JP2010104361A - Method of producing saccharified liquid using lignocellulosic biomass - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently produce a saccharified liquid using a lignocellulosic biomass. <P>SOLUTION: A method for producing the saccharified liquid includes the steps of: crushing a lignocellulosic biomass; and hydrolyzing the crushed product by using a hydrolyzing enzyme. Also, a method for producing metabolic products of microorganisms is provided by using a culture media containing the saccharified liquid. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a saccharified solution using lignocellulosic biomass.

  Since the Industrial Revolution, fossil fuels, especially oil, have been consumed in large quantities as raw materials for fuels and chemicals. As a result, global warming due to greenhouse gases such as carbon dioxide is becoming increasingly serious today, and it is predicted that the number of years that oil can be taken will be several decades. Therefore, there is an urgent need to secure new, earth-friendly resources that can replace oil.

  One solution to these problems is the research and development of biorefinery that attempts to produce raw materials for fuels and chemicals from biomass (renewable, bio-derived organic resources excluding fossil resources). It is being promoted around the world.

  Among these biorefinery technologies, biomass ethanol (hereinafter also referred to as “bioethanol”) has recently reached the stage of practical use, and starch or sugar contained in edible parts such as corn and sugarcane. Ethanol production using is carried out on a large scale in the United States and Brazil. However, there is a concern that using these food resources as raw materials for bioethanol will cause a rise in food prices and a serious shortage of food in developing countries. In view of such a situation, the present inventor has developed a biorefinery technology using lignocellulosic biomass contained in leaves and stems (soft biomass) or wood (hard biomass) which are non-edible parts of crops as raw materials. I have been working on development.

  In general, in biorefinery, a polysaccharide contained in biomass is first hydrolyzed (hereinafter also referred to as “saccharification”) to obtain a saccharified solution containing a monosaccharide. Various chemicals are obtained from this monosaccharide by fermentation using microorganisms. However, lignocellulosic biomass, such as crop leaves, stems, or wood, has a structure in which lignin and hemicellulose, which are polymers of aromatic compounds, are tightly bound to cellulose, which is a structural polysaccharide. It is known that the efficiency of the hydrolysis reaction is remarkably low compared with starch-based biomass and saccharide-based biomass whose main components are sugar and sugar.

  For these reasons, a chemical hydrolysis method using an acid or an alkali has been used in the conventional hydrolysis reaction of lignocellulosic biomass (for example, see Non-Patent Documents 1 to 4).

Masanori Ohfuka et al., “Ethanol Fermentation Characteristics of Acid-Saccharified Liquid of Starch / Cellulose Mixed Raw Materials”, Monthly Bulletin of Cold Region Civil Engineering Research Institute, 2007, 651, p. 19-20 Hajime Minato, “Effect of Sodium Hydrodide Treatment of Rice Straw on Cell Wall Composition and Digestibility of Dry Matter”, Animal Science, 77. 61-66 E. Yu Valensco, H.C. Ding, J.M. M.M. Labavich, and S.R. P. See Shoemaker, “Enzymatic Hydrology of Pretending Rice Straw”, Biosource Technology, (1997), 5, p. 109-119 Nicoleta Curreji, Mario Agelli, Brunella Pisu, Antonio Recigno, Enrico Sanjust, and August Rindi, "Complete and Efficient Enzyme Hydroslide." 937-941

  However, such a chemical hydrolysis method requires hydrolysis of lignocellulosic biomass under strongly acidic or strongly alkaline conditions, and therefore requires a large amount of acid or alkali reagent. If such a chemical hydrolysis method is performed on an industrial scale, it is not preferable from the viewpoint of environmental pollution.

  In order to solve the above-mentioned problems, the present inventor has conducted intensive research in order to efficiently produce a saccharified solution from lignocellulosic biomass. As a result, the lignocellulosic biomass was pulverized and the pulverized product was hydrolyzed using a hydrolase to find that the hydrolysis reaction proceeded with high efficiency, and the present invention was completed.

  That is, this invention is a manufacturing method of a saccharified liquid including the process of grind | pulverizing lignocellulosic biomass, and the process of hydrolyzing the obtained ground material using a hydrolase.

  According to the present invention, a saccharified solution can be efficiently produced from lignocellulosic biomass. Since the manufacturing method does not use a large amount of chemicals such as acid or alkali as in the prior art, it can contribute to the construction of environmentally friendly biorefinery technology.

  Hereinafter, preferred embodiments of the present invention will be described, but the technical scope of the present invention should be determined based on the description of the scope of claims, and is not limited to the following forms.

  This embodiment relates to a method for producing a saccharified solution, comprising a step of pulverizing lignocellulosic biomass and a step of hydrolyzing the obtained pulverized product using a hydrolase.

[Method for producing saccharified solution]
First, the process of pulverizing lignocellulosic biomass will be described.

  In the present invention, “lignocellulose-based biomass” means biomass mainly composed of cellulose, hemicellulose, and lignin. As lignocellulosic biomass, any biomass containing the above components as a main component can be used without particular limitation. Specific examples include rice straw, rice husk, wheat straw, bagasse, coconut husk, corn cob, weed, wood, Examples include pulp and paper. It can also include food industry, building industry, household waste, etc. Conventionally, most of these lignocellulosic biomass has been discarded by incineration, but according to the present invention, these lignocellulosic biomass can be effectively used as resources.

  In the step of pulverizing lignocellulosic biomass, conventionally known methods such as wet pulverization and dry pulverization can be appropriately used as the pulverization. Among these, dry pulverization is preferable because a higher saccharification rate can be achieved. Examples of the apparatus used for dry grinding include a media mill such as a ball mill and a tower mill, an impact mill such as a hammer mill and a pin mill, and a jet mill. Moreover, as an apparatus used for wet grinding, a homogenizer, a pearl mill, a mass collider, etc. are mentioned, for example. One of these pulverizers may be used alone, or two or more thereof may be used in combination. Moreover, in order to grind | pulverize efficiently, it is preferable to cut lignocellulosic biomass to about 1-5 cm previously, or to coarsely grind to about 1-10 mm.

