Method for producing sugar solution, and sugar solution produced by the method

Abstract

To effectively use cellulose-containing biomass resources and the like.SOLUTION: A method for producing a sugar solution from fibrous raw materials, includes: a culturing step for culturing an organism that produces carbohydrase; and a saccharifying step for using carbohydrase produced from the organism to saccharify the fibrous raw materials, where the saccharifying step is, either partially or entirely, conducted in the same tank as the culturing step. Optionally, the culturing step and the saccharifying step may be conducted in succession.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a method for producing a sugar solution and the sugar solution obtained thereby.

Background Art

[0002] In the conventional production of a sugar solution from a cellulosic biomass raw material, the mainstream method is to add a saccharifying enzyme produced separately from a raw material tank to the raw material, that is, to perform the process in two lines of sugar solution production and conversion enzyme production. In this method, in addition to the sugar solution production process, equipment for the conversion enzyme production process is prepared, and a mixture of a plurality of enzymes is used as necessary in order to obtain an enzyme composition suitable for efficient saccharification of a substrate as a raw material. This is a technology for mass production of a sugar solution from a homogeneous raw material.

[0003] On the other hand, non-edible resources such as cellulosic biomass resources are widely and thinly distributed, and thus a utilization method using such heterogeneous resources has attracted attention.

Summary of the Invention

Means for Solving the Problems

[0004] As a result of proceeding with technology development assuming small-scale production of a sugar solution, the inventors of the present invention constructed a simple sugar solution production process that integrates conversion enzyme production and sugar solution production. In the method of the present disclosure, in producing sugar from a cellulosic resource such as rice straw using a microorganism, enzyme production by culturing a microorganism that produces a conversion enzyme and a saccharification reaction of a raw material are carried out in two steps by changing reaction conditions in the same tank, whereby efficient production of a sugar solution can be achieved.

[0005] Therefore, the present disclosure provides the following. (Item 1) A method for producing a sugar solution from a cellulosic raw material, comprising: a step of culturing an organism that produces a saccharifying enzyme; <A step of saccharifying the fibrous raw material using a saccharifying enzyme produced from the organism, wherein at least part or all of the saccharification step is carried out in the same tank as the cultivation step. A method comprising, if necessary, the culturing step and the saccharification step being carried out in succession. (Item 2) The method according to the above item, wherein the saccharification step is carried out under conditions in which the activity of at least a part of the organism is suppressed. (Item 3) The method according to any one of the above items, wherein the saccharification step is carried out under anaerobic conditions and / or at a temperature unsuitable for the growth of the organism. (Item 4) The method according to any one of the above items, wherein the temperature unsuitable for the growth of the organism is approximately 40°C or higher. (Item 5) The saccharification step is carried out under conditions in which the organism does not die, according to the method described in any one of the above items. (Item 6) The method according to any one of the above items, wherein the organism is a filamentous fungus or an actinomycete. (Item 7) The method according to any one of the above items, wherein the filamentous fungi include the genera Trichoderma and Penicillium. (Item 8) The method according to any one of the above items, wherein the actinomycetes include the genus Streptomyces. (Item 9) The method according to any one of the above items, wherein the fibrous raw material is not added externally. (Item 10) The method according to any one of the above items, wherein the culturing step is carried out under conditions such that the organism produces a sufficient amount of saccharifying enzyme to saccharify the fibrous raw material. (Item 11) The culture step is carried out for at least about 12 hours, according to the method described in any one of the above items. (Item 12) The method according to any one of the above items, characterized in that a switch from the culturing step to the saccharification step is performed based on the sugar concentration in the culture medium, the enzyme activity of the saccharifying enzyme, the biomass, the gas concentration, the metabolic rate of the organism, or the pH of the culture medium, or any combination thereof. (Item 13) A method for producing a sugar solution from fibrous raw materials, A process of providing the fibrous raw material, A step of adding an organism that produces or possesses a saccharifying enzyme to the fibrous raw material, A step of placing a mixture of the organism and the fibrous raw material under conditions in which the saccharifying enzyme acts on the fibrous raw material. Methods that include... (Item 14) The method according to any one of the above items, wherein the conditions under which the saccharifying enzyme acts on the fibrous raw material include conditions under which the activity of at least a part of the organism is suppressed. (Item 15) The method according to any one of the above items, further comprising the step of adding the organism to the fibrous raw material and then placing the organism under conditions that produce the saccharifying enzyme. (Item 16) The method according to any one of the above items, wherein the conditions under which the saccharifying enzyme acts on the fibrous raw material include anaerobic conditions and / or temperatures unsuitable for the growth of the organism. (Item 17) The method according to any one of the above items, wherein the temperature unsuitable for the growth of the organism is approximately 40°C or higher. (Item 18) The method according to any one of the above items, wherein the conditions under which the saccharifying enzyme acts on the fibrous raw material include conditions under which the organism does not die. (Item 19) The method according to any one of the above items, wherein the organism is a filamentous fungus or an actinomycete. (Item 20) The method according to any one of the above items, wherein the filamentous fungi include the genera Trichoderma and Penicillium. (Item 21) The method according to any one of the above items, wherein the actinomycetes include the genus Streptomyces. (Item 22) The method according to any one of the above items, wherein the fibrous raw material is not externally added. (Item 23) The method according to any one of the above items, wherein the conditions for the organism to produce the saccharifying enzyme include conditions such that the organism produces a sufficient amount of saccharifying enzyme for saccharification of the fibrous raw material. (Item 24) The method according to any one of the above items, wherein the conditions for the organism to produce the saccharifying enzyme include culturing for at least about 12 hours or more. (Item 25) Based on the sugar concentration in the culture solution, the enzyme activity of the saccharifying enzyme, the biomass, the gas concentration, the metabolic amount of the organism, or the pH of the culture solution, or any combination thereof, switching from the conditions for the organism to produce the saccharifying enzyme to the conditions for the saccharifying enzyme to act on the fibrous raw material is performed, and the method according to any one of the above items. (Item 26) The sugar solution produced by the method according to any one of the above items. (Item 27) A method for recycling an organism that produces a saccharifying enzyme in the production of a sugar solution from a fibrous raw material, a step of recovering the residue by the saccharification step according to any one of the above items, a step of recycling the organism in the residue as an organism that produces a saccharifying enzyme, and a method characterized by including.

