titanium precursor compound include titanium halides, hydroxides, alkoxides, etc., and titanium alkoxides and / or titanium alkyl alkoxides (alkoxytitanium) are more preferred. The carbon number of the alkyl group forming the alkyl alkoxide is not particularly limited, but is preferably 1 to 6, and more preferably 1 to 3.

[0045] Next, a carbon element is supported or adsorbed on the surface of the obtained silica-titanium composite mesoporous body. The method for supporting or adsorbing the carbon element is not particularly limited and is as described above. A sulfo group is introduced as a surface functional group into the carbon element-supported silica-titanium composite mesoporous body thus obtained. The method for introducing the sulfo group is not particularly limited and is as described above. The silica-titanium composite mesoporous body, the carbon element-supported silica-titanium composite mesoporous body, and / or the carbon element-supported silica-titanium composite mesoporous body with a sulfo group introduced can be appropriately pulverized and crushed.

[0046] As a preferred method for producing a solid acid catalyst, a method for producing carbon element-supported mesoporous silica having a sulfo group as a surface functional group by the sol-gel method includes, in the sol-gel method, a production method similar to the above-described more preferred method for producing a solid acid catalyst, except that a silica precursor compound is used without using a titanium precursor compound.

[0047] (Step of bringing a liquid containing cellulose and water into contact with a solid acid catalyst) The method for producing a decomposition product includes bringing a liquid (reaction liquid) containing cellulose and water into contact with a solid acid catalyst. By this contact, cellulose is hydrolyzed in the presence of the solid acid catalyst to be converted into glucose, and a cellulose decomposition product can be obtained. Each component used in the step of bringing them into contact may be one kind or two or more kinds.

[0048] The hydrolysis reaction of cellulose occurring in the step of bringing them into contact is generally considered to involve a hydrolysis reaction from cellulose to oligosaccharide and a hydrolysis reaction from oligosaccharide to glucose. Therefore, in the step of bringing them into contact, as the hydrolysis treatment (saccharification treatment) of cellulose, a sugar-containing liquid containing glucose (also referred to as a "saccharified liquid") can be obtained. This sugar-containing liquid usually has the above composition (satisfies the component area ratio).

[0049] In the method for producing a decomposition product, when a suitable solid acid catalyst is used, cellulose can be efficiently hydrolyzed to obtain a cellulose decomposition product containing glucose as a main component. At this time, the conversion rate of cellulose, the selectivity of glucose (mass ratio of the content of glucose to the content of other components), etc. vary depending on the type of cellulose, the type and amount of the solid acid catalyst used, and further hydrolysis reaction conditions, etc., and cannot be uniquely determined. However, at least the yield of glucose ((amount of glucose obtained) / (charged amount of cellulose)×100 (%)) can reach 45% or more.

[0050] In the method for producing a decomposition product, a liquid (reaction liquid) containing cellulose and water is brought into contact with a solid acid catalyst under appropriate reaction conditions, usually under heating. The contacting method may be any method capable of bringing into contact the three components of cellulose, water, and a solid acid catalyst, such as a method of bringing a pre-prepared reaction solution into contact with the solid acid catalyst, or a method of bringing cellulose, water, and the solid acid catalyst into contact without pre-preparing the reaction solution (a method of bringing cellulose and the solid acid catalyst into contact in the presence of water). In the step of contacting, when the reaction solution and the solid acid catalyst are described as being contacted or introduced, etc., it includes a mode in which cellulose and water are separately contacted or introduced with the solid acid catalyst instead of the reaction solution. As the contacting step, for example, after charging the reaction solution and the solid acid catalyst into a sealed container, heating is carried out. The heating temperature (reaction temperature) of the hydrolysis reaction is not particularly limited, but can be, for example, 110 to 200°C. In terms of enhancing the hydrolysis reaction efficiency of cellulose and suppressing the by-production of other components to increase the yield of glucose, the heating temperature is preferably 120 to 180°C, and more preferably 120 to 150°C. The heating time (reaction time) is appropriately determined according to the heating temperature, the hydrolysis reaction efficiency (conversion rate) of cellulose, the glucose yield, etc., and can be, for example, 1 to 48 hours. The inside of the sealed container under heating may be at normal pressure, but usually, it becomes a pressurized state where the partial pressure of water vapor exceeds, for example, 0.1 MPa. In the present invention, the hydrolysis reaction can also be carried out in a positively pressurized environment. The pressure at this time is not particularly limited and can be, for example, exceeding 0.1 MPa and 20 MPa or less, and preferably 0.1 to 10 MPa. The reaction environment (atmosphere) is not particularly limited and can be an inert gas atmosphere, etc., but can also be an air atmosphere or a water vapor atmosphere. Considering industrial production, the reaction environment is preferably an air atmosphere and / or a water vapor atmosphere.

[0051] The usage amounts of the reaction solution and the solid acid catalyst are not particularly limited, but at least the amount of water in the reaction system (the amount of water present) should be not less than the amount required for the hydrolysis reaction of cellulose. The amount of water in the reaction system is preferably, for example, 0.1 to 1000 times by mass, and more preferably 1 to 100 times by mass, based on the amount of cellulose present, in terms of the miscibility (stirrability) and handleability of the reaction system, as well as the hydrolysis reaction efficiency and glucose yield. In the contacting step, water can also be added separately from the reaction solution. The amount of water in the above reaction system refers to the amount of water derived from the reaction solution. When water is added, the total amount of the water derived from the reaction solution and the added water is taken. The usage amount of the solid acid catalyst can be appropriately determined in consideration of reaction conditions, hydrolysis reaction efficiency, glucose yield, etc. For example, it is preferably 0.01 to 10 times by mass, and more preferably 0.1 to 5 times by mass, based on the amount of cellulose present.

