Technique for producing monosaccharide and bioethanol from lignocellulose

Abstract

To be provided is a method for producing a monosaccharide from lignocellulose under mild conditions.　Provided is a method for producing a monosaccharide, said method including: a mixing step for mixing lignocellulose and formic acid in an environment at a temperature of 60°C or lower; and a monosaccharide generation step for enzymatically saccharifying a polysaccharide contained in a lignocellulose mixture generated in the mixing step, thereby obtaining a monosaccharide containing glucose.

Description

Technology for producing monosaccharides and bioethanol from lignocellulose

[0001] The present disclosure relates to a technology for producing bioethanol. More specifically, it relates to a method for producing monosaccharides from lignocellulose and a method for producing ethanol.

[0002] There is a demand for shifting from fossil fuel-based fuels to biofuels. The production of first-generation liquid biofuels, which are mainly produced from food crops such as grains, sugar and oilseeds, is well understood. However, since the limitations of first-generation biofuels produced from food crops have recently become clear, second-generation biofuels produced from lignocellulosic feedstocks have come to be emphasized. Such lignocellulosic feedstocks include by-products (grain straw, sugarcane bagasse, forest residues), wastes (organic components of municipal solid waste), dedicated feedstocks (vegetation grasses cultivated for specific purposes, short-rotation coppice, and other energy crops), and the like. Since such lignocellulosic feedstocks contain lignin, special processes are required. Special processes for the production of second-generation biofuels include biochemical methods and thermochemical methods. In short, there are two completely different processing routes for producing biofuels from lignocellulosic feedstocks:

[0003] -Biochemical - using enzymes and microorganisms to convert the cellulose and hemicellulose components of the feedstock into sugars, which are then fermented to produce ethanol;

[0004] -Thermochemical - (also referred to as biomass-to-liquid, BTL) producing syngas (CO + H2) by pyrolysis / gasification technology, and producing ethanol therefrom based on Fischer-Tropsch conversion.

[0005] Because the biochemical route is not yet mature, it offers a greater potential for energy savings than the thermochemical route. In biochemical methods, a pretreatment step that exposes cellulose and hemicellulose for subsequent enzymatic hydrolysis is a crucial process step. Options can be classified as biological, physical, chemical, or a combination thereof. Options can be classified as biological, physical, chemical, or a combination thereof, and each has variations with different temperatures and reaction times. In recent years, there is no "best" option, and research and development continues to improve performance targets. Previous proposals have suggested pretreatments such as steam explosion, organic solvents, alkalis, acids, and especially peracids. (Non-patent document 1)

[0006] Incidentally, the advantage of the biochemical route is energy reduction. Therefore, energy reduction is paramount in this option. To achieve this, a process is needed that can operate at low temperatures throughout the entire process, where the acid used in pretreatment does not affect subsequent processes, or where the acid used in pretreatment can be removed with low energy.

[0007] If this can be achieved, it could potentially provide cheaper biofuels than thermochemical routes in the long term. However, no process has been proposed to produce ethyl alcohol from lignocellulose using such low-energy pretreatment.

[0008] Non-patent document 2 discloses a technique for obtaining bioethanol by decomposing lignin from lignocellulose using pressurized hot water treatment and then fermenting the decomposition products with acetic acid. However, this technique requires a two-stage treatment with pressurized hot water at 230°C and 270°C. Furthermore, the treated material needs to be fermented with acetic acid using a mixed microbial system. In addition, the resulting acetic acid needs to be hydrogenated to produce ethyl alcohol, which is a complicated process. Non-patent document 3 separates cellulose from lignocellulose using hardwoods (oak), conifers (cedar), and annuals (sugarcane bagasse). The sulfuric acid concentrations used were 42.5% and 44% (v / v). It is described that lignocellulose (100 mg) was treated with concentrated sulfuric acid (10 mL) for 5 hours under shaking conditions of 20°C and 120 rpm.

[0009] Patent Document 1 discloses a method for producing cellulose and / or sugar products from lignocellulosic raw materials. In this document, lignocellulosic raw materials with a low lignin content, such as washed pulp, are used. The lignocellulosic raw materials are then subjected to formic acid cooking at a temperature of 110 to 160°C with a formic acid content of 70 to 98%. Furthermore, the resulting processed material is treated with performic acid at 50 to 90°C.

[0010] Patent Document 2 describes a method for easily separating and recovering lignin and polysaccharides from lignin polysaccharide complexes such as wood, while reducing energy consumption and environmental impact. This document describes reacting a lignin polysaccharide complex with a peroxide and / or peracid in water and / or an organic solvent to obtain a liquid in which the lignin dissolves in the solvent as an oxide and / or decomposition product, and the polysaccharides dissolve and / or disperse in the solvent. The document states that the reaction is preferably carried out in the presence of an ionic liquid or an acid catalyst, and is particularly preferably carried out in the presence of a carboxylic acid, sulfonic acid, sulfuric acid, hydrochloric acid, nitric acid, or phosphoric acid.

[0011] Patent Document 3 describes a method for obtaining a lignocellulose solution. This method involves a grinding step of coarsely grinding biomass containing lignocellulose to obtain a coarse powder, a mixing step of mixing the coarse powder with an organic acid, and a dissolution step of dissolving the coarse powder in the organic acid, thereby obtaining a lignocellulose solution. It is also described that a molded article mainly composed of lignocellulose can be formed from this solution.

[0012] However, Non-Patent Document 1 does not offer any specific proposals for low-energy pretreatment.

[0013] The technology described in Non-Patent Document 2 involves a two-stage pressurized hot water treatment process, resulting in high energy consumption. Furthermore, the resulting product is acetic acid, which requires hydrogenation to convert it into bioethanol. The technology described in Non-Patent Document 3 requires the use of concentrated sulfuric acid. The treatment of waste concentrated sulfuric acid has a high environmental impact. In addition, there are limited methods for reusing concentrated sulfuric acid.

[0014] Patent Document 1 uses washed pulp as the lignocellulose material. Although washed pulp is a lignocellulose material, it is not a lignocellulose-based raw material. In other words, washed pulp is obtained by deligninizing lignocellulose-based raw materials such as wood chips, resulting in a low lignin content. More specifically, it is obtained by alkaline pulping, washing, and bleaching lignocellulose. For this reason, it has a high environmental impact. Furthermore, Patent Document 1 uses peracid. And Patent Document 1 has the problem of using a high treatment temperature with formic acid.

[0015] Furthermore, the inventors of this disclosure have found the following problems to be apparent. Specifically, when the temperature conditions for treating lignocellulose material with formic acid are high, inhibitors of saccharification and fermentation are easily generated during the subsequent production of monosaccharides by saccharifying enzymes. When the amount of inhibitors contained in the pre-treated material increases, for example, the conversion of monosaccharides to ethanol is inhibited. As a result, the efficiency of production of the target substance from monosaccharides decreases.

[0016] The technology described in Patent Document 2 requires ionic liquids, peroxides, sulfonic acid, etc. Furthermore, the technology described in Patent Document 3 does not describe how to obtain second-generation biofuel from the resulting molded product.

[0017] This disclosure provides a method for producing monosaccharides from lignocellulose under mild conditions. Furthermore, this disclosure also aims to provide an efficient method for producing ethanol using the method for producing said monosaccharides.

[0018] The inventors of this disclosure have conducted extensive research and have found that monosaccharides can be produced from lignocellulose under mild conditions using the following process: A method for producing monosaccharides comprising a mixing step of mixing lignocellulose and formic acid in an environment below a predetermined temperature, and a monosaccharide production step of enzymatically saccharifying the polysaccharides contained in the lignocellulose-containing mixture produced in the mixing step to obtain monosaccharides containing glucose.

[0019] Furthermore, through diligent research by the inventors of this disclosure, they discovered that by adopting this manufacturing method, the inhibitors in ethanol fermentation contained in the obtained monosaccharides are reduced, and ethanol can be produced from monosaccharides with high production efficiency.

[0020] This disclosure is an invention completed through further consideration based on these findings. Specifically, this disclosure provides inventions in the following embodiments.

[0021] (Method for producing monosaccharides) A ​​method for producing monosaccharides comprising: a mixing step of mixing a lignocellulose material and formic acid in an environment with a temperature of 60°C or lower; and a monosaccharide production step of subjecting the polysaccharide contained in the lignocellulose-containing mixture produced in the mixing step to enzymatic saccharification to obtain a monosaccharide containing glucose.

[0022] (Method for producing ethanol) A method for producing ethanol comprising: a step of obtaining a monosaccharide by the method for producing monosaccharides of the present disclosure; and a step of converting the monosaccharide into ethanol.