[0006] In the present disclosure, it is intended that one or more of the above features can be provided in combination in addition to the explicitly stated combinations. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading the following detailed description as necessary.

[0007] Furthermore, any other features and notable effects of this disclosure will become clear to those skilled in the art by referring to the following sections on embodiments of the invention and the drawings. [Effects of the Invention]

[0008] The method disclosed herein is extremely simple and is expected to be useful not only for industrial-scale manufacturing but also for small-scale sugar solution production, such as at the farm level. The sugar solution obtained by this method can be used in a variety of applications as a fermentation raw material for alcohol, organic acids, amino acids, nucleic acids, etc., thus contributing to the effective utilization of underutilized and unused resources such as agricultural and food waste resources. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a graph showing the recovery rates of glucose and cellooligosaccharides when using the filamentous fungus Trichoderma reesei in a method according to one embodiment of the present disclosure. [Figure 2] Figure 2 is a graph showing the recovery rates of xylose and xylooligosaccharides when using the actinomycete Streptomyces sp. in a method according to one embodiment of the present disclosure. [Figure 3] Figure 3 is a graph showing the recovery rates of glucose and xylose when using the filamentous fungus Penicillium sp. in a method according to one embodiment of the present disclosure. [Modes for carrying out the invention]

[0010] The present disclosure is described below in best form. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Accordingly, singular articles (e.g., "a," "an," "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0011] The following provides definitions of terms used specifically in this specification and / or basic technical concepts as appropriate.

[0012] In this specification, "approximately" means ±10% of the following number.

[0013] In this specification, "fibrous raw material" is interpreted broadly and refers to raw materials that contain fibrous material as their main component, such as raw materials mainly containing cellulose or hemicellulose, or aggregates of cellulose and other fibers. Examples of fibrous raw materials include waste wood, thinned wood, forest residue, construction waste, sawmill waste, rice straw, wheat straw, rice husks, sugarcane bagasse, oil palm empty fruit bunches, corn stoba, corn cobs, switchgrass, Erianthus, Napier grass, green corn, pasture grass, reeds, bamboo, aquatic plants, seaweed, algae, microcrystalline cellulose, filter paper, recycled paper, and pulp.

[0014] In this specification, "sugar solution" is interpreted broadly and refers to a solution containing sugars such as monosaccharides, disaccharides, and oligosaccharides. In this specification, sugars contained in a sugar solution include, for example, three-carbon monosaccharides (trioses) such as glyceraldehyde; four-carbon monosaccharides (tetroses) such as erythrose, threose, and erythrulose; five-carbon monosaccharides (pentoses) such as ribose, lyxose, xylose, arabinose, deoxyribose, xylulose, and ribulose; allose, talose, glucose, altrose, mannose, galactose, idose, fucose, furose, rhamnose, and psichord. Examples include monosaccharides with 6 carbon atoms (hexoses) such as sucrose, fructose, sorbose, and tagatose; monosaccharides with 7 carbon atoms (heptoses) such as sedoheptulose; disaccharides such as sucrose, lactose, maltose, trehanose, turanose, and cellobiose; trisaccharides such as raffinose, melegitose, and maltotriose; oligosaccharides such as fructooligosaccharides, galactooligosaccharides, and mannaoligosaccharides; and polysaccharides such as starch, dextrin, cellulose, hemicellulose, glucan, and pentosan. The sugar solution may also contain components other than sugars.

[0015] In this specification, "saccharifying enzyme" refers to an enzyme that has the activity to catalyze a reaction that breaks down cellulose and starch to produce sugars, and includes, for example, α-amylase, glucoamylase, prunalase, α-glucosidase, transglucosidase, isoamylase, likenase, cellobiohydrolase, endoglucanase, β-glucosidase, cellobiose dehydrogenase, xylanase, α-L-arabinofuranosidase, β-D-xylosidase, α-glucuronidase, β-glucuronidase, acetylxylanesterase, feruloylesterase, β-mannanase, β-D-mannosidase, α-galactosidase, β-galactosidase, xyloglucanase, galactanase, arabinanase, pectinase, pectin methylesterase, pectin acetylesterase, biomass swelling enzyme, etc.

[0016] In this specification, "organisms that produce saccharifying enzymes" refers to any organism that produces saccharifying enzymes, such as filamentous fungi and basidiomycetes (fungi) of the genera Trichoderma, Aspergillus, Penicillium, Rhizopus, Irpex, Fanelloquite, and Humicola, as well as actinomycetes and Gram-positive rods (bacteria) of the genera Streptomyces, Thermobifida, Cerulomonas, Bacillus, and Clostridium. Even organisms that do not originally possess or produce saccharifying enzymes are considered to be organisms that produce saccharifying enzymes if they have been genetically modified to contain or produce saccharifying enzymes.

[0017] In this specification, "organisms possessing saccharifying enzymes" refers to any organism that possesses saccharifying enzymes, such as filamentous fungi and basidiomycetes (fungi) of the genera Trichoderma, Aspergillus, Penicillium, Rhizopus, Irpex, Fanelloquite, and Humicola, as well as actinomycetes and Gram-positive rods (bacteria) of the genera Streptomyces, Thermobifida, Cerulomonas, Bacillus, and Clostridium. Even organisms that do not inherently possess saccharifying enzymes are considered to possess them if they have been artificially manipulated (e.g., introduced) to contain saccharifying enzymes, even if they do not produce saccharifying enzymes themselves.

[0018] In this specification, "at least part or all of... carried out in the same tank" means that at least part or all of one process, reaction, or treatment is carried out in the same system, container, or tank as at least part or all of another process, reaction, or treatment.

[0019] In this specification, "continuously performed" means that separate processes are performed without interruption in time and / or space, and includes cases where separate processes are performed simultaneously. For example, "process A and process B are performed continuously" includes not only cases where process B is performed immediately after process A is completed in the same space, but also cases where process A and process B are performed in parallel in the same space.

[0020] In this specification, "not added externally" means that no components, molecules, or substances are added to the system from outside the substantially closed system.

[0021] In this specification, “conditions under which the activity of an organism is suppressed” or “conditions under which the activity of at least some of an organism is suppressed” means conditions under which any activity possessed by an organism is substantially reduced compared to the organism’s growth environment, and examples include anaerobic conditions and / or temperatures unsuitable for the growth of the organism.