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

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

[0054] [Post-treatment step] In the method for producing a decomposition product, after the above contacting step, the reaction mixture 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 solid-liquid separating the reaction mixture. The reaction mixture can be subjected to solid-liquid separation without cooling, but it is preferably cooled to suppress further reaction of glucose or in consideration of workability, safety, etc. The reaction mixture can be cooled by natural cooling or by using various refrigerants. The cooling temperature of the reaction mixture is not particularly limited and can usually be 100°C or lower, preferably 80°C or lower in terms of suppressing further reaction of glucose and maintaining a high yield of glucose. Considering workability, safety, etc., it is preferably near room temperature (for example, 15 to 40°C). The lower limit temperature of the cooling temperature can be, for example, 0°C or higher, preferably 15°C or higher, from the viewpoint of workability. The cooling rate at this time can be determined appropriately and is preferably decreased by 0.1 to 10°C per minute. The cooling time can be determined appropriately according to the heating temperature, cooling rate, etc. The appropriately cooled reaction mixture is subjected to solid-liquid separation to separate a sugar-containing liquid (cellulose decomposition product) having glucose as a main component as a liquid phase and at least a solid acid catalyst and unreacted cellulose as a solid phase. The method for solid-liquid separation is not particularly limited, and examples thereof include a filtration method, a centrifugation method, and a precipitation method.

[0055] [Other steps] In the decomposition product production method, steps other than the above-mentioned contacting step and post-treatment step can also be performed. For example, a step of purifying the sugar-containing liquid separated and recovered so as to satisfy the above-mentioned component area ratio, a step of adjusting the composition of the separated and recovered sugar-containing liquid, a step of isolating the solid acid catalyst and unreacted cellulose from the separated and recovered solid phase, a step of washing and regenerating the isolated solid acid catalyst, when using the above-mentioned waste as a cellulose source, a step of removing impurities, contaminants, etc. in the waste, a step of removing lignin in the cellulose, and the like can be mentioned. The method for isolating and purifying the sugar-containing solution can be applied without particular limitation to various known isolation methods and various purification methods. Also, in the step of adjusting the composition of the separated and recovered sugar-containing solution, the amount of components can be adjusted within the range of the above component area ratio by adding glucose to the separated and recovered sugar-containing solution or the like. Further, 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, as the number of uses increases, the catalytic activity of the solid acid catalyst usually gradually decreases. Therefore, in consideration of a decrease in catalytic activity, hydrolysis reaction efficiency, or glucose yield, the solid acid catalyst can also be washed and / or regenerated. The washing and regeneration methods are not particularly limited.

[0056] The decomposition product production method can decompose cellulose into glucose by a simple and safe step of bringing the reaction solution into contact with a solid acid catalyst, and desirably obtain a cellulose decomposition product that satisfies the above 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 step. Further, since the occurrence and promotion of each side reaction can be suppressed during the hydrolysis reaction of cellulose, glucose can be obtained with high selectivity (high purity), and a cellulose decomposition product that satisfies the above composition can be obtained. In this regard, 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 the cellulose source, the hydrolysis reaction efficiency of cellulose and the yield of glucose can be further increased, and the effective utilization of non-edible biomass resources becomes possible. Therefore, the decomposition product production method can efficiently produce glucose from cellulose at low cost, and is also suitable for the industrialization of the cellulose hydrolysis reaction and the bioethanol production method.

[0057] [Step of contacting the liquid containing glucose with microorganisms (fermentation step)] The production method of the present invention involves bringing a liquid containing glucose having the above-described composition (hereinafter sometimes referred to as a glucose solution) into contact with a microorganism. In this contacting step, the water content and pH are appropriately adjusted so as to be conditions suitable for synthesis methods of various organic compounds, for example, ethanol fermentation. Usually, a microorganism is inoculated into the glucose solution, and glucose is converted into an organic compound such as ethanol by the action of the microorganism. By mixing the glucose solution and the microorganism and culturing for a certain period, the liquid in which part or all of the glucose is converted into an organic compound such as ethanol is conveniently referred to as a "fermented liquid".

[0058] <Microorganism> The microorganism used in the production method of the present invention only needs to have the properties and characteristics of synthesizing and producing various organic compounds, and the microorganism used in the preferred production method B of the present invention only needs to have ethanol productivity. Such microorganisms are not particularly limited, and examples include yeast, eubacteria, or archaebacteria.

[0059] Examples of the microorganisms used in Production Method A of the present invention include, for example, the genus Clostridium, the genus Zymomonas, the genus Escherichia, the genus Salmonella, the genus Serratia, the genus Erwinia, the genus Klebsiella, the genus Shigella, the genus Rhodococcus, the genus Pseudomonas, the genus Bacillus, the genus Lactobacillus, the genus Lactococcus, the genus Enterococcus, the genus Alcaligenes, the genus Klebsiella, the genus Paenibacillus, the genus Arthrobacter, the genus Corynebacterium, the genus Brevibacterium, the genus Schizosaccharomyces, the genus Issatchenkia, 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, and the like. Further, when the organic compound to be produced in Production Method A of the present invention is alcohol, particularly ethanol, the microorganisms described below used in the preferred Production Method B of the present invention can also be used. As the microorganism used in Production Method A of the present invention, for example, a microorganism capable of producing the organic compound to be produced can be appropriately selected from the above-mentioned microorganisms and used.

[0060] In the preferred production method B of the present invention, the yeast used is preferably capable of fermenting saccharides (hexoses and pentoses). Specifically, examples of the yeast include yeasts belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts belonging to the genus Pichia such as Pichia stipitis, yeasts belonging to the genus Candida such as Candida shihatae, yeasts belonging to the genus Pachysolen such as Pachysolen tannophilus, yeasts belonging to the genus Issatchenkia such as Issatchenkia orientalis, and yeasts belonging to the genus Kluyveromyces such as Kluyveromyces marxianus. Preferably, yeasts belonging to the genus Saccharomyces and the genus Issatchenkia are used, and more preferably, Saccharomyces cerevisiae and Issatchenkia orientalis are used. In addition, genetically modified yeasts produced using genetic recombination technology can also be used. As the genetically modified yeast, any yeast capable of fermenting saccharides (hexoses and pentoses) can be used without particular limitation, and preferably, a yeast capable of fermenting hexoses and pentoses simultaneously can be used.