  Although there is no restriction | limiting in particular in the particle size of the ground material which grind | pulverized lignocellulosic biomass, Preferably it is 0.01-1.0 mm, More preferably, it is 0.1-1.0 mm. By grinding lignocellulosic biomass to such a particle size, the efficiency of the subsequent hydrolysis reaction is significantly improved. The lower limit of the particle size is not particularly limited, but is preferably 0.01 mm or more from the viewpoint of ease of handling. Similarly, the particle size of the pulverized product is preferably as uniform as possible from the viewpoint of improving the efficiency of the hydrolysis reaction, but considering the industrially pulverization of a large amount of lignocellulosic biomass, the total mass of the pulverized product The particle size of the pulverized product is 50% by mass or more, preferably 70% by mass or more, more preferably 80% by mass or more, still more preferably 90% by mass or more, and particularly preferably 98% by mass or more, based on (dry weight conversion) Grind to be less than 1.0 mm. In the present invention, “particle size” is a value measured by the method used in the following examples.

  Subsequently, a process of hydrolyzing the pulverized product using a hydrolase (hereinafter also simply referred to as “enzyme”) will be described.

  In the present invention, the hydrolase plays a role of cleaving a glycosidic bond of polysaccharides (including oligosaccharides) contained in lignocellulosic biomass to hydrolyze into monosaccharides. Various forms of hydrolase can be used. For example, it may be in the form of an enzyme preparation in which a hydrolase produced in a living body such as a microorganism is separated from the living body, or an enzyme by culturing a microorganism or the like while contained in the living body. You may make it react.

  Lignocellulosic biomass includes polysaccharides (including oligosaccharides) such as cellulose, hemicellulose, and starch. Cellulose is a linear polysaccharide in which glucose is polymerized by β-1,4-glycosidic bonds. Hemicellulose is a polysaccharide other than cellulose that forms a plant cell wall together with cellulose, and is a generic name for homopolysaccharide and heteropolysaccharide. Examples of hemicellulose include mannan, glucomannan, glucoxylan, galactan, xylan, arabinan, arabinoxylan, arabinogalactan, pectin, chitin, galactoglucomannan, clonoxylan, and xyloglucan. Starch is a polysaccharide having a helical or network structure in which glucose is polymerized by α-1,4-glycoside bonds and / or α-1,6-glycoside bonds. Monosaccharides obtained by hydrolyzing polysaccharides contained in the above lignocellulosic biomass include monosaccharides such as glucose, galactose, mannose, fructose, xylose, and arabinose, and uronic acids such as galacturonic acid and glucuronic acid. An ester derivative in which a hydroxyl group is esterified and an alkyl derivative in which the hydroxyl group is derivatized with a methyl group or the like are generated.

  Examples of the polysaccharide contained in the lignocellulosic biomass include cellulose, hemicellulose, and starch as described above. The cellulose hydrolase (cellulase) is not particularly limited as long as it is an enzyme capable of cleaving the β-1,4-glycoside bond of cellulose. For example, endo-β-1,4-glucanase, exo-β- Examples include 1,4-glucanase, β-glucosidase, and carboxymethyl cellulase. The hemicellulose hydrolase (hemicellulase) can be used without particular limitation as long as it can cleave the glycosidic bond of hemicellulose. For example, β-xylanase, β-xylosidase, α-arabinofuranosidase, mannosidase , Mannanase, arabinase, galactanase, pectinase and the like. Any starch hydrolyzing enzyme (amylase) can be used without particular limitation as long as it is an enzyme capable of cleaving the glycosidic bond of starch. For example, α-1,4-glycoside bond and / or α-1,6- Examples include α-amylase, β-amylase, glucoamylase, isoamylase, and pullulanase that can cleave glycosidic bonds. These hydrolases may be used singly or in combination of two or more, but lignocellulosic biomass contains polysaccharides having various glycosidic bonds as described above. Therefore, it is preferable to use a combination of two or more hydrolases having different reactivities.

  The hydrolase can be produced in a living body such as a microorganism capable of hydrolyzing lignocellulosic biomass. In order to distinguish from microorganisms that produce microbial metabolites described later, hereinafter, microorganisms capable of hydrolyzing lignocellulosic biomass are also referred to as “hydrolyzing enzyme-producing microorganisms”. Usually, hydrolase-producing microorganisms produce several types of hydrolases having different reactivities as described above, and therefore, an enzyme group containing these several types of hydrolases is used for the hydrolysis reaction. It is known that the composition of each hydrolase component contained in the enzyme group varies depending on the type of the hydrolase-producing microorganism to be produced. The hydrolysis reaction of various glycosidic bonds contained in lignocellulosic biomass depends on the composition of hydrolase capable of cleaving the various glycosidic bonds, thus efficiently cleaving various glycosidic bonds contained in lignocellulosic biomass. It is preferable to use an enzyme group having such an enzyme composition. Examples of the enzyme-producing microorganism that produces the above hydrolase include Acremonium, Trichoderma, Aspergillus, Fusarium, Penicillium, and Schizophyllum. Microorganisms belonging to the genus Clostridium, Ruminococcus, and Thermomonospora are known. Of these, Acremonium cellulolyticus, Acremonium thermophilum, Trichoderma virides, Trichoderma virides, which produce hydrolase with high lignocellulosic biomass resolution. (Aspergillus accureatus), Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus niger (Fusarium solani), Fusarium solanium (Shufirum commo ly) It is preferable to use a hydrolase produced by a hydrolase-producing microorganism selected from the group consisting of serum (Clostridium thermocellum), Luminococcus albus, and Thermomonospora curvata. In addition, a hydrolase produced by a mutant of these hydrolase-producing microorganisms can be used as long as it does not adversely affect the lignocellulose-based biomass resolution.