[0022] In this specification, "conditions under which a saccharifying enzyme acts on a fibrous raw material" means conditions under which a saccharifying enzyme can catalyze a saccharification reaction using a fibrous raw material as a substrate, and includes conditions under which a saccharification reaction using a fibrous raw material as a substrate is substantially occurring.

[0023] In this specification, "filamentous fungi" is interpreted broadly and refers to all organisms that live as filamentous hyphae, commonly known as "molds." Filamentous fungi include genera such as Trichoderma, Aspergillus, Penicillium, and Rhizopus.

[0024] In this specification, "actinomycetes" is interpreted broadly to refer to prokaryotic microorganisms (such as bacteria) that produce branched, filamentous cells or hyphae, similar to mold. Actinomycetes include genera such as Streptomyces, Thermobifida, and Cellulomonas.

[0025] In this specification, "conditions for producing a sufficient amount of saccharifying enzyme to saccharify a fibrous raw material" means conditions under which a living organism produces a quantity and / or quality of saccharifying enzyme sufficient to substantially saccharify a fibrous raw material when the saccharifying enzyme carries out a saccharification reaction using the fibrous raw material as a substrate, and includes, for example, the temperature at which the organism producing the saccharifying enzyme can survive or grow, or the temperature or time at which the saccharifying enzyme can be produced. "The fibrous raw material is saccharified" means that at least a portion of the fibrous raw material is substantially saccharified.

[0026] In this specification, "switching from the culturing process to the saccharification process" means that the culturing of an organism that produces saccharifying enzymes is substantially interrupted or stopped, and the saccharification reaction of fibrous raw materials using the saccharifying enzymes produced from said organisms is substantially initiated.

[0027] In this specification, "reuse" of organisms that produce saccharifying enzymes means recovering organisms that produce saccharifying enzymes that were alive or present in one system and utilizing them in another system.

[0028] (Preferred embodiment) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and the scope of the Disclosure should not be limited to the descriptions below. It will be apparent that those skilled in the art can make appropriate modifications within the scope of the Disclosure, taking into consideration the descriptions herein. Furthermore, the embodiments of the Disclosure below can be used individually or in combination.

[0029] In one aspect of the present disclosure, a method is provided for producing a sugar solution from a fibrous raw material, comprising the steps of culturing an organism that produces a saccharifying enzyme, and saccharifying the fibrous raw material using the saccharifying enzyme produced from the organism, wherein at least part or all of the saccharifying step is carried out in the same tank as the culturing step, and the culturing step and the saccharifying step are carried out sequentially as necessary.

[0030] In other aspects of this disclosure, a method is provided for producing a sugar solution from a fibrous raw material, comprising the steps of: providing the fibrous raw material; adding an organism that produces or has a saccharifying enzyme to the fibrous raw material; and placing a mixture of the organism and the fibrous raw material under conditions that allow the saccharifying enzyme to act on the fibrous raw material.

[0031] Sugars such as glucose are used as raw materials for the fermentation production of various industrial raw materials. Furthermore, in light of recent global environmental issues and the efficient use of resources, there is a need to develop efficient sugar solution production processes from non-edible resources such as cellulose-containing biomass resources. On the other hand, these non-edible resources are not being fully utilized due to challenges such as their wide and thin distribution, high water content making them bulky and difficult to handle, the high costs of collection and transportation, and the difficulty in producing and supplying saccharifying enzymes that are appropriate for each type of resource due to their diverse nature.

[0032] In one embodiment of the present disclosure, the method of the present disclosure provides a simple sugar solution production process in which the production of a saccharifying enzyme and the production of a sugar solution from a fibrous raw material using the enzyme are carried out in the same tank. In one embodiment, by carrying out the sugar solution production process from the fibrous raw material under conditions that inactivate microbial metabolism, sugar consumption by microorganisms producing the saccharifying enzyme can be avoided, and the recovery rate of the sugar solution can be improved. Furthermore, by optimizing the timing of changes in conditions between the production of the saccharifying enzyme and the production of the sugar solution, efficient sugar recovery of carbohydrates in the raw material becomes possible. In addition, by devising a method for changing conditions, it is possible to omit the raw material and other addition steps in the sugar solution production process, in which case a reduction in the risk of microbial contamination and simplification of equipment can be expected.

[0033] <1> Fibrous raw materials As fibrous raw materials used in the method disclosed herein, for example, raw materials mainly containing cellulose or hemicellulose can be used. Examples of cellulosic raw materials include plant biomass. Examples of plant biomass include woody biomass and herbaceous biomass. Specific examples of woody biomass include waste wood, thinned wood, forest residue, construction waste, and sawmill waste. Specific examples of herbaceous biomass include rice straw, wheat straw, rice husks, sugarcane bagasse, oil palm empty fruit bunches, corn stober, corn cob, switchgrass, eryanthus, napier grass, green corn, pasture grass, reeds, and bamboo. Other examples of cellulosic raw materials include microcrystalline cellulose (Avicel), filter paper, recycled paper, and pulp. In one embodiment, one type of raw material may be used as the fibrous raw material, or two or more types of raw materials may be used in combination.

[0034] Fibrous raw materials such as plant biomass may be subjected to saccharification treatment as is, or after appropriate pretreatment. Examples of pretreatment include acid treatment, alkali treatment, hydrothermal treatment, subcritical water treatment, fine grinding treatment, steaming treatment, and microbial treatment. These pretreatments may be used individually or in combination. For example, plant biomass may be ground to approximately 5 mm or less and subjected to hydrothermal decomposition treatment.

[0035] Fibrous raw materials are generally composed of components such as cellulose, hemicellulose, and lignin. However, through hydrothermal decomposition of fibrous raw materials, the lignin and hemicellulose components migrate from the fibrous raw material into the hot water, while the cellulose component remains as a solid.

[0036] The reaction temperature (temperature of the pressurized hot water) for the hydrothermal decomposition treatment can be, for example, about 175 to 240°C, preferably about 200 to 230°C. Of the components of the fibrous raw material, the hemicellulose component dissolves at about 140°C or higher, the cellulose component dissolves at about 230°C or higher, and the lignin component dissolves at about 140°C or higher. Therefore, by performing the hydrothermal decomposition treatment at the above temperature range, the cellulose component can be sufficiently separated from the other components. After that, the hot water and solid components can be separated as needed and used as raw materials for sugar solution production.