[0023] This disclosure provides a method for producing monosaccharides from lignocellulose material under mild conditions. Furthermore, this disclosure provides an efficient and low-energy consumption method for producing ethanol, i.e., second-generation biofuels, utilizing this method for producing monosaccharides.

[0024] The graph in Figure 1a shows the relationship between the amount of glucose produced (Glucose from pulp (mg)) and the reaction time (reaction time (days)) for enzymatic saccharification using the solid content obtained in Example 1 as a sample. The graph in Figure 1b shows the relationship between the amount of xylose produced (Xylose from pulp (mg)) and the reaction time (reaction time (days)) for enzymatic saccharification using the filtrate components obtained in Example 1 as a sample. The graph in Figure 2b shows the relationship between the amount of xylose produced (Xylose from filtrate (mg)) and the reaction time (reaction time (days)) for enzymatic saccharification using the filtrate components obtained in Example 1 as a sample. The graph in Figure 3a shows the relationship between the amount of glucose produced (Glucose from pulp (mg)) and the reaction time (reaction time (days)) for enzymatic saccharification using the solid content obtained in Example 2 as a sample. The graph in Figure 3b shows the relationship between the amount of xylose produced (Xylose from pulp (mg)) and the reaction time (reaction time (days)) for enzymatic saccharification using the filtrate components obtained in Example 2 as a sample. The graph in Figure 4b shows the relationship between the amount of xylose produced (Xylose from filtrate (mg)) and the reaction time (reaction time (days)) for enzymatic saccharification using the filtrate components obtained in Example 2 as a sample. The graph in Figure 5a shows the relationship between the amount of glucose produced (Glucose from pulp (mg)) and the reaction time (reaction time (hours)) for enzymatic saccharification using the solid content obtained in Example 3 as a sample.The graph in Figure 5b shows the relationship between the amount of xylose produced (Xylose from pulp (mg)) and the reaction time (reaction time (days)). Each photograph in Figure 6 is a photograph of the solid material from Example 4. These are photographs of the solid obtained by mixing lignocellulose and formic acid aqueous solution for 1 day, 2 days, 3 days, 4 days, 5 days, and 7 days, and then filtering. The graph in Figure 7a shows the results of the enzymatic saccharification measurement using the solid material obtained in Example 5 as a sample. It shows the relationship between the amount of glucose produced (Glucose from pulp (mg)) and the reaction time (reaction time (hours)). The graph in Figure 7b shows the results of the relationship between the amount of xylose produced (Xylose from pulp (mg)) and the reaction time (reaction time (days)). The graph in Figure 8a shows the results of the enzymatic saccharification measurement in Example 6 using a polysaccharide-containing film (eucalyptus film) as a sample. This graph shows the relationship between the amount of monosaccharides produced (glucose and xylose (Sugar yield (mg))) and reaction time (h) measured. The graph in Figure 8b shows the results of measurements taken when a polysaccharide-containing film was used as a sample without being immersed in an aqueous sodium carbonate solution. This graph shows the relationship between the amount of monosaccharides produced (glucose and xylose (Sugar yield (mg))) and reaction time (h) measured when subjected to enzymatic saccharification. The graph in Figure 9 shows the results of enzymatic saccharification using a polysaccharide-containing film (eucalyptus film) as a sample in Example 7. This graph shows the relationship between the amount of monosaccharides produced (glucose and xylose (Sugar yield (mg))) and reaction time (h) measured. The graph in Figure 10 shows the relationship between the amount of monosaccharides produced (glucose and xylose (Sugar yield (mg))) and reaction time (h) measured in the enzymatic saccharification of Example 8.The graph in Figure 11 shows the relationship between the amounts (mg) of glucose, xylose, and ethanol during yeast fermentation in Example 10 and the reaction time (h). The graph in Figure 12 shows the case with and without sodium carbonate treatment. In the graph in Figure 12a, the Y-axis represents the amount of glucose and the X-axis represents the reaction time. In the graph on the right in Figure 12, the Y-axis represents the amount of xylose and the X-axis represents the reaction time. The square markers indicate the case without sodium carbonate treatment. The circular markers indicate the case with sodium carbonate treatment.

[0025] The present disclosure will be described in detail below based on preferred embodiments. However, the scope of the present disclosure is not limited to these descriptions, and other embodiments may be implemented in a manner that does not impair the essence of the present disclosure. Furthermore, the present disclosure is not limited to the embodiments described below, and various modifications are possible within the scope of the claims. In addition, other embodiments obtained by appropriately combining the technical means disclosed for each of the multiple embodiments are also included in the technical scope of the present disclosure.

[0026] In the numerical ranges described in stages in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Alternatively, the upper and lower limits, upper and lower limits, or lower and lower limits described separately may be combined to form numerical ranges. Furthermore, in the numerical ranges described in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the values ​​shown in the examples. Each element of this disclosure can be combined with one another. The upper and lower limits of each component of the configuration of this disclosure can each be combined with the upper and lower limits of the other components. In this specification, "X to Y" indicating a range means "X or greater and Y or less".

[0027] Furthermore, unless otherwise noted, all test temperatures are room temperature (20°C ± 5°C), and weight percent refers to weight percentage, not mass concentration. Also, the unit of mass "t (ton)" refers to "metric ton."

[0028] (Lignocellulose-based raw materials and lignocellulose) Lignocellulose is a complex plant material composed of three main components: cellulose, hemicellulose, and lignin. These components form the structural framework of plant cell walls. In particular, lignin is strongly bound to hemicellulose and cellulose.

[0029] Lignocellulose is abundant and holds potential as a raw material for producing biofuels. However, its complex structure makes it difficult to break down and convert into useful products.

[0030] Lignocellulose-based raw materials are raw materials obtained by crushing, pulverizing, or otherwise processing lignocellulose. In this disclosure, lignocellulose and lignocellulose-based raw materials are treated as having the same meaning.

[0031] Furthermore, some edible grains derived from plants contain no lignin at all. For example, wheat flour is one such example. This is not included in lignocellulosic raw materials. On the other hand, threshed hulls (for example, rice and wheat hulls) contain lignocellulose and correspond to lignocellulose in this disclosure.

[0032] Furthermore, kraft pulp, which is produced by chemically processing wood chips to break down lignin and hemicellulose and free up cellulose fibers, contains a small amount of lignin but is not a lignocellulosic raw material. Pulp may be a lignocellulosic material, but it is not a lignocellulosic raw material. This is because the strong bonds between hemicellulose, cellulose, and lignin in pulp are almost completely free.

[0033] The ligno-cellulosic raw materials are not particularly limited and include, for example, at least one selected from the group consisting of woody biomass, herbaceous biomass, crops, unused parts of crops, foliage, fruit peels, cotton, and hemp. The amount of plant material used in the mixing process may be one type or two or more types.

[0034] Lignocellulosic raw materials may be woody biomass. Woody biomass may be coniferous trees (such as Japanese cedar, cypress, and Japanese red pine) or broad-leaved trees (such as eucalyptus and beech). Two or more types may be used in combination. From the viewpoint of effectively utilizing declining forest resources, woody biomass that was previously discarded can be suitably used. For example, wood powder generated during lumbering, or small pieces (chips) that cannot be used as scraps, may be used as plant raw materials. Woody biomass may be natural wood or sawn timber cut from natural wood. There are no particular limitations on the shape of the woody biomass; for example, it can be used in the form of boards, chips, powder, etc., as appropriate. Examples of herbaceous biomass include sugarcane bagasse, rice straw, wheat, wheat bran, tomato, onion, and Moso bamboo.

[0035] Lignocellulose forms a complex higher-order structure in which cellulose, hemicellulose, and lignin are intricately intertwined. Specifically, cellulose, a linear polymer, forms a crystalline structure through intramolecular and intermolecular hydrogen bonds, constituting strong microfibrils (cellulose microfibrils). Hemicellulose such as xylan and glucomannan are intertwined with these microfibrils, and lignin, an irregular aromatic polymer, fills the voids in this polysaccharide matrix.

[0036] In lignocellulose, the total content (or content as lignocellulose) of cellulose, hemicellulose, and lignin is not particularly limited. The total content of cellulose, hemicellulose, and lignin may be 90% by weight or more, 95% by weight or more, 98% by weight or more, or 100% by weight. In the mixing process, some or all of the hydroxyl groups of the cellulose, hemicellulose, and lignin may be formylated.