  The hydrolase in the present invention may be in the form of an enzyme preparation obtained by separating the enzyme produced by the above-mentioned hydrolase-producing microorganism from the living organism of the microorganism, or may be in the form of an immobilized enzyme. Good. Examples of hydrolases separated from the above hydrolase-producing microorganisms include Meicelase (manufactured by Meiji Seika Co., Ltd.), Sumiteam C (manufactured by Shin Nippon Chemical Industry Co., Ltd.), Sumiteam X (manufactured by Shin Nippon Chemical Industry Co., Ltd.), Hemi Cellulase “Amano” 90 (manufactured by Amano Enzyme Co., Ltd.), Green Amil H (manufactured by Danisco Japan Co., Ltd.), pento bread (manufactured by Novozymes Japan Co., Ltd.), and the like. Among these, it is preferable to use Mecellase and Sumiteam C having particularly high hydrolytic ability.

  The hydrolysis reaction in the present invention can be carried out by appropriately adopting conventionally known methods. First, a pulverized product of the above lignocellulosic biomass is suspended in an aqueous medium to prepare a suspension. The aqueous medium is not particularly limited as long as the hydrolase is not deactivated, but water, a buffer solution, an acidic aqueous solution, or an alkaline aqueous solution is preferably used. The density | concentration of the ground material of lignocellulosic biomass contained in suspension is 50-300 g / L normally with respect to an aqueous medium, Preferably it is 100-200 g / L. For example, in the case of producing a saccharified solution by culturing a hydrolase-producing microorganism, the pH is usually from 3 to 9, and preferably from 4 to 7. Moreover, culture | cultivation temperature is 20-50 degreeC normally, Preferably it is 30-37 degreeC. The culture time is usually 12 to 144 hours, preferably 24 to 72 hours. The hydrolysis reaction can also be performed by adding a hydrolase to a suspension of a pulverized product of lignocellulosic biomass and incubating the suspension. The amount of enzyme added to the suspension is preferably 0.1 to 1% by mass with respect to the mass of the pulverized lignocellulosic biomass. By using such an amount of the enzyme, the hydrolysis reaction can be completed usually in 12 to 144 hours, preferably in 24 to 72 hours. Moreover, it is preferable to use the optimal conditions of the enzyme to be used for hydrolysis conditions. The pH is usually 3-8, preferably 4-6. Moreover, reaction temperature is 30-60 degreeC normally, Preferably it is 40-50 degreeC.

  The saccharified solution after completion of the hydrolysis reaction may be appropriately filtered for insoluble matter in the saccharified solution, or may be used for production of the next microbial metabolite as it is. From the viewpoint of improving stirring efficiency and yield, it is preferable to filter insolubles in the saccharified solution in advance.

  Moreover, it is preferable that the ground product of lignocellulosic biomass is steamed before being subjected to a hydrolysis reaction from the viewpoint of improving reaction efficiency. A conventionally well-known method can be used for a steaming process. Furthermore, an acid or alkali treatment may be appropriately performed before the hydrolysis reaction.

  The mass of monosaccharides (hereinafter also referred to as “total sugar amount”) contained in the saccharified solution produced by the above production method is preferably relative to the total mass of the pulverized product of lignocellulosic biomass subjected to the hydrolysis reaction. Is 10-90 mass%, More preferably, it is 20-80 mass%, More preferably, it is 30-70 mass%. According to this production method, as shown in the following examples, a monosaccharide can be obtained from lignocellulosic biomass at a high saccharification rate of 60% or more.

[Method of producing microbial metabolite]
Next, a method for producing a microbial metabolite including a step of culturing a microorganism in a medium containing a saccharified solution produced by the above production method will be described.

  The medium used in the present invention contains a saccharified solution produced by the above production method. The medium generally contains a carbon source, a nitrogen source, and an inorganic salt, but when these nutrient sources necessary for cultivation are sufficiently contained in the saccharified solution, the concentration is adjusted appropriately. Only the saccharified solution can be used, and if necessary, a medium can be prepared by adding a nutrient source in addition to the saccharified solution.

  Examples of the carbon source added to the medium include monosaccharides such as glucose, galactose, fructose, and xylose, disaccharides such as maltose, lactose, and sucrose, and sugars such as sugar alcohols such as glycerin and xylitol. It can be used alone or in combination of two or more. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium nitrate, amino acids such as urea and L-glutamic acid, and inorganic or organic nitrogen compounds such as uric acid can be used. Furthermore, nitrogen-containing natural products such as peptone, polypeptone, meat extract, yeast extract, soybean hydrolysate, soybean powder, milk casein, casamino acid, corn steep liquor and the like may be used as the nitrogen source. Among these, amino acids such as ammonium chloride, ammonium sulfate, urea, and L-glutamic acid, and inorganic or organic nitrogen compounds such as uric acid, nitrogen-containing natural products such as peptone, meat extract, and yeast extract are preferable. These nitrogen sources can be used alone or in combination of two or more. Examples of inorganic substances include sodium chloride, potassium chloride, calcium chloride, potassium phosphate, sodium phosphate, magnesium sulfate, ammonium sulfate, and other phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron, and zinc. Hydrochloride, sulfate, acetate and the like are used. In addition, vitamins such as thiamine and biotin, and if necessary, nucleic acid-related substances such as adenine and uracil may be used. These inorganic substances can be used alone or in combination of two or more.