[0037] When performing alkaline treatment, a dilute alkaline treatment effect can be achieved by reacting crushed and pulverized fibrous raw materials with a mixture mainly composed of calcium hydroxide and water at room temperature or under heating, and then neutralizing by passing carbon dioxide through and pressurizing as needed (Patent No. 5633839). In one embodiment, alkaline treatment can also be performed by mixing livestock manure combustion ash with fibrous raw materials in the presence of water and reacting them (Japanese Patent Publication No. 2021-170981).

[0038] <2> Glycation enzymes In this disclosure, "saccharifying enzyme" refers to an enzyme that has the activity to catalyze a reaction that breaks down cellulose or starch to produce sugar. Examples of saccharifying enzymes include α-amylase, glucoamylase, prunalase, α-glucosidase, transglucosidase, isoamylase, likenase, cellobiohydrolase, endoglucanase, β-glucosidase, cellobiose dehydrogenase, xylanase, α-L-arabinofuranosidase, β-D-xylosidase, α-glucuronidase, β-glucuronidase, acetylxylanesterase, feruloylesterase, β-mannanase, β-D-mannosidase, α-galactosidase, β-galactosidase, xyloglucanase, galactanase, arabinanase, pectinase, pectin methylesterase, pectin acetylesterase, and biomass swelling enzymes. As the saccharifying enzyme, one type of enzyme may be used, or a combination of two or more enzymes may be used. The saccharifying enzyme can be selected and used appropriately depending on the type of polysaccharide (such as cellulose or hemicellulose) contained in the fibrous raw material to be saccharified.

[0039] In one embodiment, saccharifying enzymes can be obtained by culturing organisms that produce saccharifying enzymes or cells derived from such organisms. For example, saccharifying enzymes can be obtained by culturing microorganisms or by culturing cells derived from animals or plants.

[0040] In one embodiment, the microorganism that produces saccharifying enzymes may be one that inherently produces saccharifying enzymes, or it may be one that has been modified to produce saccharifying enzymes. Microorganisms that produce saccharifying enzymes include filamentous fungi such as Trichoderma (e.g., Trichoderma reesei), Penicillium (e.g., Penicillium sp. IT-9 strain (Pb_IT_9) (NITE AP-03649) (NITE Patent Microorganism Depository Center (NPMD), received May 20, 2022)), and Acremonium (e.g., Acremonium cellulolyticus), actinomycetes such as Streptomyces (e.g., Streptomyces sp. K5 strain (K5 strain (Spores)) (NITE AP-03650), Streptomyces mexicanus P22A1, Streptomyces thermoviolaceus H33), and Thermobifida (e.g., Thermobifida cellulosilytica S3B10), and bacteria such as Clostridium thermocellum. Examples of Trichoderma reesei include Trichoderma reesei QM9414 (ATCC26921), Trichoderma reesei RutC-30 (ATCC56765), Trichoderma reesei PC-3-7 (ATCC66589), and Trichoderma reesei M2-1. When using fibrous raw materials, it is preferable to use Trichoderma species with excellent cellulase production capacity, and glucose repression-releasing mutants are even more preferable. Examples of glucose repression-releasing mutants include Trichoderma reesei M2-1 and Trichoderma reesei RutC-30, and an example of a partially glucose repression-releasing mutant is Trichoderma reesei PC-3-7.Other examples of filamentous fungi include Penicillium citrinum ATCC9849, Penicillium funiculosum ATCC11797, Penicillium oxalicum GZ-2, Aspergillus oryzae ATCC14895, Aspergillus nidulans ATCC62041, Acremonium cellulolyticus C1, and Rhizopus oryzae ATCC20344. Examples of actinomycetes include Streptomyces lividans 1326, Streptomyces albus ATCC3005, Thermobifida fusca ATCC27730, and Cellulomonus fimi ATCC484. Examples of bacteria include Clostridium thermocellum ATCC27405 and Bacillus licheniformis ATCC14580.

[0041] While not strictly theoretical, using glucose-repression-releasing mutant strains allows for the production of saccharifying enzymes even in the presence of glucose. As a result, the pre-culture medium using glucose-repression-releasing mutant strains contains a small amount of saccharifying enzymes in addition to the bacterial cells. By inoculating this pre-culture medium as the bacterial solution for the main culture, the saccharifying enzymes act as a kind of booster, accelerating the release of sugars from fibrous raw materials. Consequently, the growth of organisms (e.g., microorganisms) is accelerated, and the culture time required to produce saccharifying enzymes can be shortened.

[0042] Furthermore, microorganisms that produce saccharifying enzymes can be obtained, for example, by introducing a gene encoding a saccharifying enzyme into a host microorganism so that it can be expressed. Examples of host microorganisms include bacteria belonging to the Enterobacteriaceae family, such as Escherichia bacteria, and Corynebacterium bacteria. Preferred host microorganisms include Escherichia coli and Corynebacterium glutamicum. Gene introduction can be achieved, for example, by introducing a vector containing the gene into the microorganism, or by introducing the gene onto the chromosome of the microorganism. The amino acid sequences of various saccharifying enzymes and the base sequences of the genes encoding them can be obtained, for example, from public databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / ). In one embodiment, saccharifying enzymes can also be produced by expressing a gene encoding a saccharifying enzyme in a cell-free protein synthesis system.

[0043] In one embodiment, the production of saccharifying enzymes by organisms or microorganisms can be carried out by placing the organisms or microorganisms under conditions that produce saccharifying enzymes. The culture conditions for organisms or microorganisms that produce saccharifying enzymes are not particularly limited, as long as the organisms, such as microorganisms, can grow and a sufficient amount of saccharifying enzymes are produced for the saccharification of fibrous raw materials. In one embodiment, microorganisms that produce saccharifying enzymes can be cultured under the usual conditions for culturing microorganisms such as filamentous fungi and actinomycetes. For specific culture conditions, conventionally known culture conditions for various microorganisms that produce saccharifying enzymes, such as Trichoderma reesei, can be referred to. In one embodiment, the culture of organisms such as microorganisms can be carried out for at least about 6 hours, about 9 hours, about 12 hours, about 18 hours, or about 24 hours or more, as long as a sufficient amount of saccharifying enzymes is produced for the saccharification of fibrous raw materials.