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

[0062] As the microorganism used in the production method of the present invention, in addition to the above, those described in paragraph

[0043] of Patent Document 1 can also be used, and the content thereof is incorporated as it is into the description of this specification as a part thereof. In the production method of the present invention, one or more kinds of microorganisms can be used, and it is preferably used as a culture solution. The culture solution of the microorganism can be prepared by ordinary methods and conditions.

[0063] The glucose solution contains a cellulose decomposition product. In addition to the cellulose decomposition product, it may also contain nutrient sources necessary for the growth and activity of microorganisms. The nutrient source is not particularly limited, and examples thereof include yeast extract and polypeptone. In addition, as the nutrient source, those described in paragraph

[0043] of Patent Document 1 can be used, and the content thereof is incorporated as it is into the description of this specification as a part thereof. In addition, the glucose solution may contain components usually used according to the microorganisms to be used, such as a pH adjuster, a buffer, a chelating agent, an antibiotic, an expression inducer, and an antifoaming agent. In the fermentation process, since the influence of acetic acid bacteria can be suppressed, acetic acid bacteria may be mixed in the glucose solution. The acetic acid bacteria are not particularly limited, and various known acetic acid bacteria such as the genus Acetobacter and the genus Gluconacetobacter can be mentioned. However, the acetic acid bacteria that may be mixed in the glucose solution are preferably about 0.2% by mass or less as the inoculation amount of the acetic acid bacteria culture solution (OD660 = 2).

[0064] In the fermentation process, the method and conditions (for example, fermentation method, fermentation conditions) for bringing the glucose solution into contact with the microorganism are not particularly limited, and the atmosphere, temperature, pH, time, etc. can be appropriately selected and set according to the microorganism to be used. In Production Method A of the present invention, as the method and conditions for bringing the glucose solution into contact with the microorganism, for example, the following methods and conditions in Production Method B of the present invention can be applied.

[0065] For example, regarding the method and conditions of contacting in the preferred production method B of the present invention (including the aspect of producing ethanol in the production method A of the present invention), reference can be made to known ethanol fermentation methods and conditions, specifically the methods and conditions described in Patent Document 1, etc., and they can be appropriately selected and determined. An example thereof will be described below.

[0066] The glucose solution usually contains glucose and water derived from the glucose solution. The content (concentration) of glucose in the glucose solution usually coincides with the above content of glucose in the cellulose decomposition product. However, when appropriately adding water to adjust the concentration in the glucose solution, the concentration of glucose in the glucose solution will be lower than the concentration of glucose in the cellulose decomposition product. The content of glucose in the glucose solution at this time can be, for example, 0.1 to 10% by mass, and preferably 1 to 5% by mass in terms of efficiently producing ethanol. The water content in the glucose solution can be, for example, 90 to 99.9% by mass, and preferably 95 to 99% by mass in terms of efficiently producing ethanol.

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

[0068] The ethanol content produced in the fermentation broth is not particularly limited and is appropriately determined by factors such as the ethanol-producing ability of the microorganisms 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 content in the fermentation broth cannot be increased to such an extent that it affects the survival and activity of the microorganisms. For example, preferably, the upper limit is 150 g / L or less.

[0069] The atmosphere of the fermentation process can be appropriately determined according to the microorganisms used. It can be an aerobic atmosphere during contact, but it can also be an anaerobic atmosphere during fermentation.

[0070] The fermentation process may be carried out once or divided into multiple times. When carried out in multiple times, the fermentation conditions in each fermentation process may be the same or different. The fermentation process can be carried out batchwise or continuously using a fermentation tank. As the fermentation tank, those of any shape can be used. For example, various fermentation tanks such as stirred type, air-lift type, bubble column type, loop type, open bond type, and photobioreactor type can be used.

[0071] As described above, a fermentation mixture containing microorganisms and organic compounds such as ethanol can be obtained. Regarding the manufacturing method of the present invention, when focusing on the manufacturing process, it can also be said to be a method for manufacturing organic compounds such as ethanol from cellulose, including a step of contacting with the above-mentioned decomposition product manufacturing method.

[0072] [Post-treatment process] In the production method of the present invention, the reaction mixture (fermentation mixture) obtained after the above-described contacting step can be post-treated to obtain an organic compound such as ethanol. Examples of the post-treatment of the fermentation mixture include a step of separating the solid and liquid of the fermentation mixture and a step of isolating ethanol. The fermentation mixture may be directly subjected to a step of isolating an organic compound such as ethanol. However, usually, the solid and liquid are separated to obtain a liquid containing an organic compound such as ethanol as the liquid phase and a solid content containing microorganisms as the solid phase. The method for separating the solid and liquid is not particularly limited, and examples thereof include a filtration method, a centrifugation method, and a precipitation method. In the step of isolating an organic compound such as ethanol, usually, the liquid containing the organic compound such as ethanol separated from the solid and liquid can be isolated by distillation. As the distillation method and conditions, a normal distillation method can be applied, and examples thereof include atmospheric distillation and vacuum distillation.

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

[0074] The production method A of the present invention can produce a target organic compound by microorganisms from the cellulose decomposition product satisfying the above component area ratio, preferably in a high yield. Moreover, the preferred production method B of the present invention can produce ethanol efficiently (in high yields) from a cellulose decomposition product that satisfies the above component area ratio by allowing ethanol fermentation by microorganisms to proceed or be promoted while suppressing the occurrence and progression of side reactions. Even if the fermentation time is long, ethanol can be produced efficiently. In addition, a cellulose decomposition product prepared using a preferred solid acid catalyst satisfies the above component area ratio and can further promote the progress of ethanol fermentation more efficiently. 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 when acetic acid bacteria coexist during ethanol fermentation, and can suppress the conversion of ethanol converted from the hydrolyzate of cellulose into 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 as a cellulose source is used, a cellulose decomposition product with an improved glucose content can be obtained while satisfying the above component area ratio, enabling the effective utilization of non-edible biomass resources. 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 the method for producing bioethanol.