In the present invention, a microbial metabolite is produced by adding and culturing a microorganism capable of producing a microbial metabolite to the medium. In the present invention, the “microbial metabolite” means a substance obtained by converting an organic substance or an inorganic substance taken by the microorganism from the outside world for life support using a life function. The microbial metabolite is not particularly limited as long as it can be used as a chemical or a raw material thereof. For example, organic acids, amino acids, alcohols, nucleosides, nucleotides, nucleobases, lipids, saturated and unsaturated fatty acids, carbohydrates , Aromatic compounds, vitamins, and enzymes. Among these, the organic acid is not particularly limited as long as it is an organic compound showing acidity, but is preferably a compound having a carboxyl group as an acidic group. Specific examples of such organic acids include lactic acid, fumaric acid, itaconic acid, malic acid, pyruvic acid, tartaric acid, succinic acid, maleic acid, glutaric acid, levulinic acid, propionic acid, gluconic acid, and aconitic acid. Etc. Of these organic acids, lactic acid, fumaric acid, itaconic acid, malic acid, or pyruvic acid is preferable. Lactic acid is also useful as a biodegradable plastic raw material, pharmaceutical raw material, food additive, and feed additive. Fumaric acid can be used as a polyester resin raw material, a sizing raw material, a perfume raw material, a food additive, and a feed additive. Itaconic acid can be used as a modifier such as a synthetic resin and latex, a coating material, and a food additive. Malic acid can be used as a sour agent, a pH adjuster, and an emulsifier for beverages and foods. Pyruvate can be used as a raw material for fragrances, cosmetics, agricultural chemicals and the like. The alcohol is not particularly limited as long as it is a compound having 1 to 6 hydroxy groups in the molecule. Specifically, methanol, ethanol, propanol, isopropanol, butanol, ethanediol, propanediol, butanediol, Examples include glycerol, sorbitol, mannitol, xylitol, and arabinitol. Of these alcohols, ethanol and xylitol are preferred. Ethanol is expected as the next generation automobile fuel. Xylitol can be used as a caries inhibitory sweetener and as a sweetener for diabetics. Other amino acids include lysine, glutamic acid, methionine, phenylalanine, aspartic acid, and threonine; nucleotides include nicotinamide adenine dinucleotide (NAD) and adenosine monophosphate (AMP); gamma-linolenic acid, arachidonic acid, and docosahexaenoic acid; hyaluronic acid and trehalose as carbohydrates; aromatic amines, vanillin, and indigo as aromatic compounds; ascorbic acid, vitamin B ₆ , vitamin B ₁₂ as vitamins Examples of the enzyme include cellulase, hemicellulase, protease, and amylase.

  The microbial metabolites can be produced by culturing microorganisms that can produce them. Examples of these microorganisms include, for example, Rhizopus, Pachisolen, Aspergillus, Toluropsis, Candida, Acremonium (Acremonium), Corynebacterium, Bacillus, Ashbya, Escherichia, Alcaligenes, Actinobacillus, Anavirospyrum. Propionibacterium (Propio Ibacterium), and although a microorganism belonging to the genus Clostridium (Clostridium) are exemplified, but the invention is not limited thereto. Specifically, Pachisolen tannofilus, Aspergillus terreus, Rhizopus oryzis blas tros, Alpergillus flavis tros (Candida tropicalis), Corynebacterium glutamicum, Bacillus subtilis, Ashbya gossippi, Escherichia bischer llus niger), Rhizopus oryzae (Rhizops oryzae), Alcaligenes Ratasu (Alcaligenes latus), Ana erotic Biot spin Rirumu-succinyl professional Dew sense (Anaerobiospirillum succiniproducens), Actinobacillus Sukushinogenesu (Actinobacillus succinogenes), Lactobacillus del Burukki (Lactobacillus delbruckii), Lactobacillus leichmannii, Propionibacterium arabinosum, Propionibacterium shamani (Propi) nibacterium schermanii), Propionibacterium flow Den Lei Qi (Propionibacterium freudenreichii), Clostridium Puropionikamu (Clostridium propionicum), and Clostridium acetobutylicum (Clostridium acetobutlicum) and the like. Among them, Risopus genus that produces lactic acid, Aspergillus terreus that produces itaconic acid, Pachysolen tannophilus that produces ethanol, Rhizopus oryzae that produces fumaric acid, Aspergillus flavus that produces malic acid, and pilulic acid that produces pilulic acid. It is preferable to use at least one selected from the group consisting of Candida tropicalis that produces, and Acremonium thermophilum that produces cellulase.

  The culture conditions can be appropriately adjusted by those skilled in the art without particular limitation as long as the growth of the microorganism of the present invention is not substantially inhibited and can produce a microbial metabolite, but the culture temperature is usually 20-40. ° C, preferably 30-35 ° C. Moreover, although the pH of a culture medium is 4-7 normally, in order to suppress the proliferation of various germs in the culture medium after sterilization, it is preferable that it is 4-5. When the pH is less than 4 before or during the culture, it is preferable to control the pH using ammonia or the like. Conversely, when the pH becomes 10 or more, the pH is controlled using sulfuric acid or hydrochloric acid. It is preferable.

  Although the culture method varies depending on the type of microorganism, any one of anaerobic conditions and aerobic conditions can be appropriately employed. A conventionally known culture tank can be appropriately used as the culture tank for culturing, but it is preferable to use a culture tank equipped with a stirrer to improve the production rate of microbial metabolites. Examples of such a culture apparatus include an aeration and stirring type culture tank, a bubble column type culture tank, a packed bed culture tank, and a fluidized bed culture tank.

  In the above description, a method for producing a microbial metabolite by performing a step of producing a saccharified solution and a step of producing a microbial metabolite as separate steps has been described. According to the present invention, lignocellulosic biomass can be obtained. It is also possible to carry out the hydrolysis reaction and microbial metabolite in one step. For example, a microorganism metabolite can also be produced by simultaneously adding a hydrolase-producing microorganism and a microorganism producing a microbial metabolite to a suspension containing a pulverized product of lignocellulosic biomass and performing mixed culture. it can.

  After completion of the above culture, a desired microbial metabolite can be separated from the medium containing the microbial metabolite by a conventionally known technique. Examples of the separation method include column chromatography, distillation, and crystallization. These separation methods may be used alone or in combination of two or more.

  The effect of this invention is demonstrated using a following example and a comparative example. However, the technical scope of the present invention is not limited only to the following examples.

<Crushing rice straw>
[Production Example 1]
After threshing, the rice straw stored in the indoor warehouse for about 2 months was cut into about 1 to 5 cm with household scissors and coarsely pulverized with a home mixer for 5 minutes to obtain a pulverized product A.