[0044] In one embodiment, the culture medium for culturing an organism (e.g., a microorganism) that produces saccharifying enzymes in the method of the present disclosure is not particularly limited, as long as it allows the organism, such as a microorganism, to grow and produces a sufficient amount of saccharifying enzyme to saccharify the fibrous raw material. In one embodiment, the culture medium used in the method of the present disclosure may also contain saccharifying enzymes beforehand. In this case, the saccharifying enzymes contained in the culture medium beforehand may be the same as or different from the saccharifying enzymes produced by the organism (e.g., a microorganism) cultured in the culture medium. Although not bound by theory, by including saccharifying enzymes in the culture medium when culturing an organism (e.g., a microorganism), the saccharifying enzymes act as a booster, so to speak, accelerating the release of sugars from the fibrous raw material, and as a result, the growth of the organism (e.g., a microorganism) can be accelerated, and the culture time required to produce saccharifying enzymes can be shortened.

[0045] The produced saccharifying enzyme can be used, for example, while still contained in the culture medium, in the subsequent saccharification process of fibrous raw materials. In one embodiment, the method of this disclosure allows at least part or all of the cultivation of an organism that produces saccharifying enzymes and the saccharification process of fibrous raw materials using the saccharifying enzymes produced from that organism to be carried out in the same tank, and the cultivation and saccharification processes can be carried out continuously. Therefore, the saccharifying enzyme can be used in the saccharification reaction without being subjected to treatments such as concentration, dilution, drying, fractionation, purification, or formulation.

[0046] In one embodiment, the switch from culturing organisms that produce saccharifying enzymes to the saccharification reaction of fibrous raw materials can be appropriately set according to various conditions such as the type of organism or microorganism, the type of saccharifying enzyme, the type and amount of fibrous raw materials, and the reaction temperature. In one embodiment, the switch to the saccharification reaction can be performed based on the sugar concentration in the culture medium, the enzyme activity of the saccharifying enzyme, the biomass, the gas concentration, the metabolic rate of the organism, or the pH of the culture medium, or any combination thereof, and such switching conditions can be appropriately set based on preliminary experiments.

[0047] <3> Sugar solution production In one embodiment, a sugar solution can be produced using the fibrous raw material and saccharifying enzyme described above. The production of a sugar solution realized by the method of this disclosure can be carried out by culturing organisms such as microorganisms that produce saccharifying enzymes, and the culturing and saccharification can be carried out continuously in the same tank as the culturing, at least part or all of which is the same tank. In one embodiment, the saccharification reaction may be carried out by standing still, or by stirring or shaking. In one embodiment, the saccharification step using enzymes is preferably carried out under conditions in which the activity of at least part of the organisms such as microorganisms that produce saccharifying enzymes is suppressed so that the organisms do not assimilate the fibrous raw material that will be used as raw material for the sugar solution. Examples of such conditions include temperature conditions unsuitable for the growth of microorganisms that produce saccharifying enzymes and anaerobic conditions. Temperatures unsuitable for the growth of microorganisms that produce saccharifying enzymes include, for example, temperatures above approximately 38°C, 39°C, 40°C, 42°C, 45°C, 47°C, 50°C, 60°C, 70°C, 80°C, and 90°C.

[0048] Conditions that suppress the activity of at least some of the organisms producing saccharifying enzymes, and temperatures unsuitable for their growth, can be appropriately set according to various conditions such as the type of organism or microorganism, the type of saccharifying enzyme, the type of fibrous raw material, and the desired saccharification rate. In other words, reaction conditions can be appropriately set, for example, by referring to the reaction conditions of saccharifying enzymes or based on preliminary experiments.

[0049] In the method of this disclosure, at least part or all of the process of culturing an organism that produces saccharifying enzymes and the process of saccharifying a fibrous raw material using the saccharifying enzymes produced from that organism are carried out in the same tank. Therefore, in one embodiment of this disclosure, in order to reuse the organism in other systems, it is preferable that the saccharification process using the enzyme obtained from the organism that produces saccharifying enzymes be carried out under conditions that do not kill the organism.

[0050] Therefore, in one aspect of this disclosure, in such cases, a method is provided for reusing organisms that produce saccharifying enzymes in the production of a sugar solution from fibrous raw materials, characterized by comprising the steps of: recovering the residue from a saccharification process using enzymes obtained from organisms that produce saccharifying enzymes; and reusing the organisms in the residue as organisms that produce saccharifying enzymes. When the sugar solution is recovered after the saccharification process is completed, the saccharifying enzymes coexist in the culture medium or attached to the residue. Therefore, in one embodiment of this disclosure, not only the organisms that produce saccharifying enzymes, but also the saccharifying enzymes contained in the sugar solution or residue can be recovered and reused.

[0051] In one embodiment of the present disclosure, the method of the present disclosure preferably carries out at least part or all of the cultivation of an organism that produces saccharifying enzymes and the saccharification process of a fibrous raw material using the saccharifying enzymes produced from that organism in the same tank, and the cultivation and saccharification processes can be carried out continuously, so it is preferable not to add the fibrous raw material that will be used as the raw material for the sugar solution from outside. In this way, cultivation and saccharification can be carried out in a substantially closed environment, and contamination such as contamination by other microorganisms can be prevented.

[0052] The sugars contained in the sugar solution produced by the method of this disclosure are not particularly limited and may be so-called sugars in general. Examples of sugars contained in the sugar solution produced by the method of this disclosure include: three-carbon monosaccharides (trioses) such as glyceraldehyde; four-carbon monosaccharides (tetroses) such as erythrose, threose, and erythrulose; five-carbon monosaccharides (pentoses) such as ribose, lyxose, xylose, arabinose, deoxyribose, xylulose, and ribulose; allose, talose, gross, glucose, altrose, mannose, galactose, idose, fucose, fuculose, rhamnose, Examples include six-carbon monosaccharides (hexoses) such as psicose, fructose, sorbose, and tagatose; seven-carbon monosaccharides (heptoses) such as sedoheptulose; disaccharides such as sucrose, lactose, maltose, trehanose, turanose, and cellobiose; trisaccharides such as raffinose, melegitose, and maltotriose; oligosaccharides such as fructooligosaccharides, galactooligosaccharides, and mannaoligosaccharides; and polysaccharides such as starch, dextrin, cellulose, hemicellulose, glucan, and pentosan. In one embodiment of the present disclosure, the sugar solution of the present disclosure may contain one of the above sugars alone or two or more.