[0037] The pulverized lignocellulose can be used as the lignocellulose itself. That is, the lignocellulose can be pulverized to the desired size before mixing it with formic acid. The size of the pulverized material is not particularly limited, and the maximum diameter in plan view may be 50 mm or less, 40 mm or less, or 30 mm or less. From the viewpoint of dust prevention, the size of the lignocellulose is preferably 0.1 mm or larger in maximum diameter in plan view.

[0038] [Method for producing monosaccharides] The method for producing monosaccharides according to this disclosure includes at least the following mixing step and monosaccharide production step. Mixing step: A lignocellulose material (hereinafter sometimes simply referred to as "plant material") and formic acid are mixed in an environment with a temperature of 60°C or lower. Monosaccharide production step: The polysaccharides contained in the lignocellulose-containing solution produced in the mixing step are subjected to enzymatic saccharification to obtain a monosaccharide containing glucose.

[0039] (Mixing process) In the mixing process, the lignocellulose material and formic acid are mixed in a low-temperature environment of 60°C or below.

[0040] The concentration of formic acid mixed with lignocellulose is not particularly limited as long as it promotes the deligninization of lignocellulose, and is, for example, 30% by mass or more, 40% by mass or more, 50% by mass or more, 55% by mass or more, 76% by mass or more, preferably 80% by mass or more, more preferably 88% by mass or more, even more preferably 92% by mass or more, and even more preferably 98% by mass or more. Upper limits include, for example, 99% by mass or less, 98% by mass or less.

[0041] For 40%, 76%, and 85% formic acid, commercially available silage modifiers such as Metax (trademark) can be used. Furthermore, at a concentration of 76%, the formic acid can be distilled without the use of an azeotrope.

[0042] Formic acid is mixed with lignocellulose, for example, in the form of an aqueous solution. Through delignification, at least a part of the lignin present in the solid lignocellulose migrates into the solution. As delignification progresses, polysaccharides such as cellulose and hemicellulose in lignocellulose are suitably converted into monosaccharides by enzymatic saccharification in the monolayer formation step described below.

[0043] The mixing ratio of formic acid relative to 100 parts by mass of lignocellulose, calculated as formic acid volume, is preferably 2 parts by volume or more, more preferably 3 parts by volume or more, still more preferably 4 parts by volume or more, and is preferably 30 parts by volume or less, more preferably 20 parts by volume or less. A preferable range is about 2 to 30 parts by volume.

[0044] In the present disclosure, the temperature of the mixing step is 60°C or lower. In the method for producing a monosaccharide of the present disclosure, the mixing temperature of lignocellulose and formic acid is set to a mild condition of 60°C or lower. This makes it possible to suppress the generation of alcohol conversion inhibitors while delignifying lignocellulose. By suppressing the generation of inhibitors in the lignocellulose-containing solution obtained in the mixing step, the enzymatic saccharification reaction in the subsequent monosaccharide generation step is not inhibited by the inhibitors, and monosaccharides can be produced efficiently.

[0045] In the mixing step, in addition to lignocellulose and formic acid, a solvent may be further mixed. When an aqueous formic acid solution is used as formic acid, water serves as the solvent. The solvent is not particularly limited as long as it does not inhibit the monosaccharide generation step described below, and examples thereof include ethyl alcohol and the like.

[0046] The temperature of the mixing step is preferably lower than 60°C, more preferably 55°C or lower, still more preferably 50°C or lower, and is preferably 30°C or higher, more preferably 35°C or higher, still more preferably 40°C or higher. A preferable range is about 40 to 50°C. When the temperature is low, energy efficiency is improved, and when the temperature is high, the duration of the mixing step can be shortened. Low-temperature waste heat can be easily utilized when the temperature is around 50°C.

[0047] If the temperature in the mixing step exceeds 60°C, the degree of formyl esterification of OH groups present in cellulose, hemicellulose and lignin will increase. As a result, the amount of formic acid chemically bound in the residual solid after evaporating formic acid from the mixture for enzymatic saccharification increases, which interferes with enzymatic saccharification. In addition, since the production amount of furfurals increases, the inhibition of enzymatic saccharification by furfurals is also accelerated.

[0048] The mixing time in the mixing step is appropriately adjusted according to the type of lignocellulose, that is, the type of ligno-cellulosic raw material, formic acid concentration, the mixing amount of each component supplied to the mixing step, and the like. For example, it is 1 day or more, 2 days or more, 7 days or more, and for example, it is 27 days or less, 21 days or less, 14 days or less, and a preferable range includes 2 to 7 days. When the temperature of the mixing step is low, the mixing time may be set longer.

[0049] The mixing step can be performed, for example, under atmospheric pressure, or the pressure may be reduced to degas the mixed solution as described below.

[0050] From the viewpoint of more suitably exerting the effect of the invention of the present disclosure, it is preferable to apply a frictional force to lignocellulose in the mixing step. As a method of applying a frictional force to the lignocellulosic material, in the process of stirring and mixing lignocellulose and formic acid using a stirring body, adjustment is performed such that the stirring body (stirrer bar (rotor), stirring blade, etc.) defibrates the lignocellulosic material (for example, increasing the solid content concentration of the plant material), or adjustment is performed such that the plant material is sandwiched between the stirring body and the wall surface of the container, and stirring and mixing by the stirring body are performed.

[0051] In order to more suitably improve the effect of the invention of the present disclosure, it is preferable to degas the mixed solution of lignocellulose and formic acid in the mixing step. Degassing the mixed solution promotes delignification of lignocellulose and promotes enzymatic saccharification in the monosaccharide production step.

[0052] There are no particular limitations on the method for degassing the mixed solution; for example, placing the mixed solution in a reduced pressure environment (e.g., an environment with a pressure of -0.02 to -0.06 MPa). There are no particular limitations on the degassing time, but for example, it could be 5 to 30 minutes.

[0053] In the manufacturing method disclosed herein, a lignocellulose-containing solution containing polysaccharides is produced by a mixing step, and the polysaccharides are subjected to a monosaccharide production step.

[0054] In the manufacturing method disclosed herein, a step to remove formic acid may be further provided between the mixing step and the monosaccharide production step. This can be used to adjust the pH of the enzymatic saccharification in the monosaccharide production step and thereby promote the enzymatic saccharification reaction in the monosaccharide production step.

[0055] Methods for removing formic acid include, for example, solid-liquid separation of the lignocellulose-containing solution produced in the mixing process, reducing the pressure, and adding a base. These methods may be used individually or in combination of two or more.

[0056] One method for solid-liquid separation is to filter the lignocellulose-containing solution to separate it into solid and filtrate components. Formic acid migrates to the liquid component (filtrate component). Water or other liquids may be used as a washing solution for solid-liquid separation.

[0057] Furthermore, in the manufacturing method disclosed herein, even if the purpose is not to remove formic acid, the lignocellulose-containing solution produced in the mixing step may be subjected to solid-liquid separation to separate it into solid and liquid components. As described later, polysaccharides may be contained in both the solid and liquid components. In addition, the polysaccharides contained in the solid and the polysaccharides contained in the liquid components generally have different compositions.

[0058] Alternatively, formic acid can be removed by evaporating it from the lignocellulose-containing solution by placing it under reduced pressure. The reduced pressure conditions (pressure, temperature, etc.) should be adjusted so that the formic acid evaporates and is removed. In this way, the lignocellulose-containing solution can be solidified by removing the formic acid without performing solid-liquid separation. This solid contains the composition originating from the polysaccharides contained in the lignocellulose.

[0059] Furthermore, formic acid can also be removed by reacting it with a base by mixing the lignocellulose-containing solution with a base. Examples of bases include sodium carbonate, potassium carbonate, sodium hydroxide, and sodium bicarbonate. Among these, sodium carbonate is particularly preferred. For example, when formic acid reacts with sodium carbonate, sodium formate is contained in the liquid phase. Only one type of base may be used, or two or more types may be used. As described later, the base can also be used to convert the formyl group of the polysaccharide obtained in the mixing step into a hydrogen atom, and during this conversion, it can also react with formic acid to remove it.

[0060] In the manufacturing method of this disclosure, a step of converting the formyl groups contained in the polysaccharide into hydrogen atoms may be further included between the mixing step and the monosaccharide production step. The method of converting the formyl groups contained in the polysaccharide into hydrogen atoms is not particularly limited. One example is a method of reacting the polysaccharide with a base. Specifically, one method of converting the formyl groups contained in the polysaccharide into hydrogen atoms is to mix the lignocellulose-containing solution produced in the mixing step with a base. Another example is to subject the lignocellulose-containing solution produced in the mixing step to solid-liquid separation described later, and mix the polysaccharide contained in the obtained solid (e.g., film) with a base. As for the base, as mentioned above, for example, sodium carbonate is an example. Among these, sodium carbonate is particularly preferred. Only one type of base may be used, or two or more types may be used. The amount of base used should be adjusted appropriately to the extent that the formyl groups contained in the polysaccharide are removed.