[0075] [[Composition for Microbial Culture]] The composition for microbial culture of the present invention consists of a liquid containing glucose. The liquid containing glucose is a hydrolyzate of cellulose obtained by bringing a liquid containing cellulose and water into contact with a solid acid catalyst. When the liquid containing glucose is subjected to LC analysis using a refractive index detector, the total area value of the peaks derived from components with a lower molecular weight than glucose is 0.01 to 2.0 times the area value of the peak derived from glucose. The cellulose decomposition product constituting the composition for microbial culture of the present invention is the same as the cellulose decomposition product used in the production method of the present invention, for example, the production method A of the present invention. The composition for culturing microorganisms of the present invention may contain components commonly used depending on the microorganisms to be used, components commonly used in culture (medium), and the like. Examples of such other components include nutrient sources necessary for the growth and activity of microorganisms, pH adjusters, buffers, chelating agents, antibiotics, expression inducers, antifoaming agents, and the like. Each component is not particularly limited and is as described above. Further, since the composition for culturing microorganisms can suppress the influence of acetic acid bacteria, the above-mentioned acetic acid bacteria may be mixed therein.

[0076] The total content of the cellulose degradation product in the composition for culturing microorganisms is not particularly limited and can be appropriately determined in consideration of the production efficiency of organic compounds and the like. For example, it can be 0.1% by mass or more. Among them, the content of glucose in the composition for culturing microorganisms can be 0.1 to 10% by mass in terms of the production efficiency of organic compounds and the like. The total content of other components in the composition for culturing microorganisms is not particularly limited and can be appropriately determined in consideration of the production efficiency of organic compounds and the like. The content of acetic acid bacteria in the composition for culturing microorganisms is preferably about 0.2% by mass or less as the inoculation amount of the acetic acid bacteria culture solution (OD660 = 2).

[0077] Since the composition for culturing microorganisms of the present invention contains a cellulose degradation product having the above specific component area ratio and can produce various organic compounds, it is suitably used as a medium in the production method of the present invention.

Examples

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

[0079] [Preparation of solid acid catalyst] <Experimental Example 1. Preparation of solid acid catalyst for cellulose degradation> (1) Preparation of silica porous body (First step) 625.5 g of an aqueous solution of 16 mass% hexadecyltrimethylammonium hydroxide was stirred, and a mixed solution of 9.25 g of titanium tetraisopropoxide and 50.0 g of 2-propanol was added dropwise thereto at room temperature (25 °C). After stirring for 30 minutes, 190.5 g of tetramethoxysilicate was added dropwise. After adding 5.0 g of 2-propanol, stirring was continued for 3 hours, and as a result, a precipitate was formed.

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

[0081] (Third step) 20 g of the dried solid obtained in the second step was placed in a flask, and a mixed solution of 200 mL of methanol and 10 g of concentrated hydrochloric acid (content 36 mass%) was added (acid treatment). Heating was continued at the reflux temperature for 1 hour while stirring, and after allowing to cool, 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 finally filtered solid was dried under reduced pressure at 120 °C and 10 mmHg for 1.5 hours.

[0082] (Fourth step) The solid obtained in the third step was heat-treated (calcined) at 600 °C for 3 hours under a flowing air atmosphere.

[0083] (Fifth step) The calcined solid obtained in the fourth step was pulverized 8 times with a hammer mill to obtain a powdery silica porous body (silica-titanium composite mesoporous body) having an average particle diameter of 10 μm. The average particle diameter was measured using a laser diffraction / scattering particle size distribution analyzer (LA-950 manufactured by Horiba, Ltd.) with ion-exchanged water as the dispersion medium and calculated on a volume basis.

[0084] (2) Preparation of silica porous body-carbon composite 3 g of the silica porous body and 30 g of propylene oxide (reagent manufactured by Fujifilm Wako Pure Chemical Corporation) serving as a carbon source were added into an autoclave equipped with a stirrer. The inside of the autoclave was pressurized with nitrogen up to 1 MPa-G, and the operation of depressurizing to normal pressure was repeated three times to replace the inside of the autoclave with nitrogen. The autoclave was immersed in an oil bath, the rotation speed of the stirrer was set to 500 rpm, and while stirring the inside of the autoclave, the temperature of the oil bath was adjusted so that the internal temperature of the autoclave became 100 °C. After stirring for 6 hours after the internal temperature of the autoclave reached 100 °C, the solid obtained by filtration was air-dried, and further in an electric furnace, under a nitrogen atmosphere, the temperature was raised to 500 °C at 6 °C / min and heat-treated at 500 °C for 2 hours, thereby preparing 3.2 g of a silica porous body-carbon composite as a carbon element-supported silica-titanium composite mesoporous body.

[0085] (3) Preparation of solid acid catalyst 1 g of the silica porous body-carbon composite and 20 mL of 95 mass% sulfuric acid were added to an autoclave and treated at 150 °C for 15 hours. After the treatment, washing with water was performed at 50 °C until the pH of the washing liquid showed the same value 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 element-supported silica-titanium composite mesoporous body having a sulfo group as a surface functional group.

[0086] <Experimental Example 2. Pore analysis of silica porous body> The powdery silica porous body (silica-titanium composite mesoporous body) 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 the total pore volume were measured by measuring the nitrogen adsorption isotherm at the liquid nitrogen temperature with BELSORP MINI X (manufactured by MicrotracBEL) after degassing the silica porous body at 120 °C for 2 hours under vacuum, and were determined from the BET method and the value of the nitrogen adsorption amount near the relative pressure (P / P0 = 0.96), respectively. The average pore diameter was calculated by the following (Equation 1) from the value of the total pore volume and the value of the specific surface area. (Equation 1): Average pore diameter (nm) = 4 × total pore volume (mL / g) / specific surface area (m2 / g)×1000

[0087] In addition, when the Ti concentration in the powdery silica porous body was analyzed by XRF, it was 1.5 mass%.