[0053] In one aspect of this disclosure, a sugar solution produced by the method of this disclosure is provided. In one embodiment, the sugar solution produced by the method of this disclosure is not particularly limited as long as it contains the sugars described above, however, it is preferable to remove solids derived from fibrous raw materials by filtration, adsorption, etc. Furthermore, the sugar solution produced by the method of this disclosure can be used by diluting it with water to lower the sugar concentration, or by increasing the sugar concentration by adding sugars or concentrating it, depending on the purpose of use. [Examples]

[0054] (Example 1: Sugar solution production using the filamentous fungus Trichoderma reesei) <Preparation of raw materials> In a 1L medium bottle, 100g of dried rice straw powder (variety: Koshihikari), 15g of calcium hydroxide, and 900mL of pure water were added and thoroughly mixed. The mixture was then heated in an autoclave at 120°C for 1 hour (alkaline treatment of rice straw). After cooling, HCl was added while stirring until the pH of the suspension reached approximately 3. After standing overnight at 4°C, the solids were recovered by centrifugation (neutralization step). Pure water was added to the solids to suspend them, and the mixture was stirred for approximately 30 minutes. The solids were then recovered by centrifugation. This process was repeated until the pH of the suspension reached approximately 7 (washing step). Pure water was added to the recovered solids to a final concentration of 10% (w / w) to obtain a pre-treated rice straw raw material slurry.

[0055] In a 100 mL Erlenmeyer flask, 15 g of the above raw material slurry (equivalent to 1.5 g dry weight), 3 g of nutrient solution, 3 g of 0.5 M tartrate buffer (pH 4.0), and 9 g of pure water were placed and sterilized by autoclaving (121°C, 20 minutes) to obtain the "raw material slurry for saccharification." The composition of the nutrient solution was 14 g / L (NH4)2SO4, 20 g / L KH2PO4, 3 g / L CaCl2·2H2O, 3 g / L MgSO4·7H2O, 10 g / L Polypepton (Wako), 5 g / L Yeast Extract (Difco), 10 g / L Tween 80, 0.6 mg / L H3BO3, 2.6 mg / L (NH4)6Mo7O 24 ·4H2O, 10mg / L FeCl3·6H2O, 4mg / L CuSO4·5H2O, 0.8mg / L MnCl2·4H2O, 20mg / L ZnCl2.

[0056] <Preculture> In a 500mL Erlenmeyer flask, add preculture medium (20g / L Glucose, 1.4g / L (NH4)2SO4, 2.0g / L KH2PO4, 0.3g / L CaCl2·2H2O, 0.3g / L MgSO4·7H2O, 1.0g / L Polypepton (Wako), 0.5g / L Yeast Extract(Difco), 1.0g / L Tween 80, 0.06mg / L H3BO3, 0.26mg / L (NH4)6Mo7O 24100 mL of the following culture medium was added: 4H2O, 1.0 mg / L FeCl3·6H2O, 0.4 mg / L CuSO4·5H2O, 0.08 mg / L MnCl2·4H2O, 2.0 mg / L ZnCl2) and sterilized by autoclaving (121°C, 20 minutes). After cooling, the culture medium was mixed with conidia (1 × 10⁴) of the filamentous fungus Trichoderma reesei M2-1 strain. 8 The culture medium was inoculated and incubated in a rotary shaker (TITEC G / BR-200) at 28°C and 180 rpm for 72 hours. This culture medium was used as the pre-culture medium.

[0057] <Sugar liquid manufacturing process> To the aforementioned saccharification raw material slurry, 1.5 g of pre-culture solution was inoculated and the mixture was rotated and shaken at 28°C and 150 rpm in a rotary shaker (TITEC BR-23UM) (enzyme production phase). The incubation time at 28°C, i.e., the duration of the enzyme production phase, was set to 0 hours, 9 hours, 24 hours, 48 ​​hours, and 120 hours (B1, B2, B3, B4, and B5 respectively in the graph in Figure 1). After each set time, the flask was transferred to a rotary shaker (TITEC BR-23UM) set to 50°C and rotated and shaken at 150 rpm (saccharification phase). Rotation and shaking were continued until the total incubation period (enzyme production phase + saccharification phase) reached 120 hours. A 0.2g slurry sample was taken daily, and glucose and cellooligosaccharides in the supernatant were measured according to the method described in M. Ike, et al., Journal of Applied Glycoscience 60, 177-185 (2013). Three flasks were used for each condition, and the average values ​​were calculated and shown in Figure 1.

[0058] (Example 2: Sugar solution production using actinomycetes Streptomyces sp.) 200g of slaked lime was added to 2kg of dried rice straw (variety: Koshihikari) and tap water was added to adjust the overall moisture content to 60% (w / w), after which it was treated with a fiberizer. The defibrated sample was placed in an autoclave bag and heated in an autoclave at 95°C for 1 hour. After cooling, 20 times the amount of HCl (1N) relative to the wet weight was added and shaken for 1 hour to dissolve the calcium. After neutralization with sodium bicarbonate (powder), it was dewatered using a manual press. Next, the same amount of tap water was added instead of HCl and shaken for 1 hour to perform a washing operation. After dewatering, the same operation was repeated three times to obtain HCl-washed pre-treated rice straw (HW-PRS).

[0059] Four 300 mL Erlenmeyer flasks were each filled with 2.5 g of HW-PRS (equivalent to dry weight (DM)) in a buffer solution (NaNO3 24 mM, KCl 79 mM, Na2HPO4 11 mM, MgSO4-7H2O 0.6 mM, FeSO4-5H2O 0.16 mM, MES (2-(N-morpholino)ethanesulfonic acid) 20 mM, pH=6.0) (total 50 g), and autoclaved at 120°C for 15 minutes. On the other hand, 0.01 mL of Streptomyces sp. K5 strain (K5 strain (Spores)) (Patent Microorganism Depository Center (NPMD), received May 20, 2022, NITE AP-03650) strain spore slurry was added to 50 mL of LB (Luria-Broth) medium, and the culture was shaken at 200 rpm and 50°C for 24 hours to prepare a starter culture. 0.5 mL of the starter culture was inoculated into a buffer containing 2.5 g of the above HW-PRS, and after sampling 1 mL of the supernatant (d0 in Figure 2), the flask was cultured under aerobic conditions with shaking at 200 rpm and 50°C for 24 hours, using a permeable silicone stopper (Shin-Etsu Polymer, model C-30) as a lid. After sampling 1 mL of the supernatant (d1 in Figure 2), the lids of the two flasks were replaced from silicone stoppers to silicone stoppers for anaerobic culture. Two vent tubes were inserted into the silicone stopper for anaerobic culture. Nitrogen gas was purged from one tube for 2 minutes, and then exhausted through the check valve on the other tube to purge the inside of the flask with nitrogen gas. After venting, the tubes were closed with pinchcocks to prevent the inflow of outside air, maintaining anaerobic conditions, and shaking culture was continued under the same conditions. The remaining two flasks were cultured with the silicone stopper as a lid and served as a control group. After 24 hours, 1 mL of the supernatant was sampled (d2 in Figure 2). The flask under anaerobic conditions was purged with nitrogen gas using the method described above, and culture was continued for another 24 hours before 1 mL of the supernatant was sampled (d3 in Figure 2).The total xylan (free TX) concentration in the culture supernatant and the total xylan (substrate TX) in 2.5 g of HW-PRS were measured according to the method described in M. Ike, et al., Journal of Applied Glycoscience 60, 177-185 (2013), and the xylan release rate was evaluated using the following formula: Total xylan (free TX) concentration in the culture supernatant (mg / ml) × 47.5 (mL) / Total xylan weight in 2.5 g of HW-PRS (mg). The average values ​​for the anaerobic condition group (2 flasks) and the aerobic condition group (2 flasks) were calculated, and the results are shown in Figure 2.