[0061] As described above, the manufacturing method of the present disclosure may further include a step of subjecting the lignocellulose-containing solution produced in the mixing step to solid-liquid separation such as filtration to separate it into solid components and liquid components (filtrate components). Both the solid components and liquid components can be subjected to the monosaccharide production step. That is, in the manufacturing method of the present disclosure, at least the polysaccharides contained in the solid components can be subjected to the monosaccharide production step. In addition, the polysaccharides contained in the liquid components can also be subjected to the monosaccharide production step.

[0062] Furthermore, as described above, the manufacturing method of the present disclosure may further include an evaporation step in which the entire lignocellulose-containing solution produced in the mixing step is evaporated to obtain a solid without solid-liquid separation. In this case, in the monosaccharide production step described later, the polysaccharides contained in the solid can be subjected to enzymatic saccharification to obtain a monosaccharide containing glucose.

[0063] It is preferable to further include a step after the evaporation step in which the formyl groups of the polysaccharides contained in the solid are converted into hydrogen atoms. The method for converting the formyl groups of the polysaccharides contained in the solid to hydrogen atoms is the same as the method of reacting the polysaccharide with a base described above. For example, the method of reacting the polysaccharide contained in the solid with a base can be cited. The same bases as described above are exemplified, and sodium carbonate is particularly preferred. Only one type of base may be used, or two or more types may be used. The amount of base used should be appropriately adjusted to the extent that the formyl groups of the polysaccharides contained in the solid are removed.

[0064] (Monosaccharide Production Process) The monosaccharide production process, which is a method for producing monosaccharides according to the present disclosure, is described below. The monosaccharide production process is a process in which monosaccharides are obtained by enzymatic saccharification of polysaccharides contained in the lignocellulose-containing solution produced in the mixing process. As described above, the lignocellulose-containing solution can be subjected to solid-liquid separation, and at least one of the solid component containing polysaccharides and the liquid component containing polysaccharides can be enzymatically saccharified. Alternatively, the polysaccharides contained in the lignocellulose-containing solution produced in the mixing process can be solidified and then enzymatically saccharified.

[0065] The enzymatic saccharification of polysaccharides is not particularly limited as long as the polysaccharide is converted into a monosaccharide containing glucose by the enzymatic saccharification reaction, and known methods can be employed. In this disclosure, examples of polysaccharides include glucose, xylose, mannose, galactose, arabinose, and pectin. Note that lignin is not a sugar. The monosaccharide produced by the enzymatic saccharification of polysaccharides only needs to contain at least glucose. In addition to glucose, other monosaccharides may also be included. To produce ethanol from xylose, specific microorganisms can be used. For example, xylose can be converted to ethanol by using genetically modified E. coli or yeast (Saccharomyces cerevisiae). To convert mannose to ethanol, white rot organisms such as Schizophyllum commune can be used. Furthermore, galactose can be converted to ethanol using yeast (Saccharomyces cerevisiae).

[0066] In this disclosure, the polysaccharide preferably contains at least one of cellulose and hemicellulose, and more preferably contains cellulose. When the polysaccharide contains cellulose, the enzyme used in the enzymatic saccharification reaction is a saccharifying enzyme that converts cellulose to monosaccharides (glucose). When the polysaccharide contains hemicellulose, the enzyme used in the enzymatic saccharification reaction is a saccharifying enzyme that converts hemicellulose to monosaccharides (such as xylose).

[0067] In this disclosure, it is preferable to use saccharifying enzymes that convert cellulose and hemicellulose into monosaccharides. Specific examples of saccharifying enzymes include Cellic® Novozymes® CTec2 (manufactured by Novozymes A / S). The enzymes used in the monosaccharide production process may be one type or two or more types.

[0068] In the monosaccharide production process, the temperature conditions are adjusted as appropriate depending on the type of enzyme, etc. The temperature may be room temperature, or it may be around 30°C. In the monosaccharide production process, the pH conditions are also adjusted as appropriate depending on the type of enzyme, etc. It should be adjusted to the optimal pH for each process.

[0069] In the monosaccharide production process, the reaction time for the enzymatic saccharification reaction is adjusted as appropriate depending on the type of enzyme, the reaction scale, etc. For example, it may be 10 to 20 hours.

[0070] In the monosaccharide production process, the pressure conditions for the enzymatic saccharification reaction can be, for example, carried out under atmospheric pressure.

[0071] The monosaccharide production process is preferably carried out while stirring the polysaccharide and saccharifying enzyme. The stirring method is not particularly limited as long as it ensures that the polysaccharide and saccharifying enzyme are uniformly mixed. For example, this can be done by rotating a stirring device (such as a stirrer bar or stirring blade).

[0072] As described above, in the manufacturing method of this disclosure, the lignocellulose-containing solution produced in the mixing step is subjected to solid-liquid separation such as filtration. At least one of the resulting solid component and liquid component (filtrate component) is then subjected to a monosaccharide production step. That is, in the manufacturing method of this disclosure, there is at least a mode in which monosaccharides are produced from polysaccharides contained in the solid component. There is also a mode in which monosaccharides are produced from polysaccharides contained in the liquid component.

[0073] Furthermore, the manufacturing method of this disclosure may further include an evaporation step in which the entire lignocellulose-containing solution produced in the mixing step is evaporated to obtain a solid without solid-liquid separation. In this case, in the monosaccharide production step described later, the polysaccharides contained in the solid can be subjected to enzymatic saccharification to obtain monosaccharides including glucose. The monosaccharide thus obtained can be subjected to the ethanol conversion step described later, while still in solid form. In this respect, the formic acid used in this disclosure has a low boiling point, making it easy to employ the evaporation step.

[0074] The solid component may be in the form of a film, for example. Specifically, a film containing polysaccharides can be formed, and this film can be enzymatically saccharified to produce monosaccharides containing glucose. The monosaccharides obtained in this way can then be subjected to the conversion to ethanol step described later, while still in the form of film.

[0075] The film thickness is, for example, 200 μm or less, preferably 100 μm or less, more preferably 70 μm or less, and also, for example, 20 μm or more, preferably 40 μm or more, more preferably 50 μm or more, with a preferred range being 40 to 120 μm. If the thickness is too thin, the film will lack strength and become difficult to handle. If it is too thick, reactions such as formylation will take a long time.

[0076] In the monosaccharide production process, lignin degradation products may coexist with polysaccharides. When a lignocellulose-containing solution is subjected to solid-liquid separation, some of the lignin degradation products contained in the lignocellulose migrate into the liquid component, but in many cases, lignin and its degradation products are also present in the solid component. If lignin degradation products are present along with polysaccharides in the monosaccharide production process, lignin degradation products will also be present after monosaccharides have been produced from polysaccharides.

[0077] Furthermore, after the conversion reaction from polysaccharide to monosaccharide in the monosaccharide production process, furfural may be present along with the monosaccharide. However, furfural is an inhibitor of the conversion of monosaccharide to ethanol, as described later. For this reason, it is preferable that the content of furfural coexisting with monosaccharide is reduced. The content of furfural per 100 parts by mass of monosaccharide produced in the monosaccharide production process is preferably 1 part by mass or less, more preferably 0.7 parts by mass or less, and even more preferably 0.5 parts by mass or less. It is also possible that furfural is substantially absent. That is, the preferred range for the content is 0 to 1 part by mass, 0 to 0.7 parts by mass, 0 to 0.5 parts by mass, etc.

[0078] [Ethanol Manufacturing Process] The ethanol manufacturing process of this disclosure includes a step of converting the monosaccharide obtained by the monosaccharide manufacturing process of this disclosure described above into ethanol.

[0079] Various methods are known for the process of converting monosaccharides, including glucose, into ethanol. In this disclosure, the reaction is not particularly limited, and various methods can be employed.

[0080] One example is alcoholic fermentation, a reaction that produces ethanol from glucose, carried out by enzymes in microorganisms such as yeast.

[0081] Furthermore, in this disclosure, other monosaccharides such as xylose can also be produced by the monosaccharide manufacturing process described above. Therefore, in the ethanol manufacturing process of this disclosure, not only glucose but also xylose can be used as a raw material for ethanol.

[0082] Yeasts that convert glucose and xylose to ethanol include, for example, SK-FGGX A2 / 5 and SK-N2, and these known yeasts can be used. In addition, for example, yeast genetically modified to convert xylose according to the procedure in the non-patent document below can also be used.