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

[0089] <Experimental Example 4. Measurement of sulfonic group content of solid acid catalyst> The sulfonic group content of the solid acid catalyst was measured by titration using a potentiometric automatic titrator (manufactured by Kyoto Electronics). 50 mg of the solid acid catalyst was weighed into a screw tube bottle, 30 mL of an NaCl aqueous solution prepared to 0.05 N 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, 10 mL of the filtrate was collected using a whole pipette and transferred to a 50 mL beaker. 2 drops of phenolphthalein solution were added to the 50 mL beaker using a Pasteur pipette, and 0.005 N NaOH aqueous solution was added dropwise by 0.1 mL each for titration. From the equivalence point of the titration result, the Na + exchange amount was calculated to determine the sulfonic group content. The sulfonic group content of the thus obtained solid acid catalyst was 0.09 mmol / g.

[0090] <Experimental Example 5. Method for obtaining and preparing raw materials (board pulp, pulp sludge)> The board pulp as the raw material was obtained by dehydrating the raw material used in the tissue paper making machine in the papermaking process. The pulp sludge as the raw material was obtained by adding sulfuric acid band, anionic flocculant, and cationic flocculant to the slurry-like solution of pulp discharged from the tissue paper making machine in the papermaking process to solidify it and then dehydrating it.

[0091] <Experimental Example 6. Pretreatment method of raw materials> After dehydrating the sheet pulp obtained in the above Experimental Example 5., it was dried at 60 °C for 12 hours and then vacuum dried at 120 °C for 12 hours. After vacuum drying, it was finely cut with scissors to obtain the pretreated sheet pulp. After dehydrating the pulp sludge obtained in the above Experimental Example 5., it was dried at 60 °C for 12 hours and then vacuum dried at 120 °C for 12 hours. After vacuum drying, it was finely cut with scissors to obtain the pretreated pulp sludge.

[0092] <Experimental Example 7. Preparation of Cellulose Hydrolysate> (1) Hydrolysis Reaction of Cellulose As the cellulose of the reaction substrate, the one treated as follows was used. 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. It was set on a tabletop pot mill turntable and ball milled at 200 rpm for 120 hours. This operation was carried out in 2 batches, and 9 g of ball milled cellulose was obtained. Next, the ball milled cellulose, a solid acid catalyst, and purified water were added to an autoclave equipped with a stirrer. While stirring at 300 rpm, the temperature was raised from room temperature to 150 °C in about 30 minutes, and then the hydrolysis reaction of cellulose was carried out in 2 batches at 150 °C for 24 hours (internal pressure 0.5 MPa). In the first batch of ball milling, the addition amounts of cellulose, solid acid catalyst, and purified water were 2 g, 2 g, and 25 g, respectively. Similarly, in the second batch of ball milling, the addition amounts of cellulose, solid acid catalyst, and purified water were 3 g, 3 g, and 38 g, respectively. After the reaction was completed, the autoclave was cooled to room temperature. Then, the reaction solution was filtered to separate it into a liquid and a solid, and the two batches were mixed to obtain a cellulose hydrolysate and a solid residue. In each experimental example, when simply referred to as "cellulose hydrolysate", it means the cellulose hydrolysate prepared in this Experimental Example 7.(1).

[0093] (2) Hydrolysis Reaction of Sheet Pulp As the sheet pulp of the reaction substrate, the one treated as follows was used. 1 kg of zirconia balls with a diameter of 1 cm and 5 g of the pretreated (finely chopped) plate pulp obtained in the above Experimental Example 6 were placed in a ceramic pot mill. It was set on a desktop pot mill turntable and ball milled at 200 rpm for 120 hours. This operation was performed in multiple batches, and the plate pulp adhering to the wall surface of the ceramic bottle was collected as a sample, and 9 g of the ball milled plate pulp was obtained. Next, the ball milled plate pulp, a solid acid catalyst, and purified water were added to an autoclave equipped with a stirrer, and while stirring at 300 rpm, the temperature was raised from room temperature to 150 °C in about 30 minutes, and then the hydrolysis reaction of the plate pulp was carried out at 150 °C for 24 hours (internal pressure 0.5 MPa) in 3 batches. In the first batch of ball milling treatment, the addition amounts of the plate pulp, the solid acid catalyst, and the purified water 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 solution was filtered to separate it into a liquid and a solid. The solid obtained by filtration separation was vacuum dried at 120 °C for 12 hours, 3 g of the newly ball milled plate pulp and 37.5 g of purified water were added, and the hydrolysis reaction of the plate pulp was carried out to carry out the hydrolysis reaction of the second batch of plate pulp. By repeating the same operation as in the second batch, the hydrolysis reaction of the third batch of plate pulp was carried out. The liquids for 3 batches were mixed to obtain a plate pulp decomposition solution. The plate pulp decomposition solution was concentrated at 50 °C until it became 1 / 3 of the volume ratio to obtain "plate pulp decomposition product" as a cellulose decomposition product.