[Production Example 2]
The pulverized product A prepared in Production Example 1 was further pulverized for 12 hours with a ball mill (ANZ-60S, manufactured by Nippon Ceramic Science Co., Ltd.) to obtain a pulverized product B.

[Production Example 3]
The pulverized product A prepared in Production Example 1 was once suspended in tap water, and this was wet pulverized with a homogenizer (Polytron PT2100, KINEMATICA AG) for 1 minute to obtain a pulverized product C.

(Measurement of particle size distribution of pulverized product)
The particle size distribution of the pulverized products A and B prepared in Production Examples 1 and 2 was measured using two types of test sieves (nominal dimensions 1.0 mm and 0.1 mm, manufactured by SANPO). Specifically, two types of sieves of 1.0 mm and 0.1 mm are stacked in order from the top, 5 g of the sample is put on the top and sieved intermittently for 5 minutes, and the mass of the powder remaining in each stage is measured. The mass percentage (%) relative to 5 g is shown. In addition, since the pulverized product C of Production Example 3 was suspended in water, measurement was impossible. The results are shown in Table 1.

<Effects of grain size of pulverized rice straw on saccharification>
[Example 1]
1.0 g of the pulverized product B prepared in Production Example 2 and 10 mL of tap water were suspended in a 100 mL Erlenmeyer flask and adjusted to pH 4.5 by adding 1N sulfuric acid. To the suspension, 1% by mass of Mecelase (Meiji Seika Co., Ltd., hereinafter the same) as a hydrolase was added to the pulverized product B, and on a rotary shaker (MMS-110, Tokyo Riken Kikai Co., Ltd.) Saccharification was performed by shaking (200 rpm) at 50 ° C. for 24 hours.

[Comparative Example 1]
Saccharification was performed in the same manner as in Example 1 except that the pulverized product C prepared in Production Example 3 was used instead of the pulverized product B. The pulverized product C was sufficiently dried and weighed as a dry mass.

[Comparative Example 2]
Saccharification was performed in the same manner as in Example 1 except that the pulverized product A prepared in Production Example 1 was used instead of the pulverized product B.

(Quantification of total sugar)
The total sugar was quantified by Michinori Nakamura, Junji Kakinuma, “Biochemical Experimental Method (25) Starch / Related Glycoenzyme Experimental Method”, published by the Society Press Center, published in October 1989, page 206. It was done according to the law.

(Quantification of glucose)
Glucose was quantified using Wako Pure Chemical Industries, Ltd., glucose CII test Wako.

  Table 2 shows the results of the total sugar concentration, glucose concentration, and saccharification rate (mass percentage of the total sugar amount with respect to the dry mass of the crushed rice straw).

<Influence of hydrolase>
[Example 2]
2.0 g of the pulverized product B prepared in Production Example 2 and 10 mL of tap water were suspended in a 100 mL Erlenmeyer flask and adjusted to pH 4.5 by adding 1N sulfuric acid. Saccharification was performed by adding 1% by mass of Mecellase as a hydrolase to the pulverized product B and shaking (200 rpm) at 50 ° C. for 24 hours on a rotary shaker.

[Example 3]
Saccharification was performed in the same manner as in Example 2 except that Sumiteam C (manufactured by Shin Nippon Chemical Industry Co., Ltd.) was used as the hydrolase instead of Meicelase.

[Example 4]
As a hydrolase, 1.0% (v / w) of the liquid agent cellulase (XL-522, manufactured by Nagase ChemteX Corporation) with respect to the mass of the pulverized product B was used instead of 1% by mass of Mecellase. Except for this, saccharification was performed in the same manner as in Example 2.

[Example 5]
Saccharification was performed in the same manner as in Example 2 except that hemicellulase “Amano” 90 (manufactured by Amano Enzyme Co., Ltd.) was used as the hydrolase instead of mecellase.

[Comparative Example 3]
By changing to hydrolase and adjusting the pH to 1.0 by adding 1N sulfuric acid and treating it at autoclave at 121 ° C. for 30 minutes, hydrolyzing the sulfuric acid, and then adding 1N aqueous sodium hydroxide solution Saccharification was carried out in the same manner as in Example 2 except that the pH was adjusted to 4.5. The saccharified solution after the reaction was colored black brown.

  Table 3 shows the results of total sugar concentration, glucose concentration, and saccharification rate of Examples 2 to 5 and Comparative Example 3.

<Influence of the amount of hydrolase added>
[Example 6]
2.0 g of the pulverized product B prepared in Production Example 2 and 10 mL of tap water were suspended in a 100 mL Erlenmeyer flask and adjusted to pH 4.5 by adding 1N hydrochloric acid. Saccharification was performed by adding 1% by mass of Mecellase as a hydrolase to the pulverized product B and shaking (200 rpm) at 50 ° C. for 24 hours on a rotary shaker.

[Example 7]
Saccharification was carried out in the same manner as in Example 6 except that the amount of mecelase added was 0.5% by mass.

[Example 8]
Saccharification was performed in the same manner as in Example 6 except that the amount of mecelase added was 0.25% by mass.

  Table 4 shows the results of total sugar concentration, glucose concentration, and saccharification rate of Examples 6 to 8.

<Influence of pretreatment conditions>
[Example 9]
In Example 2 above, as a pretreatment, the pulverized product B was suspended in tap water, adjusted to pH 4.5 by adding 1N sulfuric acid, and autoclaved (HV-50, manufactured by Hirayama Seisakusho Co., Ltd.) at 121 ° C. After steaming for 20 minutes, saccharification was performed in the same manner as in Example 2, except that mecelase was added to the suspension.

[Example 10]
In Example 2 above, as a pretreatment, the pulverized product B was suspended in tap water, adjusted to pH 1.04 by adding 1N sulfuric acid, cooked at 121 ° C. for 20 minutes, and then added with 1N sodium hydroxide aqueous solution. Then, the pH was adjusted to 4.5, and saccharification was performed in the same manner as in Example 2 except that mecelase was added to the suspension.