[0083] The conditions for the process of converting monosaccharides to ethanol vary depending on the type of reaction and can be appropriately selected for each reaction. (Non-patent literature: Khattab, SMR, Watanabe, T., Efficient conversion of glycerol to ethanol by an engineered Saccharomyces cerevisiae strain, Applied and Environmental Microbiology, 87, e00268-21 (2021). DOI10.1128 / aem.00268-21 8)

[0084] For example, in the case of alcoholic fermentation using yeast, the temperature conditions are adjusted appropriately depending on the type of yeast, etc. For example, 20°C or higher, 30°C or higher, or 38°C or lower, with preferred ranges being 20-38°C or 30-38°C.

[0085] Furthermore, the pH conditions are adjusted as appropriate depending on the type of yeast, for example, between 4 and 6.

[0086] Furthermore, the reaction time for alcoholic fermentation is appropriately adjusted depending on the type of yeast, reaction scale, etc., and can be, for example, 12 hours or more, 24 hours or more, 72 hours or less, 100 hours or less, and preferred ranges include 12 to 100 hours, 12 to 72 hours, 24 to 100 hours, and 24 hours to 72 hours.

[0087] Alcoholic fermentation can be carried out, for example, under atmospheric pressure.

[0088] Alcoholic fermentation is preferably carried out while stirring the monosaccharides and yeast. The stirring method is not particularly limited as long as it ensures that the monosaccharides and yeast are uniformly mixed, and examples include rotating a stirring device (such as a stirrer bar or stirring blade).

[0089] The present disclosure will be described in detail below with reference to examples and comparative examples. However, the present disclosure is not limited to the examples.

[0090] [Example 1] At room temperature (25°C) for monosaccharide production, five 50 ml vials (Mighty Vial No. 7) were prepared, and 400 mg of eucalyptus wood powder (pulverized lignocellulose (particle size 500-1000 μm)), which is lignocellulose, was added to each. A magnetic stirrer bar was added. Formic acid (98% purity, manufactured by Nacalai Tesque) and distilled water were mixed to prepare formic acid aqueous solutions with concentrations of 40% by mass, 60% by mass, 80% by mass, and 98% by mass, respectively.

[0091] A mixed solution was obtained by adding 20 mL of these formic acid aqueous solutions of different concentrations to each vial. For one sample, 20 mL of water without formic acid (0% formic acid concentration) was added to the mixed solution. Next, the mixed solution was degassed for 10 minutes using a vacuum pump and a desiccator for reduced pressure, and then left in the air for 30 minutes. Next, the mixed solution was stirred for 7 days at a temperature of 50°C using a magnetic stirring hot stirrer to obtain a lignocellulose-containing solution. During this time, the stirrer bar was rotated at the end of the vial so that frictional force (frictional force due to being sandwiched between the vial wall and the stirrer bar) was applied to the eucalyptus powder.

[0092] Next, the obtained lignocellulose-containing solution was subjected to filtration (Buchner funnel, filter paper: Advantec 55 mm No. 1) to separate it into solid and filtrate components. The obtained solid was washed with 40 mL of water to remove formic acid. 20 mL of the washing solution obtained by washing was added to the filtrate component, and the remainder was discarded. For the filtrate component, formic acid was removed by volatilization using an evaporator, and 10 mL of distilled water was added to the residue to suspend it. Through these steps, the polysaccharides contained in the lignocellulose-containing solution were separated into solid and filtrate components. The filtrate component was subjected to a rotary evaporator to remove the solvent formic acid and water to obtain a residue. After drying the solid and residue, their weights were measured, and the weights of the solid (mg) and filtrate component (mg) were as shown in Table 1.

[0093] Next, the entire amount of the solid component containing polysaccharides and the residue obtained by concentrating and drying the filtrate components were used as samples and subjected to enzymatic saccharification under the following conditions. This sample was mixed with MilliQ water (10 mL), and 10 mL of succinic acid aqueous solution (100 mM pH 5), 8 μL of 5% sodium azide (Sigma-Aldrich), and 10 FPU of saccharifying enzyme (Cellic® Ctec2) were added to a 100 mL Erlenmeyer flask, which was then stoppered with a rubber stopper and reacted under conditions of stirring speed of 150 rpm and temperature of 50°C.

[0094] Figure 1a shows the graph of the relationship between the amount of glucose produced (Glucose from pulp (mg)) and the reaction time (reaction time (days)) for enzymatic saccharification of solid samples using high-performance liquid chromatography (HPLC). Figure 1b shows the graph of the relationship between the amount of xylose produced (Xylose from pulp (mg)) and the reaction time (reaction time (days)). In Figure 1, for example, "80% FA" indicates the measurement results when an 80% by mass aqueous solution of formic acid was used, and "0% FA" indicates the measurement results when an aqueous solution without formic acid was used.

[0095] The conditions for high-performance liquid chromatography (HPLC) were as follows: Column: Aminex HPX-87H (1 column) + guard column, Mobile phase: 5 mM H2SO4, Flow rate: 0.4 mL / min, Column temperature: 65°C, Measurement time: 30 min / run, Injection volume: 3 μL.

[0096] For enzymatic saccharification using filtrate components as samples, the relationship between the amount of glucose produced (Glucose from filtrate (mg)) and the reaction time (reaction time (days)) is measured and shown in the graph in Figure 2a. Similarly, the relationship between the amount of xylose produced (Xylose from filtrate (mg)) and the reaction time (reaction time (days)) is measured and shown in the graph in Figure 2b.

[0097] The graph in Figure 1a shows that when using 80% formic acid in enzymatic saccharification of solid components as a sample, the efficiency of glucose production, a monosaccharide, is high. Similarly, the graph in Figure 2b shows that when using 80% formic acid in enzymatic saccharification of filtrate components as a sample, the efficiency of xylose production, a monosaccharide, is high. Furthermore, the graphs in Figures 1 and 2 reveal that in enzymatic saccharification of solid components as a sample, glucose is the main monosaccharide produced, while in enzymatic saccharification of filtrate components as a sample, xylose is the main monosaccharide produced. Therefore, by filtering the lignocellulose-containing solution produced in the mixing step and then separating it into solid and filtrate components for enzymatic saccharification, it is possible to separate the main monosaccharide components produced into glycol and xylose.

[0098] [Example 2] Production of monosaccharides (examination of enzyme amount) Monosaccharides were produced in the same manner as in Example 1, except that the concentration of the formic acid aqueous solution used was fixed at 80% by mass and the amount of enzyme used was increased from 10 FPU to 20 FPU, 30 FPU, or 40 FPU. The relationship between the amount of glucose produced (Glucose (mg)) and the reaction time (days) and the relationship between the amount of xylose produced (Xylose (mg)) and the reaction time (days) were measured.

[0099] In Example 2, the relationship between the amount of glucose produced (Glucose from pulp (mg)) and the reaction time (reaction time (days)) for enzymatic saccharification using solid matter as a sample is shown in the graph in Figure 3a. Similarly, the relationship between the amount of xylose produced (Xylose from pulp (mg)) and the reaction time (reaction time (days)) is shown in the graph in Figure 3b. The high-performance liquid chromatography (HPLC) conditions were the same as in Example 1.

[0100] Furthermore, in Example 2, the relationship between the amount of glucose produced (Glucose from filtrate (mg)) and the reaction time (reaction time (days)) was measured in the same manner for the enzymatic saccharification of the filtrate component sample, and the results are shown in the graph in Figure 4a. The relationship between the amount of xylose produced (Xylose from filtrate (mg)) and the reaction time (reaction time (days)) is also shown in the graph in Figure 4b.

[0101] The results from Example 2 confirmed that increasing the amount of enzyme used also increased the production of glucose and xylose.

[0102] <Measurement of lignin content when lignocellulose and formic acid are mixed> A lignocellulose-containing solution was obtained in the same manner as in Example 1. The obtained lignocellulose-containing solution was then subjected to filtration to separate the polysaccharides contained in the lignocellulose-containing solution into solid and filtrate components.

[0103] The masses of the obtained solids and filtrate components were measured, and the amount of lignin contained in the solids and filtrate components was determined by the Klason method. The results are shown in Table 1 below.

[0104]

[0105] The results shown in Table 1 indicate that when lignocellulose and formic acid are mixed in an environment with a temperature of 60°C or lower, the lignin and hemicellulose of the lignocellulose are separated, and the lignin is eluted into the filtrate.