[0094] (3) Hydrolysis reaction of pulp sludge As the reaction substrate of the pulp sludge, the one treated as follows was used. 1 kg of zirconia balls with a diameter of 1 cm and 5 g of the pretreated (finely chopped) pulp sludge obtained in the above Experimental Example 6 were placed in a ceramic pot mill. It was set on a desktop pot mill turntable and ball milled at 200 rpm for 120 hours. This operation was performed in multiple batches, and the plate pulp adhering to the wall surface of the ceramic bottle was collected as a sample, and 21 g of the ball milled plate pulp was obtained. Next, ball-milled pulp sludge, a solid acid catalyst, and purified water were added to an autoclave equipped with a stirring device, and while stirring at 300 rpm, the temperature was raised from room temperature to 150 °C in about 30 minutes. Then, the hydrolysis reaction of the pulp sludge was carried out for 24 hours at 150 °C (internal pressure 0.5 MPa) in 7 batches. In the first batch of ball-milling treatment, the addition amounts of pulp sludge, solid acid catalyst, and purified water 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 solution was filtered to separate it into a liquid and a solid. The solid obtained by filtration 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, and the hydrolysis reaction of the pulp sludge was carried out to perform the hydrolysis reaction of the pulp sludge in the second batch. By repeating the same operation as in the second batch, the hydrolysis reactions of the pulp sludge in the 3rd, 4th, 5th, 6th, and 7th batches were carried out. The liquids from 7 batches were mixed to obtain a pulp sludge decomposition solution. The pulp sludge decomposition solution was concentrated at 50 °C until it reached 1 / 7 in volume ratio to obtain "pulp sludge decomposition product" as a cellulose decomposition product.

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

[0096]

Table 1

[0097]

Table 2

[0098]

Table 3

[0099] As a result of HPLC analysis using a differential refractive index detector for the cellulose degradation product, board pulp degradation product, and pulp sludge degradation product, the peak area values of glucose and components with a lower molecular weight than glucose that elute after the retention time of glucose in each obtained chromatogram are shown in Tables 4 to 6 below. "Others 1" to "Others 10" shown in Tables 4 to 6 are all components with a lower molecular weight than glucose and are neither cellobiose nor fructose. Note that "Others n" (n is an integer of 1 or 2 in Table 4, an integer from 1 to 6 in Table 5, and an integer from 1 to 10 in Table 6) in each table indicates the peaks derived from unidentified substances in each chromatogram in the order of detection using n, and "Others n" with the same numerical value does not mean a peak derived from a common substance in all chromatograms (Tables 4 to 6). The waveform processing parameters used for peak detection were calculated for the area value under the conditions that the slope was 200 μV / min, the width was 5 seconds, and the minimum area was 1000 counts as the minimum area / height.

[0100]

Table 4

[0101]

Table 5

[0102]

Table 6

[0103] The area ratio (component area ratio) was calculated from the area values shown in Tables 4 to 6. Specifically, in Table 4, the area value of glucose was 10750862, and the total area value of the peaks derived from the components with a lower molecular weight than glucose (other 1 + other 2 + levoglucosan + HMF + furfural) was 783106. Therefore, in Table 4 (the cellulose decomposition product obtained in the above (1)), the area ratio of the total area value of the components with a lower molecular weight than glucose to the area value of glucose was 0.072 times. Similarly, the area ratios were also determined for the pulp decomposition product and the pulp sludge decomposition product obtained in the above (2) and (3), respectively. 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 a lower molecular weight than glucose" (Table 5: Others 1 + Others 2 + Others 3 + Others 4 + Others 5 + Others 6 + Levoglucosan + HMF, Table 6: Others 1 + Others 2 + Others 3 + Others 4 + Others 5 + Others 6 + Others 7 + Others 8 + Others 9 + Others 10 + Levoglucosan + HMF + Furfural) were 8387038 and 8357042, respectively. Therefore, the area ratios of the total area value of "components with a lower molecular weight than glucose" to the area value of "glucose" in Tables 5 and 6 were 0.826 times and 0.767 times, respectively.

[0104] <Experimental Example 8-1. Cultivation of Microorganisms Using Cellulose Decomposition Product as Carbon Source: Ethanol Fermentation> The materials for this experimental example were as follows. (Medium) - Medium for yeast (carbon source: cellulose decomposition product obtained in Experimental Example 7.(1)) - The components were mixed in ultrapure water to the following concentrations, adjusted to pH 5.6, and then autoclaved. Cellulose decomposition product in an amount corresponding to 20 g / L of glucose, 20 g / L polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 10 g / L yeast extract (manufactured by Thermo Fisher Scientific) - Medium for acetic acid bacteria (carbon source: cellulose decomposition product obtained in Experimental Example 7.(1)) - The components were mixed in ultrapure water to the following concentrations, adjusted to pH 7.0, and then autoclaved. Cellulose decomposition product in an amount corresponding 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 potassium dihydrogen phosphate (manufactured by Nacalai Tesque), 0.5 g / L magnesium sulfate heptahydrate (manufactured by Nacalai Tesque)

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

[0106] (Method for culturing microorganism: Ethanol fermentation) From a glycerol stock (a frozen microbial preservation solution obtained by adding glycerol to a microbial culture solution to a final concentration of 30% by mass and freezing), 5.0 mL of the medium / tube (each tube containing 5.0 mL of each medium) was inoculated with the microorganism corresponding to the medium and cultured overnight at 30°C (hereinafter referred to as "pre-culture"). 0.1 mL of each obtained pre-culture solution was added to 5.0 mL of the medium / tube (a tube filled with the same medium as the pre-culture solution), and cultured at 30°C and 200 rpm for 48 hours (hereinafter referred to as "main culture").

[0107] (Measurement of turbidity of the main culture solution) During the process of the main culture, samples were taken from each main culture solution 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 at 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 using cellulosic hydrolysate as the glucose source.

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

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

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

[0111] <Experimental Example 8-2. Cultivation of microorganisms using glucose as a carbon source: Ethanol fermentation> Except for using the following materials in the yeast medium and acetic acid bacteria medium in Experimental Example 8-1., the turbidity measurement of the main culture solution, the glucose concentration measurement in the main culture solution, and the ethanol concentration measurement in the main culture solution were each carried out in the same manner as in Experimental Example 8-1. The turbidity changes at 0 hour, 24 hours, and 48 hours after the main culture are shown in FIGS. 2 and 3. In FIGS. 2 and 3, "●" indicates the results of Experimental Example 8-2. using commercially available glucose as the glucose source.