[Example 11]
In Example 2 above, as pretreatment, the pulverized product B is suspended in tap water, adjusted to pH 10.1 by adding 1N ammonia water, cooked at 121 ° C. for 20 minutes, and then added with 1N sulfuric acid. The saccharification was carried out in the same manner as in Example 2 except that the pH was adjusted to 4.5 and mecelase was added to the suspension.

[Example 12]
In Example 2 above, as a pretreatment, the pulverized product B is suspended in tap water, adjusted to pH 10.5 by adding 1N ammonia water, cooked at 121 ° C. for 20 minutes, and then added with 1N sulfuric acid. The saccharification was carried out in the same manner as in Example 2 except that the pH was adjusted to 4.5 and mecelase was added to the suspension.

[Example 13]
In Example 2 above, as a pretreatment, the pulverized product B was suspended in tap water, adjusted to pH 10.5 by adding a 1N sodium hydroxide aqueous solution, cooked at 121 ° C. for 20 minutes, and then added with 1N sulfuric acid. Then, the pH was adjusted to 4.5, and saccharification was performed in the same manner as in Example 2 except that mecelase was added to the suspension.

  Table 5 shows the results of total sugar concentration, glucose concentration, and saccharification rate of Examples 2 and 9-13.

<Influence of initial pH in saccharification>
[Examples 14 to 22]
1.0 g of pulverized product B prepared in Production Example 2 and 10 mL of tap water were suspended in a 100 mL Erlenmeyer flask, and the pH was adjusted by adding 1N sulfuric acid or 1N sodium hydroxide aqueous solution as shown in Table 6. Saccharification was performed by adding 1% by mass of Mecellase as a hydrolase to the pulverized product B and shaking (200 rpm) at 50 ° C. for 24 hours on a rotary shaker.

  Table 6 shows the results of total sugar concentration, glucose concentration, and saccharification rate of Examples 14-22.

<Optimization of lactic acid production medium>
[Example 23]
The pulverized product B prepared in Production Example 2 was suspended at 300 g / L with respect to tap water, and 1N sulfuric acid was added to adjust the pH to 4.5. 1 mass% of mecerase as a hydrolase was added to the suspension with respect to the pulverized product B, and saccharification was performed on a rotary shaker at 200C for 24 hours (200 rpm). , Referred to as “saccharified solution A”). The concentration of total sugar contained in the saccharified solution A was 120 g / L. As a medium for producing lactic acid, 60 mL of saccharified solution A was placed in a 300 mL Erlenmeyer flask and sterilized at 121 ° C. for 25 minutes using an autoclave to prepare a medium. Pre-culture medium a (glucose 50 g / L, corn starch (manufactured by Nippon Corn Starch Co., Ltd., hereinafter the same)) 50 g / L, ammonium sulfate 1.35 g / L, potassium dihydrogen phosphate 0.30 g / L, magnesium sulfate Heptahydrate 0.25 g / L, zinc sulfate heptahydrate 0.04 g / L, calcium carbonate 1 g / L, sterilized in autoclave at 121 ° C. for 25 minutes) pre-culture (170 rpm, 30 ° C. on rotary shaker) Rhizopus sp. 3 mL of MK96-1156 (accession number: FERM BP-6777) was added, and lactic acid fermentation was performed by shaking (170 rpm) at 30 ° C. for 96 hours on a rotary shaker. In addition, 1 g of calcium carbonate sterilized separately was added for pH adjustment during lactic acid fermentation.

[Example 24]
The same method as in Example 23, except that yeast extract (221750, manufactured by Wako Pure Chemical Industries, Ltd., hereinafter the same) was added to saccharified solution A as 3 g / L as the medium for lactic acid production. Lactic acid fermentation was performed.

[Example 25]
Except that corn steep liquor (CLS) (034-16775, manufactured by Wako Pure Chemical Industries, Ltd., hereinafter the same) was added to saccharified solution A to 3 g / L as a medium for lactic acid production. Lactic acid fermentation was performed in the same manner as in Example 23.

[Example 26]
Lactic acid fermentation was performed in the same manner as in Example 23, except that saccharified solution A added with ammonium sulfate at 3 g / L was used as the medium for lactic acid production.

[Example 27]
Lactic acid fermentation was performed in the same manner as in Example 23, except that saccharified solution A added with ammonium nitrate at 3 g / L was used as the medium for lactic acid production.

[Comparative Example 4]
A culture medium (120 g glucose, 1.35 g ammonium sulfate, 0.3 g potassium dihydrogen phosphate, 0.25 g magnesium sulfate heptahydrate, 0.04 g zinc sulfate heptahydrate per 1 L of the medium ( Lactic acid fermentation was performed in the same manner as in Example 23, except that standard medium X) was used as the medium for lactic acid production.

  Table 7 shows the results of Examples 23 to 27 and Comparative Example 4. In all Examples and Comparative Examples, the residual sugar contained in the medium after the end of lactic acid fermentation was extremely small.

  From the results in Table 7, it was shown that according to the method for producing a saccharified solution of the present invention, L-lactic acid equivalent to the standard medium X can be produced only by adding a small amount of nitrogen source.

<Production of L-lactic acid using rice straw saccharified solution>
[Example 28]
Add 1.0L of tap water to a 3L jar fermenter (3MDL, manufactured by Maruhishi Bioengineer Co., Ltd., the same shall apply hereinafter), and add 200g of crushed rice straw obtained in the same manner as in Production Example 2 while stirring. The solution became cloudy, and 1N sulfuric acid was added thereto to adjust the pH to 4.5. Mecelase was added to the suspension in an amount of 1.0% by mass based on the pulverized rice straw, and saccharification was performed at 50 ° C. for 72 hours. The total saccharide concentration, glucose concentration, and saccharification rate of the saccharified solution during the saccharification reaction were measured. The results are shown in Table 8.