[0106] [Example 3] Investigation of formylation A 50 ml vial (Mighty Vial No. 7) was prepared at room temperature (25°C), and 400 mg of eucalyptus wood powder (pulverized lignocellulose particle size 500-1000 μm), which is lignocellulose, was added. A magnetic stirrer tip was also added. Formic acid (Nacalai Tesque: concentration 98%) and distilled water were mixed to prepare an 80% by mass aqueous formic acid solution, and 20 mL of the aqueous formic acid solution was added to the vial to obtain a mixed solution. Next, the mixed solution was degassed for 10 minutes using a vacuum pump and a desiccator for reduced pressure, and then left in the air for 30 minutes. Next, the mixed solution was stirred for 7 days at a temperature of 50°C using a magnetic hot stirrer to obtain a lignocellulose-containing solution.

[0107] Next, the obtained lignocellulose-containing solution was subjected to filtration (Buchner funnel, filter paper: Advantec 55 mm No. 1) to separate it into solid and filtrate components. An 80% by mass aqueous solution of formic acid was added to the obtained solid, and this was washed with 10 mL of water to remove the formic acid. Through these steps, the polysaccharides contained in the lignocellulose-containing solution were separated into solid and filtrate components.

[0108] Next, at room temperature (25°C), the solid containing polysaccharides was immersed in 10 mL of a 50 g / L sodium carbonate aqueous solution for 20 minutes, and then washed with distilled water until the resulting distilled water was neutral. This process involves immersing the polysaccharide-containing solid in a sodium carbonate aqueous solution to convert the formyl groups, which are formed when the hydroxyl groups of the polysaccharides react with formic acid, into hydrogen atoms.

[0109] The solid content after washing was used as a sample and subjected to enzymatic saccharification under the following conditions. The amount of saccharifying enzyme (Cellic® Ctec2) charged was set to 10 FPU, 20 FPU, 30 FPU, and 40 FPU, respectively. 10 mL of a mixture of the sample and pure water (MilliQ water), 10 mL of succinic acid (100 mM pH 5), 8 μL of a 5 wt% solution of commercially available sodium azide (FUJIFILM Wako Pure Chemical Corporation), and the saccharifying enzyme (Cellic® Ctec2) were placed in a 100 mL Erlenmeyer flask, which was then stoppered with a rubber stopper and reacted under conditions of stirring speed of 150 rpm and temperature of 50°C.

[0110] Figure 5a shows the graph of the relationship between the amount of glucose produced (Glucose from pulp (mg)) and the reaction time (reaction time (hours)) for enzymatic saccharification of solid matter samples using high-performance liquid chromatography (HPLC). Figure 5b shows the graph of the relationship between the amount of xylose produced (Xylose from pulp (mg)) and the reaction time (reaction time (hours)). In Figure 5, for example, 40 FPU represents the measurement results when the amount of enzyme charged was 40 FPU. The conditions for high-performance liquid chromatography (HPLC) were the same as in Example 1.

[0111] A comparison of the graphs in Figure 3 and Figure 5 shows that the enzymatic saccharification rate is significantly improved by performing a step to convert the formyl group, which is generated when the hydroxyl group of a polysaccharide reacts with formic acid, into a hydrogen atom before enzymatic saccharification. Furthermore, Figure 12a shows data comparing the amount of glucose produced when the enzyme input amount was 30 FPU in Example 1 (Figure 3) and Example 3 (Figure 5), and Figure 12b shows data comparing the amount of xylose produced. From Figure 12, it can also be seen that the enzymatic saccharification rate is significantly improved when the step to convert the formyl group to a hydroxyl group is performed.

[0112] [Example 4] Investigation of mixing time At room temperature (25°C), a 50 ml vial (Mighty Vial No. 7) was prepared, and 400 mg of eucalyptus wood powder (pulverized lignocellulose particle size 500-1000 μm), which is lignocellulose, was added. An 80% by mass formic acid aqueous solution (manufactured by Nacalai Tesque) was prepared, and 20 mL of the formic acid aqueous solution was added to the vial to obtain a mixed solution. Next, the mixed solution was degassed for 10 minutes using a vacuum pump and a desiccator for reduced pressure, and then left in the air for 30 minutes. Next, using a hot stirrer, the mixed solution was stirred for 1 day, 2 days, 3 days, 4 days, 5 days, and 7 days, respectively, at a temperature of 50°C and a motor of 1.8-2.0, to obtain a lignocellulose-containing solution. In each case, the stirrer bar was rotated at the end of the vial so that frictional force (frictional force due to being sandwiched between the vial wall and the stirrer bar) was applied to the eucalyptus powder.

[0113] Next, the obtained lignocellulose-containing solution was subjected to filtration (Buchner funnel, filter paper: Advantec 55mm No.1) to separate the solid and filtrate components. An 80% by mass aqueous solution of formic acid was added to the obtained solid, and this was washed with 10 mL of water to remove the formic acid. Through these steps, the polysaccharides contained in the lignocellulose-containing solution were separated into solid and filtrate components. Through these steps, the polysaccharides contained in the lignocellulose-containing solution were separated into solid and filtrate components.

[0114] Next, the solid was immersed in 10 mL of a 50 g / L sodium carbonate aqueous solution for 20 minutes at room temperature (25°C). The solid was then washed with distilled water. Washing was continued until the washing solution became neutral. This step is a deformylation process, which converts the formyl groups formed by the reaction of hydroxyl groups of polysaccharides with formic acid into hydrogen atoms. Figure 6 shows a photograph of the solid obtained by mixing lignocellulose with formic acid aqueous solution. It can be seen that the amount of powder decreases as the mixing period lengthens.

[0115] The filtrate obtained by filtration was subjected to evaporative removal of formic acid using an evaporator to obtain the residue.

[0116] The entire amount of residue obtained by volatilizing and removing the solid components and polysaccharide-containing filtrate components after washing was used as a sample and subjected to enzymatic saccharification under the following conditions. The amount of saccharifying enzyme (Cellic® Ctec2) charged was set to 10 FPU, 20 FPU, 30 FPU, and 40 FPU, respectively. 10 mL of a mixture of the sample and pure water (MilliQ water), 10 mL of succinic acid (100 mM pH 5), 8 μL of 5 wt% sodium azide aqueous solution, and the saccharifying enzyme (Cellic® Ctec2) were placed in a 100 mL Erlenmeyer flask, stopped with a rubber stopper, and reacted under conditions of stirring speed of 150 rpm and temperature of 50°C.

[0117] [Example 5] Production of Monosaccharides (Verification of the Effects of Degassing and Friction) Monosaccharides were produced in the same manner as in Example 3, except that the mixed solution was not degassed in the step of mixing lignocellulose and formic acid aqueous solution as in Example 3, and the stirrer bar was rotated in the center of the vial to prevent frictional force (frictional force due to being sandwiched between the vial wall and the stirrer bar) from being applied to the eucalyptus powder. The relationship between the amount of glucose produced (Glucose (mg)) and the reaction time (reaction time (days)), and the relationship between the amount of xylose produced (Xylose (mg)) and the reaction time (reaction time (days)) were measured.

[0118] In Example 5, the relationship between the amount of glucose produced (Glucose from pulp (mg)) and the reaction time (reaction time (hours)) for enzymatic saccharification using solid content as a sample is shown in the graph in Figure 7a. Similarly, the relationship between the amount of xylose produced (Xylose from pulp (mg)) and the reaction time (reaction time (hours)) is shown in the graph in Figure 7b. The high-performance liquid chromatography (HPLC) conditions were the same as in Example 1.

[0119] The results from Example 5 confirmed that degassing the mixed solution during the mixing process and applying frictional force to the lignocellulose improve the efficiency of glucose and xylose production through enzymatic saccharification of polysaccharides.

[0120] [Example 6] Production of monosaccharides using film A lignocellulose-containing solution was obtained in the same manner as in Example 3. Next, the obtained lignocellulose-containing solution was transferred to a polypropylene tray (5 cm long, 8 cm wide, 1 cm high), and the formic acid was evaporated in a fume hood to obtain a film (eucalyptus film) with a thickness of 50 μm. This method will be referred to as the casting method below.

[0121] Next, at room temperature (25°C), the obtained film (containing polysaccharides) was immersed in 10 mL of a 50 g / L sodium carbonate aqueous solution for 20 minutes, and then washed with distilled water until the distilled water after washing was neutral to remove any remaining solids. As described above, this step is a deformylation step.