[0060] (Example 3: Sugar solution production using the filamentous fungus Penicillium sp.) <Preparation of raw materials> In a 1L medium bottle, 100g of dried rice straw powder (variety: Koshihikari), 15g of calcium hydroxide, and 900mL of pure water were added and thoroughly mixed. The mixture was then heated in an autoclave at 120°C for 1 hour (alkaline treatment of rice straw). After cooling, HCl was added while stirring until the pH of the suspension reached approximately 3. After standing overnight at 4°C, the solids were recovered by centrifugation (neutralization step). Pure water was added to the solids to suspend them, and the mixture was stirred for approximately 30 minutes. The solids were then recovered by centrifugation. This process was repeated until the pH of the suspension reached approximately 7 (washing step). Pure water was added to the recovered solids to a final concentration of 10% (w / w) to obtain a pre-treated rice straw raw material slurry.

[0061] In a 100 mL Erlenmeyer flask, 15 g of the above raw material slurry (equivalent to 1.5 g dry weight), 3 g of nutrient solution, 3 g of 0.5 M tartrate buffer (pH 4.0), and 9 g of pure water were placed and sterilized by autoclaving (121°C, 20 minutes) to obtain the "raw material slurry for saccharification." The composition of the nutrient solution was 14 g / L (NH4)2SO4, 20 g / L KH2PO4, 3 g / L CaCl2·2H2O, 3 g / L MgSO4·7H2O, 10 g / L Polypepton (Wako), 5 g / L Yeast Extract (Difco), 10 g / L Tween 80, 0.6 mg / L H3BO3, 2.6 mg / L (NH4)6Mo7O24 ·4H2O, 10mg / L FeCl3·6H2O, 4mg / L CuSO4·5H2O, 0.8mg / L MnCl2·4H2O, 20mg / L ZnCl2.

[0062] <Preculture> In a 500mL Erlenmeyer flask, add preculture medium (20g / L Glucose, 1.4g / L(NH4)2SO4, 2.0g / L KH2PO4, 0.3g / L CaCl2·2H2O, 0.3g / L MgSO4·7H2O, 1.0g / L Polypepton (Wako), 0.5g / L Yeast Extract(Difco), 1.0g / L Tween 80, 0.06mg / L H3BO3, 0.26mg / L (NH4)6Mo7O 24 100 mL of the following mixtures were added: 4H2O, 1.0 mg / L FeCl3, 6H2O, 0.4 mg / L CuSO4, 5H2O, 0.08 mg / L MnCl2, 4H2O, 2.0 mg / L ZnCl2) and sterilized by autoclaving (121°C, 20 minutes). After cooling, the culture medium was mixed with conidia (1 × 10¹⁴) of the filamentous fungus Penicillium sp. IT-9 strain (Pb_IT_9) (Patent Microorganism Depository Center (NPMD), received May 20, 2022, NITE AP-03649). 8 The culture medium was inoculated and incubated in a rotary shaker (TITEC G / BR-200) at 28°C and 180 rpm for 72 hours. This culture medium was used as the pre-culture medium.

[0063] <Sugar liquid manufacturing process> To the aforementioned saccharification raw material slurry, 1.5 g of pre-culture solution was inoculated and the mixture was rotated and shaken at 150 rpm in a rotary shaker (TITEC BR-23UM) at 28°C (enzyme production phase). The incubation time at 28°C, i.e., the duration of the enzyme production phase, was set to 0 hours, 9 hours, 24 hours, 48 ​​hours, and 120 hours (P1, P2, P3, P4, and P5 in the graph in Figure 3, respectively). After each set time, the flask was transferred to a rotary shaker (TITEC BR-23UM) set to 50°C and rotated and shaken at 150 rpm (saccharification phase). Rotation and shaking were continued until the total incubation period (enzyme production phase + saccharification phase) reached 120 hours. After the process was completed, the amounts of glucose and xylose contained in the supernatant were measured according to the method described in M. Ike, et al., Journal of Applied Glycoscience 60, 177-185 (2013), and the results are shown in Figure 3.

[0064] (Example 4: Example 1 of switching between the culture process and the saccharification process) Similar to the <sugar solution production process> in Example 1, 1.5 g of pre-culture solution is inoculated into the saccharification raw material slurry, and the mixture is rotated and shaken at 28°C and 150 rpm using a rotary shaker (TITEC BR-23UM) to produce the saccharifying enzyme. The pH is monitored during the culture time at 28°C, and when a decrease in pH (for example, a decrease of about 0.1) is observed, the temperature is switched to 50°C, and the saccharification process using the obtained saccharifying enzyme is initiated to obtain the sugar solution.

[0065] During the incubation period at 28°C, as the bacterial strain grows actively, it consumes sugar and the pH decreases. The degree of pH decrease that serves as a guideline for switching temperatures can be appropriately set depending on other initial incubation conditions and the microorganisms used, or the setting value that maximizes sugar yield can be determined through preliminary experiments.