  The obtained saccharified solution was filtered with Nutsé to remove insoluble components, and tap water was added to adjust the liquid volume to 1.5 L, which was then subjected to subsequent lactic acid fermentation. To the saccharified solution, 0.3 g / L of potassium dihydrogen phosphate and 0.25 g / L of magnesium sulfate were added and sterilized at 121 ° C. for 10 minutes using an autoclave to prepare a medium. Rhizopus sp. Was pre-cultured in the pre-culture medium a in advance (cultured at 170 rpm on a rotary shaker at 30 ° C. for 24 hours). 100 mL of MK96-1156 (accession number: FERM BP-6777) was added and cultured at 300 rpm, aeration volume 0.5 vvm, and culture temperature 30 ° C. for 72 hours. During the culture, the pH of the medium was monitored as needed with a pH sensor (GS-8405, manufactured by Toa DKK Corporation), and the pH was maintained at 6.0 by adding 25% aqueous ammonia. Table 9 shows the changes over time in the lactic acid concentration, total sugar concentration, and glucose concentration after the start of culture. The yield of L-lactic acid relative to the total sugar was 67% by mass, and the yield of L-lactic acid relative to the crushed rice straw used was 47% by mass.

[Comparative Example 5]
Add 1.0 L of tap water to a 3 L jar fermenter, add 200 g of pulverized rice straw obtained in the same manner as in Production Example 2 with stirring, suspend the mixture, and add tap water to add the total volume of the suspension. Was 1.5 L. 1N sulfuric acid was added thereto to adjust the pH to 1.5, and acid saccharification was carried out by applying pressure at 121 ° C. for 2 hours in an autoclave. The obtained saccharified solution had a total sugar concentration of 40.2 g / L and a glucose concentration of 23.5 g / L. After adding separately sterilized potassium dihydrogen phosphate to 0.3 g / L and magnesium sulfate to 0.25 g / L to the saccharified solution, 25% ammonia water was also sterilized while monitoring with a pH sensor. The pH was returned to 6.0. Rhizopus sp. Was pre-cultured in the pre-culture medium a in advance (cultured at 170 rpm on a rotary shaker at 30 ° C. for 24 hours). MK96-1156 (accession number: FERM BP-6777) was added, and the cells were cultured at 300 rpm, aeration volume 0.5 vvm, and culture temperature 30 ° C. for 48 hours. During the culture, the pH of the medium was monitored with a pH sensor as needed, and the pH was maintained at 6.0 by adding 25% aqueous ammonia. Table 10 shows the changes over time in the lactic acid concentration, total sugar concentration, and glucose concentration during the culture.

  Referring to Table 10, 18.7 g / L of L-lactic acid was produced in 48 hours, and the yield was calculated to be 47% by mass with respect to the total sugar and 14% with respect to the pulverized rice straw. %. These yields are remarkably low as compared with Example 28 using the saccharified solution using the enzyme, and the obtained L-lactic acid culture solution was colored black brown, so that purification and recovery were very difficult. It was thought that.

<Production of L-lactic acid by mixed culture>
[Example 29]
60 mL of a medium containing 30 g / L of glucose, 10 g / L of arbocel (manufactured by Miki Sangyo), 60 g / L of soybean flour (Yosei Oil), and 3.2 g / L of ammonium nitrate was placed in a 300 mL Erlenmeyer flask and sterilized at 121 ° C. for 25 minutes. One platinum loop of Acremonium thermophilum ATCC 24622 on slant (YPD agar medium) was inoculated into the medium, and cultured at 30 ° C. for 72 hours using a rotary shaker (200 rpm). On the other hand, crushed rice straw obtained by the same method as in Production Example 2 was 100 g / L, ammonium sulfate was 1.35 g / L, potassium dihydrogen phosphate was 0.3 g / L, and magnesium sulfate heptahydrate was 0.00. 25 g / L, zinc sulfate heptahydrate was added to a concentration of 0.04 g / L, 1 L with tap water was placed in a 3 L jar fermenter, and sterilized at 121 ° C. for 25 minutes using an autoclave. A. 60 mL of a thermophilum culture solution was added and cultured at 35 ° C. for 24 hours with stirring. A. After confirming the growth of thermophilum with a microscope, Rhizopus sp. was cultured with shaking in a 300 mL Erlenmeyer flask containing 60 mL of the above-mentioned preculture medium a separately at 30 ° C. for 24 hours using a rotary shaker. MK96-1156 (Accession number: FERM BP-6777) was added in its entirety, and A.M. thermophilum and R.W. sp. Mixed culture with MK96-1156 (Accession Number: FERM BP-6777) was performed. The aeration rate was 0.5 vvm, and the dissolved oxygen (DO) concentration was maintained at 20% or more while monitoring with a DO sensor (OX-2500, manufactured by Maruhishi Bioengineering Co., Ltd.). The pH was maintained at 6.0 using 25% aqueous ammonium. A. in culture. thermophilum and R.W. sp. Table 11 shows the changes over time in the viable cell count, total sugar concentration, glucose concentration, and lactic acid concentration of MK96-1156 (Accession Number: FERM BP-6777). The yield of L-lactic acid based on the pulverized rice straw was 25% by mass.

  From the above results, A.I. thermophilum and R.W. sp. It was shown that saccharification and lactic acid fermentation can be performed simultaneously by mixed culture of MK96-1156 (Accession No .: FERM BP-6777).

<Production of ethanol using rice straw saccharified solution>
[Example 30]
Add 1.0 L of tap water to a 3 L jar fermenter, add 200 g of pulverized rice straw obtained in the same manner as in Production Example 2 while stirring, suspend it, and add tap water to make the volume 1.5 L. did. Next, 25% hydrochloric acid was added to the suspension to adjust the pH to 4.5, and 1% by mass of mecerase was added to the pulverized rice straw, followed by saccharification at 50 ° C. for 24 hours. The resulting saccharified solution was adjusted to pH 6.0 by adding a 1N aqueous sodium hydroxide solution. The saccharified solution was sterilized at 121 ° C. for 15 minutes using an autoclave to obtain a medium. Separately, YPD medium (glucose 20 g / L, polypeptone (394-0015, manufactured by Nippon Pharmaceutical Co., Ltd., hereinafter the same) 20 g / L, yeast extract (221750, manufactured by Wako Pure Chemical Industries, Ltd., hereinafter the same) 10 g / L) was added with 100 mL of Pachysolen tanophilus NBRC 1007 that had been reciprocally shake-cultured at 30 ° C. for 2 days in advance, and aerated and agitation culture was performed at a culture temperature of 30 ° C. while maintaining the dissolved oxygen concentration at 20%. Table 12 shows changes in culture progress.