[0112] (Medium) - Yeast medium (carbon source: glucose) - In the "medium for yeast (carbon source: cellulose degradation product obtained in Experiment Example 7.(1))" of Experiment Example 8-1, a medium in which glucose (reagent manufactured by Nacalai Tesque) was used at 20 g / L instead of the cellulose degradation product. - Medium for acetic acid bacteria (carbon source: glucose) - In the "medium for acetic acid bacteria (carbon source: cellulose degradation product obtained in Experiment Example 7.(1))" of Experiment Example 8-1, a medium in which glucose (reagent manufactured by Nacalai Tesque) was used at 20 g / L instead of the cellulose degradation product.

[0113] <Experiment Example 8-3. Evaluation of ethanol fermentation> (Determination of microbial growth degree) In the measurement of the turbidity of the main culture solution in Experiment Example 8-2, taking the turbidity value of the experiment example cultured for 48 hours with glucose as the carbon source as the cell concentration of 100%, the relative cell concentration (%) in each main culture solution (cultured for 48 hours) was calculated and judged according to the following criteria. The results are shown in Table 7 together with the values of the relative cell concentration. - Judgment - A: Relative cell concentration ≧ 75% B: 75% > Relative cell concentration ≧ 18% C: 18% > Relative cell concentration

[0114]

Table 7

[0115] From the results of Table 7 and the results of Figures 2 and 3, when glucose was used as the carbon source, an increase in the cell concentration of both yeast and acetic acid bacteria was observed. Specifically, when yeast was used (Figure 2), under the conditions in the above experiment example, the turbidity became constant in about 24 hours and the growth of yeast stopped. 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 it can be seen that the turbidity increased and acetic acid bacteria grew after exceeding 24 hours. In contrast, when using cellulosic hydrolysate as the carbon source, an increase in the cell concentration of yeast was observed, but no increase in the cell concentration of acetic acid bacteria was observed. Moreover, when using cellulosic hydrolysate, the relative cell concentration was significantly improved compared to the case of using glucose. Specifically, when using yeast (Figure 2), under the conditions in the above Experimental Example 8-1, the turbidity continued to increase even after 24 hours, indicating that the growth of yeast continued. When using acetic acid bacteria (Figure 3), the turbidity hardly changed, indicating that the growth of acetic acid bacteria and acetic acid fermentation (ethanol decomposition) were suppressed.

[0116] (Determination of Glucose Assimilation Rate) The value of the glucose residual rate (%) calculated by the above (Equation 2) was used as the glucose assimilation rate and judged according to the following criteria. The results are shown in Table 8 together with the values of the glucose residual rate. - Judgment - A: Glucose residual rate ≤ 25% B: 25% < Glucose residual rate ≤ 75% C: 75% < Glucose residual rate

[0117]

Table 8

[0118] From the results in Table 8, when using glucose as the carbon source, both yeast and acetic acid bacteria assimilated glucose. In contrast, when using cellulosic hydrolysate as the carbon source, yeast assimilated the glucose contained in the cellulosic hydrolysate, but acetic acid bacteria did not assimilate the glucose contained in the cellulosic hydrolysate.

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

[0120]

Table 9

[0121] From the results in Table 9, ethanol was detected at a similar level whether cellulose degradation products or glucose were used as the carbon source. Moreover, even when cellulose degradation products were used, the ethanol concentration (fermentation efficiency) was almost the same as when glucose was used alone. This indicates that efficient production of ethanol by yeast is possible by using cellulose degradation products.

[0122] <Experimental Example 9. Cultivation of Yeast Using Cellulose Degradation Products, Board Pulp Degradation Products, and Pulp Sludge Degradation Products as Carbon Sources: Ethanol Fermentation> The materials used in this experimental example were as follows. (Medium) A medium prepared by mixing each component in ultrapure water to the following concentrations, adjusting the pH to 5.6, and then filter-sterilizing 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 Corporation), 6.7 g / L YNB, w / Ammonium Sulfate, 790 mg / L CSM, Powder - Yeast medium (carbon source: cellulose degradation products obtained in Experimental Example 7.(1)) - Cellulose hydrolyzate in an amount equivalent to 20 g / L in terms of glucose amount, 6.7 g / L YNB, w / Ammonium Sulfate, 790 mg / L CSM, Powder - Medium for yeast (carbon source: board pulp hydrolyzate obtained in Experimental Example 7.(2)) - Board pulp hydrolyzate in an amount equivalent to 20 g / L in terms of glucose amount, 6.7 g / L YNB, w / Ammonium Sulfate, 790 mg / L CSM, Powder - Medium for yeast (carbon source: pulp sludge hydrolyzate obtained in Experimental Example 7.(3)) - Pulp sludge hydrolyzate in an amount equivalent to 20 g / L in terms of glucose amount, 6.7 g / L YNB, w / Ammonium Sulfate, 790 mg / L CSM, Powder

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

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

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

[0126] (Determination of yeast growth degree) In the measurement of the turbidity of this culture broth, taking the turbidity value of the experimental example cultured for 48 hours with glucose as the carbon source as 100% of the cell concentration, the relative cell concentration (%) in each culture broth (cultured for 48 hours) was calculated and judged according to the following criteria. The results are shown in Table 10 together with the values of the relative cell concentration. - Judgment - A: Relative cell concentration ≥ 75% B: 75% > relative cell concentration ≥ 18% C: 18% > relative cell concentration

[0127]

Table 10

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

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

[0130] (Determination of ethanol productivity) Based on the ethanol concentration in the culture solution, the following criteria were used for determination. - Judgment - S: > Ethanol concentration ≥ 9.0 g / L A: 9.0 g / L > Ethanol concentration ≥ 3.0 g / L B: 3.0 g / L > Ethanol concentration ≥ 0.2 g / L C: 0.2 g / L > Ethanol concentration

[0131]

Table 11

[0132] From the results in Table 11, ethanol was detected in all cases where glucose, cellulose degradation products, board pulp degradation products, and pulp sludge degradation products were used as carbon sources. When cellulose degradation products were used as the carbon source, the ethanol concentration (fermentation efficiency) was almost the same as when glucose was used as the carbon source. When board pulp degradation products were used as the carbon source, a significantly higher ethanol concentration (fermentation efficiency) was shown compared to when glucose was used as the carbon source.