[0122] Next, the washed film was used as a sample and subjected to enzymatic saccharification under the following conditions. 10 mL of a mixture of the sample and pure water (MilliQ water), 10 mL of succinic acid (100 mM pH 5), 8 μL of 5 wt% sodium azide solution, and 30 FPU of saccharifying enzyme (Cellic® Ctec2) were placed in a 100 mL Erlenmeyer flask, which was then sealed with a rubber stopper and reacted at a stirring speed of 150 rpm and a temperature of 50°C.

[0123] In Example 6, the relationship between the amount of monosaccharides produced (glucose and xylose (Sugar yield (mg))) and the reaction time (reaction time (h)) was measured for enzymatic saccharification using a polysaccharide-containing film (eucalyptus film) as a sample. The results are shown in the graph in Figure 8a. The high-performance liquid chromatography (HPLC) conditions were the same as in Example 1.

[0124] Furthermore, in Example 6, measurements were taken when the film was not immersed in an aqueous sodium carbonate solution. That is, when deformylation was not performed. In this case, the film was directly enzymatically saccharified. The relationship between the amount of monosaccharides produced (glucose and xylose, Sugar yield (mg)) and the reaction time (h) in this case is shown in the graph in Figure 8b. The high-performance liquid chromatography (HPLC) conditions were the same as in Example 1.

[0125] It was confirmed that monosaccharides were successfully produced even when polysaccharide-containing films (eucalyptus films) were subjected to enzymatic saccharification. In particular, when the film was immersed in an aqueous sodium carbonate solution (graph in Figure 8a), the rate of glucose production by enzymatic saccharification was very fast. When the film was not immersed in an aqueous sodium carbonate solution (graph in Figure 8b), the film began to break down after three days of reaction, and the saccharification rate gradually increased.

[0126] Furthermore, when the film was directly added to the saccharification reaction solution, xylose was detected before the start of saccharification. The amount of xylose was also more than twice as high when the film was not immersed in the sodium carbonate solution. This is thought to be because xylose and xylooligosaccharides leached out of the film while it was immersed in the sodium carbonate solution.

[0127] [Example 7] Confirmation of the effect of the buffer solution Similar to Example 6, a lignocellulose-containing solution was formed into a film by the casting method to obtain a film (eucalyptus film) with a thickness of 50 μm. The film was immersed in 20 mL of succinate buffer for 30 minutes. By immersing the film in succinate buffer, xylose and xylooligosaccharides were actively eluted from the film. The succinate buffer used was prepared by adding 0.1 mol / L sodium succinate aqueous solution to 0.1 mol / L succinic acid aqueous solution to adjust the pH to 4.2.

[0128] Next, at room temperature (25°C), the film (containing polysaccharides) immersed in succinate buffer was immersed in 20 mL of a 50 g / L sodium carbonate aqueous solution for 20 minutes, and then the solids were washed with distilled water until the distilled water after washing was neutral. This step involves immersing the film containing polysaccharides in a sodium carbonate aqueous solution to convert the formyl groups, which are formed when the hydroxyl groups of the polysaccharides react with formic acid, into hydrogen atoms.

[0129] Next, the washed film was used as a sample and subjected to enzymatic saccharification under the same conditions as in Example 6.

[0130] In Example 7, the relationship between the amount of monosaccharides produced (glucose and xylose (Sugar yield (mg))) and the reaction time (reaction time (h)) was measured for enzymatic saccharification using a polysaccharide-containing film (eucalyptus film) as a sample. The results are shown in the graph in Figure 9. The high-performance liquid chromatography (HPLC) conditions were the same as in Example 1.

[0131] In Example 7, the film was immersed in succinate buffer solution before being immersed in sodium carbonate aqueous solution, resulting in an increased amount of xylose compared to Example 6.

[0132] [Example 8] Enzymatic saccharification in YEP medium Five 50 ml vials (Mighty Vial No. 7) were prepared at room temperature (25°C), and 400 mg of eucalyptus wood powder (pulverized lignocellulose particle size 500-1000 μm), which is lignocellulose, was added to each vial. An 80% by mass aqueous solution of formic acid (manufactured by Nacalai Tesque) was prepared for each vial, and 20 mL of the aqueous solution was added to each vial to obtain a mixed solution. Next, the mixed solution was degassed for 10 minutes using a vacuum pump and a desiccator for reduced pressure, and then left in the air for 30 minutes. After that, the mixture was stirred with a hot stirrer at a temperature of 50°C for 7 days to form a lignocellulose solution.

[0133] Next, a 50 μm thick film was obtained by casting using a lignocellulose-containing solution containing 2 g of eucalyptus powder (enough for five vials). The obtained film was immersed in 20 mL of succinate buffer containing 0.6 g of YEP medium (Peptone, Yeast extract, MP Biomedicals) for 30 minutes. The film and solution were separated into solid and liquid phases, and the solution was collected in a 100 mL Erlenmeyer flask that had been autoclaved to obtain the saccharified solution.

[0134] Next, the obtained film was immersed in 20 mL of a 50 g / L sodium carbonate aqueous solution for 30 minutes, and the removed film was further washed with distilled water to remove solid matter until the washing water became neutral.

[0135] Next, the washed film was placed in a 100 mL Erlenmeyer flask containing the stored succinate buffer reaction solution, sealed with a rubber stopper, and reacted at a stirring speed of 150 rpm, a temperature of 50°C, and for 144 hours to obtain a solution containing monosaccharides.

[0136] As described above, in Example 8, a polysaccharide-containing film (eucalyptus film) was used as a sample, and enzymatic saccharification was performed using YEP medium. The relationship between the amount of monosaccharides produced by enzymatic saccharification (glucose and xylose (Sugar yield (mg))) and the reaction time (h) is shown in the graph in Figure 11. The conditions for high-performance liquid chromatography (HPLC) were the same as in Example 1.

[0137] When a reaction solution containing YEP medium was used, approximately 3 days of enzymatic saccharification produced 769.6 ± 8.4 mg of glucose and 241.9 ± 10.1 mg of xylose from 2 g of eucalyptus. Thus, no adverse effects on monosaccharide production were observed when YEP medium, which is used in yeast fermentation, was used for enzymatic saccharification.

[0138] [Comparative Example 1] Measurement of Fermentation Inhibitor Content After Delignin Process Using Conventional Method Eucalyptus wood powder ground in a Willey mill was used, similar to Example 6. 400 mg of eucalyptus wood powder was added to 4 mL of 72 wt% sulfuric acid and stirred at room temperature for 30 minutes. Distilled water was then added to make up the total volume to 100 mL, and the sulfuric acid concentration was adjusted to 3 wt%. This mixture was heat-treated in an autoclave (internal temperature 120°C) for 1 hour to deligninize it. In the delignin process, cellulose and hemicellulose were completely decomposed into monosaccharides (glucose, xylose). This treated liquid was filtered to remove solids and obtain a filtrate. This filtrate was analyzed by high-performance liquid chromatography to quantify the content of glucose, xylose, HMF, and furfural. The results are shown in Table 2 below.

[0139]

[0140] [Example 9] Measurement of the content of the fermentation inhibitor (furfural) in this disclosure Five 50 ml vials (Mighty Vial No. 7) were prepared at room temperature (25°C), and 400 mg of eucalyptus wood powder (pulverized lignocellulose (particle size 500-1000 μm)), which is lignocellulose, was added to each vial. An 80% by mass aqueous solution of formic acid (manufactured by Nacalai Tesque) was prepared, and 20 mL of the aqueous solution was added to each vial to obtain a mixed solution. Next, the mixed solution was degassed for 10 minutes using a vacuum pump and a desiccator for reduced pressure, and then left in the air for 30 minutes. Next, the mixed solution was stirred for 7 days at a temperature of 50°C using a hot stirrer to obtain a lignocellulose-containing solution. The stirrer bar was rotated at the end of the vial so that frictional force (frictional force due to being sandwiched between the vial wall and the stirrer bar) was applied to the eucalyptus powder.

[0141] The resulting saccharified solution was subjected to high-performance liquid chromatography (HPLC), and the content (mg) of glucose, xylose, 5-hydroxymethylfurfural (IMF), and furfural was measured. The results are shown in Table 3 below. The conditions for high-performance liquid chromatography (HPLC) were the same as in Example 1.

[0142] The results shown in Table 2 reveal that when deligninization is performed using the conventional method, hydroxymethylfurfural (HMF) and furfural levels are high. Furfural compounds such as HMF and furfural are known to inhibit enzymatic saccharification and yeast fermentation.

[0143] On the other hand, the results in Table 3 show that mixing lignocellulose with formic acid promotes the decomposition of hemicellulose into xylose and xylooligosaccharides.