[0066] (Example 5: Example 2 of switching between the culture process and the saccharification process) In the sugar solution production process of Example 1, instead of a rotary shaker, a 5L fermenter is used to produce saccharifying enzymes by culturing at 28°C while aerating and stirring. The oxygen concentration released during the culturing time at 28°C is monitored, and when it has decreased by a certain percentage compared to the oxygen concentration at the start of culturing, the temperature is switched to 50°C, and the saccharification process using the obtained saccharifying enzyme is initiated to obtain a sugar solution.

[0067] During the incubation period at 28°C, as the bacterial strains become more active, they consume oxygen, and the oxygen concentration decreases as the number of strains increases. After that, it stabilizes, and as bacterial activity decreases, the oxygen concentration increases. Therefore, by evaluating the relationship between the degree of oxygen concentration decrease and sugar yield in advance, it can be used as an indicator for the timing of temperature changes.

[0068] (Example 6: Method for reusing organisms that produce saccharifying enzymes) In the sugar solution manufacturing process of Example 1, a portion of the suspension is collected after the entire culture period (enzyme production phase + saccharification phase) has been completed. Since this suspension contains organisms that produce saccharifying enzymes and saccharifying enzymes themselves, this suspension can be added to a newly prepared slurry of raw materials for saccharification instead of the pre-culture medium, and the saccharification process can be carried out to obtain a sugar solution.

[0069] Furthermore, the recovered suspension can be replaced with solid residue. Also, since the supernatant (including the prepared sugar solution) contains saccharifying enzymes, the sugar solution can be obtained by adding this together with the pre-culture medium.

[0070] (Note) As described above, while the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of this disclosure should be interpreted solely by the claims. Patents, patent applications and other documents cited herein should be incorporated herein by reference as if their contents were specifically described herein. [Industrial applicability]

[0071] According to this disclosure, non-edible resources such as cellulose-containing biomass resources, which are abundant resources, can be utilized in the food and other materials fields. In particular, since it is possible to produce sugar solutions on a small scale using cellulose-containing biomass resources, it is expected to be used for small-scale sugar solution production at the farm level and other locations. Furthermore, according to this disclosure, the obtained sugar solution can be used in various applications as a fermentation raw material for alcohol, organic acids, amino acids, nucleic acids, etc., making it useful in the field of non-edible materials as well.

Claims

1. A method for producing a sugar liquid from a fiber raw material, comprising the steps of: Cultivating an organism that produces a saccharifying enzyme; a step of saccharifying the fiber raw material using a saccharifying enzyme produced by the organism, the saccharification step being carried out at least in part or entirely in the same tank as the culturing step; and, if necessary, the culturing step and the saccharification step are carried out consecutively.

2. The method according to claim 1 , wherein the saccharification step is carried out under conditions in which at least a portion of the activity of the organism is inhibited.

3. The method according to claim 1 or 2, wherein the saccharification step is carried out under anaerobic conditions and / or at a temperature not suitable for the growth of the organism.

4. 4. The method of claim 3, wherein the temperature unsuitable for growth of the organism is about 40° C. or higher.

5. The method according to claim 1 or 2, wherein the saccharification step is carried out under conditions that do not kill the organism.

6. The method according to claim 1 or 2, wherein the organism is a filamentous fungus or an actinomycete.

7. The method of claim 6 , wherein the filamentous fungi include the genera Trichoderma and Penicillium.

8. The method of claim 6 , wherein the actinomycete comprises the genus Streptomyces.

9. 3. The method of claim 1 or 2, wherein the fibrous raw material is not added exogenously.

10. 3. The method of claim 1 or 2, wherein the culturing step is carried out under conditions such that the organism produces a sufficient amount of saccharification enzymes to saccharify the fibrous material.

11. The method of claim 1 or 2, wherein the culturing step is carried out for at least about 12 hours or more.

12. 3. The method according to claim 1 or 2, characterized in that the culture step is switched to the saccharification step based on the sugar concentration in the culture solution, the enzymatic activity of the saccharification enzyme, the biomass, the gas concentration, the metabolic rate of the organism, or the pH of the culture solution, or any combination thereof.

13. A method for producing a sugar liquid from a fiber raw material, comprising the steps of: Providing the fiber raw material; adding to the fiber material an organism that produces or has a saccharification enzyme; placing the mixture of the organism and the fiber material under conditions in which the saccharifying enzyme acts on the fiber material; A method comprising:

14. The method of claim 13 , wherein the conditions under which the saccharification enzyme acts on the fiber material include conditions under which at least a portion of the activity of the organism is inhibited.

15. 15. The method of claim 13 or 14, further comprising the step of, after adding the organism to the fiber feedstock, placing the organism under conditions in which it produces the saccharification enzymes.

16. 15. The method of claim 13 or 14, wherein the conditions under which the saccharifying enzyme acts on the fiber material include anaerobic conditions and / or temperatures that are not suitable for the growth of the organism.

17. 17. The method of claim 16, wherein the temperature unsuitable for growth of the organism is about 40°C or higher.

18. The method according to claim 13 or 14, wherein the conditions under which the saccharifying enzyme acts on the fiber raw material include conditions under which the organism is not killed.

19. 15. The method of claim 13 or 14, wherein the organism is a filamentous fungus or an actinomycete.

20. 20. The method of claim 19, wherein the filamentous fungi include the genera Trichoderma and Penicillium.

21. 20. The method of claim 19, wherein the actinomycete comprises the genus Streptomyces.

22. 15. The method of claim 13 or 14, wherein the fibrous raw material is not added exogenously.

23. 16. The method of claim 15, wherein the conditions under which the organism produces the saccharification enzyme include conditions under which the organism produces a sufficient amount of the saccharification enzyme to allow the fibrous material to be saccharified.

24. The method of claim 15 , wherein the conditions under which the organism produces the saccharification enzyme include culturing for at least about 12 hours or more.

25. The method according to claim 15, characterized in that the conditions under which the organism produces the saccharifying enzyme are switched to conditions under which the saccharifying enzyme acts on the fibrous raw material based on the sugar concentration in the culture solution, the enzymatic activity of the saccharifying enzyme, the biomass, the gas concentration, the metabolic rate of the organism, or the pH of the culture solution, or any combination thereof.

26. 15. A sugar liquid produced by the method according to any one of claims 1, 2, 13 or 14.

27. A method for reusing an organism that produces a saccharifying enzyme in producing a sugar liquid from a fiber raw material, comprising the steps of: A step of recovering a residue from the saccharification step according to claim 1 or 2; Reusing the organisms in the residue as organisms that produce saccharification enzymes; A method comprising:

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