[0133] <Experimental Example 10. Identification of acetic acid bacteria growth inhibitors> The materials for this experimental example were as follows. (Medium) A medium was prepared by mixing each component in ultrapure water to the following concentrations, adjusting the pH to 7.0, and then autoclaving. - Medium for acetic acid bacteria (carbon source: glucose) - 20 g / L glucose (manufactured by FUJIFILM Wako Pure Chemical Corporation), 5 g / L polypeptone (manufactured by Shiotani MS), 3 g / L yeast extract (manufactured by Biokar diagnostics), 3 g / L meat extract (manufactured by Gibco), 2 g / L ammonium sulfate (manufactured by FUJIFILM Wako Pure Chemical Corporation), 1 g / L potassium dihydrogen phosphate (manufactured by FUJIFILM Wako Pure Chemical Corporation), 0.5 g / L magnesium sulfate heptahydrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) (Candidate growth inhibitor) In Table 1, it was considered that the substance having a growth inhibitory effect on acetic acid bacteria would be either levoglucosan, HMF, or furfural. Therefore, these substances were used as candidate growth inhibitors, and their concentrations were varied as follows and added to the acetic acid bacteria medium (carbon source: glucose). · Levoglucosan (manufactured by FUJIFILM Wako Pure Chemical Corporation): 0 g / L, 0.049 g / L, 0.098 g / L, 0.195 g / L, 0.390 g / L · 5-Hydroxymethylfurfural (HMF) (manufactured by Nacalai Tesque): 0 g / L, 0.066 g / L, 0.133 g / L, 0.265 g / L, 0.530 g / L · Furfural (manufactured by FUJIFILM Wako Pure Chemical Corporation): 0 g / L, 0.035 g / L, 0.070 g / L, 0.140 g / L, 0.280 g / L, 0.560 g / L

[0134] (Microorganism) Acetic acid bacteria (Acetobacter aceti NBRC14818)

[0135] (Cultivation method of acetic acid bacteria) 20 mL of the culture medium for acetic acid bacteria (carbon source: glucose) was added to a 125 mL Erlenmeyer flask with a baffle, and inoculated with glycerol stock of acetic acid bacteria, followed by culturing at 30 °C and 225 rpm for 3 days (hereinafter referred to as "pre-culture"). The obtained pre-culture solution was collected, and the supernatant was removed by centrifugation to obtain acetic acid bacteria cells. The obtained cells were suspended in the culture medium for acetic acid bacteria (carbon source: glucose) to which each candidate growth inhibitor was added so that the inoculation concentration was OD600 = 0.084. 4 mL of each acetic acid bacteria culture solution was added to a test tube, and cultured at 30 °C and 225 rpm for 48 hours (hereinafter referred to as "main culture").

[0136] (Measurement of turbidity of the main culture solution) During the process of the main culture, samples were taken from each main culture solution at different time points (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 turbidity changes at 0 hours, 8 hours, 24 hours, and 48 hours after the main culture are shown in Figures 5 to 7.

[0137] (Judgment of the growth degree of acetic acid bacteria) In the measurement of the turbidity of the main culture solution, taking the turbidity value of the experimental example cultured for 48 hours without adding the candidate growth inhibitor as 100% of the cell concentration, the relative cell concentration (%) in each main culture solution (cultured for 48 hours) was calculated and judged according to the following criteria. The results are shown in Tables 12 to 14 together with the values of the relative cell concentration. - Judgment - A: Relative cell concentration ≥ 75% B: 75% > Relative cell concentration ≥ 20% C: 20% > Relative cell concentration

[0138]

Table 12

[0139]

Table 13

[0140]

Table 14

[0141] From the results of Tables 12 to 14 and the results of Figures 5 to 7, levoglucosan and HMF did not affect the growth of acetic acid bacteria, but furfural clearly inhibited the growth of acetic acid bacteria. Specifically, when 0.035 g / L of furfural was added to the medium for acetic acid bacteria, the relative 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 of furfural was added to the medium for acetic acid bacteria, the relative cell concentration was 6 to 7% of the experimental example without furfural addition (0 g / L). Under the conditions of 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 higher.

[0142] From the above results, it was shown that cellulose degradation products, board pulp degradation products, and pulp sludge degradation products satisfying the above component area ratio can be used as carbon sources for ethanol production by yeast. In particular, cellulose degradation products and board pulp degradation products were shown to exhibit fermentation efficiency equal to or higher than that when glucose was used alone. Also, it was shown that the hydrolysis product of cellulose 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 degradation product obtained in Experimental Example 7.(1) exhibited almost the same fermentation efficiency as when glucose was used alone, and 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 degradation product that satisfies 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 the decomposition of ethanol 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 degradation product that satisfies the above component area ratio using microorganisms 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 understood 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.

Claims

1. A method for producing an organic compound comprising contacting a liquid containing glucose with a microorganism, wherein the liquid containing glucose is a hydrolysate of cellulose obtained by contacting a liquid containing cellulose and water with a solid acid catalyst, and when the liquid containing glucose is subjected to LC analysis using a 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. A method for producing an organic compound.

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

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

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

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

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

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

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

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

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

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

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

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

14. A composition for culturing microorganisms comprising a liquid containing glucose, wherein The liquid containing glucose is a hydrolyzate of cellulose obtained by bringing a liquid containing cellulose and water into contact with a solid acid catalyst. When the liquid containing glucose 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. A composition for microorganism culture.

Images