[0144] However, as is clear from the comparison between Table 2 and Table 3, the amount of furfural produced is extremely small. Compared to conventional methods that use strong acids such as sulfuric acid and high temperature and high pressure conditions, the method disclosed herein, which mixes lignocellulose and formic acid at a low temperature of 60°C or below, produces less furfural, which inhibits yeast fermentation.

[0145] [Example 10] Yeast fermentation of monosaccharides The monosaccharide-containing solution obtained by enzymatic saccharification in Example 8 was subjected to the following yeast fermentation to produce ethanol. As the yeast, a yeast capable of assimilating xylose was prepared according to the procedure in Non-Patent Literature 4. This is a genetically modified yeast that produces ethanol not only from glucose but also from xylose and glycerol. 10 mL of YPD medium to which a portion of the colonies of this yeast was added was centrifuged in a 50 mL Falcon tube at 30°C, 200 rpm, for 16 hours. Yeast from four Falcon tubes was used for yeast fermentation.

[0146] The monosaccharide-containing solution (saccharified liquid) obtained by enzymatic saccharification in Example 8 was also subjected to centrifugation, and the supernatant of the saccharified liquid was collected and subjected to yeast fermentation.

[0147] The yeast was washed with pure water, suspended in the supernatant of the saccharification solution, and placed in a 100 mL Erlenmeyer flask. Yeast fermentation was carried out at 30°C and 200 rpm, and samples were taken periodically to obtain the OD (Oxygen Demand). 600 Measurements and HPLC analysis were performed. Figure 11 shows the relationship between the amounts (mg) of glucose, xylose, and ethanol during yeast fermentation and the reaction time (h).

[0148] The graph in Figure 11 shows that the yeast fermentation in Example 10 almost completely converted the monosaccharides (glucose and xylose) obtained by enzymatic saccharification in Example 8 into ethanol. Specifically, it can be seen that most of the glucose was consumed within 4 hours from the start of the reaction, and xylose was consumed between 10 and 20 hours from the start of the reaction, producing ethanol. Compared to the consumption rate of glucose, the consumption rate of xylose was slower. It should be noted that the amount of ethanol decreased due to volatilization, and it began to decrease after 18 hours from the start of the reaction.

[0149] [Disclosure Items] Each of the following items discloses a preferred embodiment. Item 1. A method for producing a monosaccharide, comprising: a mixing step of mixing lignocellulose and formic acid in an environment with a temperature of 60°C or lower; and a monosaccharide production step of subjecting the polysaccharide contained in the lignocellulose mixture produced in the mixing step to enzymatic saccharification to obtain a monosaccharide containing glucose. Item 2. The method for producing a monosaccharide according to Item 1, further comprising a step of removing the formic acid between the mixing step and the monosaccharide production step. Item 3. The method for producing a monosaccharide according to Item 1 or 2, further comprising a step of converting the formyl groups contained in the polysaccharide into hydrogen atoms between the mixing step and the monosaccharide production step. Item 4. The method for producing a monosaccharide according to Item 3, wherein the step of converting the formyl groups contained in the polysaccharide into hydrogen atoms is carried out using sodium carbonate. Item 5. The method for producing a monosaccharide according to any one of Items 1 to 4, wherein the mixed solution of the lignocellulose and the formic acid is degassed in the mixing step. Item 6. A method for producing a monosaccharide according to any one of claims 1 to 5, wherein a frictional force is applied to the lignocellulose in the mixing step. Claim 7. A method for producing a monosaccharide according to any one of claims 1 to 6, further comprising an evaporation step of evaporating and drying the entire amount of the lignocellulose-containing solution produced in the mixing step without solid-liquid separation to obtain a solid, wherein in the monosaccharide production step, the polysaccharide contained in the solid is subjected to the enzymatic saccharification to obtain a monosaccharide containing glucose. Claim 8. A method for producing a monosaccharide according to claim 7, further comprising a step of converting the formyl groups of the polysaccharide contained in the solid into hydrogen atoms. Claim 9. A method for producing a monosaccharide according to any one of claims 1 to 8, further comprising a step of filtration of the lignocellulose-containing solution produced in the mixing step to separate it into solid and filtrate components. Claim 10. A method for producing a monosaccharide according to claim 9, wherein at least the polysaccharide contained in the solid is subjected to the monosaccharide production step. Claim 11. A method for producing a monosaccharide according to claim 9 or 10, wherein at least the polysaccharide contained in the filtrate component is subjected to the monosaccharide production step. Claim 12. A method for producing a monosaccharide according to any one of claims 1 to 11, wherein the monosaccharide further comprises xylose. Claim 13. A method for producing ethanol, comprising the steps of: obtaining a monosaccharide by the method according to any one of claims 1 to 12; and converting the monosaccharide into ethanol.Item 14. A method for producing ethanol, comprising the steps of: obtaining a monosaccharide by the manufacturing method described in Item 7; and converting the monosaccharide into ethanol.

[0150] (Method for producing monosaccharides) A ​​method for producing monosaccharides comprising: a mixing step of mixing a lignocellulose material and formic acid in an environment with a temperature of 60°C or lower; and a monosaccharide production step of subjecting the polysaccharide contained in the lignocellulose-containing mixture produced in the mixing step to enzymatic saccharification to obtain a monosaccharide containing glucose.

[0151] (Method for producing ethanol) A method for producing ethanol comprising: a step of obtaining a monosaccharide by the method for producing monosaccharides of the present disclosure; and a step of converting the monosaccharide into ethanol.

[0152] Patent Document 1: EP2573259A1 Patent Document 2: WO2021 / 125362 Patent Document 3: WO2023 / 058662

[0153] Non-patent document 1: Bioresource Technology Volume 101, Issue 6, March 2010, Pages 1570-1580 Non-patent document 2: Journal of Applied Glycoscience Volume 56 Issue 1 Pages 1-6 2009 Non-patent document 3: J.Appl.Glyosci.,56.1-6(2009) Non-patent document 4: Khattab, SMR, Watanabe, T., Efficient conversion of glycerol to ethanol by an engineered Saccharomyces cerevisiae strain, Applied and Environmental Microbiology, 87, e00268-21 (2021). DOI10.1128 / aem.00268-21 8

Claims

A mixing step in which lignocellulose and formic acid are mixed in an environment with a temperature of 60°C or lower, A monosaccharide production step is performed in which the polysaccharides contained in the lignocellulose mixture produced in the above mixing step are subjected to enzymatic saccharification to obtain monosaccharides containing glucose, A method for producing monosaccharides, including 　 The method for producing a monosaccharide according to claim 1, further comprising a step of removing the formic acid between the mixing step and the monosaccharide production step. 　 A method for producing a monosaccharide according to claim 1 or 2, further comprising a step of converting the formyl groups contained in the polysaccharide into hydrogen atoms between the mixing step and the monosaccharide production step. 　 The method for producing a monosaccharide according to claim 3, wherein the step of converting the formyl group contained in the polysaccharide into a hydrogen atom is carried out using sodium carbonate. 　 The method for producing a monosaccharide according to claim 1 or 2, wherein the mixed solution of lignocellulose and formic acid is degassed in the mixing step. 　 A method for producing a monosaccharide according to claim 1 or 2, wherein a frictional force is applied to the lignocellulose in the mixing step. 　 The process further comprises an evaporation step, in which the lignocellulose-containing solution produced in the mixing step is evaporated and dried in its entirety to obtain a solid without separating the solid and liquid components, A method for producing a monosaccharide according to claim 1 or 2, wherein in the monosaccharide production step, the polysaccharide contained in the solid is subjected to the enzymatic saccharification to obtain a monosaccharide containing glucose. 　 The method for producing a monosaccharide according to claim 7, further comprising the step of converting the formyl group of the polysaccharide contained in the solid into a hydrogen atom. 　 A method for producing a monosaccharide according to claim 1 or 2, further comprising the step of subjecting the lignocellulose-containing solution produced in the mixing step to filtration to separate it into solid components and filtrate components. 　 A method for producing a monosaccharide according to claim 9, wherein at least the polysaccharide contained in the solid is subjected to the monosaccharide production step. 　 The method for producing a monosaccharide according to claim 9, wherein at least the polysaccharide contained in the filtrate component is subjected to the monosaccharide production step. 　 The method for producing a monosaccharide according to claim 1 or 2, wherein the monosaccharide further comprises xylose. 　 A step of obtaining a monosaccharide by the manufacturing method described in claim 1 or 2, A step of converting the monosaccharide into ethanol, A method for producing ethanol, including the method described above. 　 A step of obtaining a monosaccharide by the manufacturing method described in claim 7, A step of converting the monosaccharide into ethanol, A method for producing ethanol, including the method described above.

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