  As shown in Table 12, the ethanol-producing bacteria consumed all glucose 24 hours after the start of culture. Thereafter, saccharides other than glucose such as xylose were consumed, and finally all saccharides that could be assimilated in 144 hours after the start of culture were consumed. The yield of ethanol was 39.8% by mass with respect to the total sugar, and 26% by mass with respect to the pulverized rice straw.

<Production of itaconic acid using rice straw saccharified solution>
[Example 31]
Add 1.0 L of tap water to a 3 L jar fermenter, add 200 g of pulverized rice straw obtained in the same manner as in Production Example 2 with stirring, suspend the mixture, and add 1 N sulfuric acid to the suspension to adjust the pH. Adjusted to 4.5. To this, 1% by mass of mecerase was added to the pulverized rice straw and saccharified at 50 ° C. for 24 hours. The obtained saccharified solution was adjusted to pH 2.0 by adding 20% nitric acid. The saccharified solution was sterilized in an autoclave at 121 ° C. for 5 minutes to obtain an itaconic acid production medium. Separately, pre-culture medium b (glucose 55.5 g / L, corn steep liquor (034-16775, manufactured by Wako Pure Chemical Industries, Ltd., the same shall apply hereinafter) 3 g / L, sodium nitrate 5 g / L, magnesium sulfate hemihydrate Aspergillus terreus TN-484 (Yahiro, K., Takahama, T., T. Park, YS, Okaba M., cultivated by reciprocal shaking culture for 2 days at 30 ° C. using “Breating of Aspergillus terreus mutant TN-484 for itacic acid production with high yield.” J. Ferment. Bioeng., 79, 506-508 (1995)). It was maintained at 20% while monitoring with a sensor, and aerated and agitated culture was performed at a culture temperature of 30 ° C. The itaconic acid concentration was measured according to Capcell PaK C18 MG Lot. Analysis was performed by HPLC (LC-10A, manufactured by Shimadzu Corporation) equipped with a BS14 column (manufactured by Shiseido Co., Ltd.). Measurement was performed by using 70% acetonitrile as a mobile phase and eluting at a rate of 1 mL / min and detecting at 257 nm. The cell mass was measured by washing the cells with 0.85% physiological saline, drying at 105 ° C. for 2 hours and weighing. The time course of the culture is shown in Table 13.

  As shown in Table 13, glucose was consumed simultaneously with the start of culture, and almost all was consumed 96 hours after the start of culture. Finally, the yield of itaconic acid after 144 hours from the start of cultivation was 41% by mass with respect to the total sugar (theoretical yield was 72%) and 32% by mass with respect to the ground rice straw product. It was.

<Manufacture of fumaric acid using rice straw saccharified solution>
[Example 32]
Add 1.0 L of tap water to a 3 L jar fermenter, add 250 g of rice straw pulverized product obtained in the same manner as in Production Example 2 with stirring, and suspend it. Add 1 N sulfuric acid to the suspension to adjust the pH. Adjusted to 4.5. To this, 1% by mass of mecerase was added to the pulverized rice straw and saccharified at 50 ° C. for 24 hours. The saccharified solution is placed in a 3 L jar fermenter, to which ammonium sulfate 1.35 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.25 g / L, zinc sulfate seven water The Japanese product was added so that it might become 0.04 g / L. 1N sulfuric acid was added to the saccharified solution to adjust the pH to 4.5, and tap water was added to adjust the volume to 1.5 L, followed by sterilization at 121 ° C. for 25 minutes in an autoclave to produce fumaric acid. A medium was used. Separately, in a sterilized 500 mL Erlenmeyer flask, preculture medium c (glucose 55.5 g / L, ammonium sulfate 1.35 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 90 mL of 0.25 g / L, zinc sulfate heptahydrate 0.04 g / L, calcium carbonate 15 g / L) was dispensed. 10 mL of sterile 0.85% saline was added to a slant for culturing Rhizopus oryzae NBRC 4707 (IFO 4707) to suspend spores. Then, the suspension was inoculated into the preculture medium, and reciprocal shaking culture was performed on a rotary shaker at 220 rpm and 32 ° C. for 18 hours. After adding 100 mL of the preculture solution to 1.5 L of the fumaric acid production medium, the culture was performed at a temperature of 32 ° C., a rotation speed of 300 rpm, and an aeration rate of 0.5 vvm. While monitoring the pH during the culture using a pH sensor, the pH was maintained at 6.0 with 12.5% aqueous ammonia. In addition, the measurement of the fumaric acid density | concentration and microbial cell density | concentration in a culture solution was performed using the following method. Table 14 shows the time course of the culture.

(Measurement of fumaric acid concentration)
The fumaric acid concentration was measured using HPLC (CL-10A, manufactured by Shimadzu Corporation). A liquid chromatograph LC-10A, a column oven CTO-6A, an ultraviolet detector SPD-6A, and a chromatopack C-R6A (manufactured by Shimadzu Corporation) were used. CAPCELL PAK C18 (4.6 mmφ × 250 mm) was used as the column, and CAPCELL PAK C18 (4.6 mmφ × 35 mm) (manufactured by Shiseido Co., Ltd.) was used as the guard column. The mobile phase is a 0.45 μm membrane filter (JH, Nippon Millipore Co., Ltd.), a mixture of 2.5% acetonitrile for high performance liquid chromatography (Wako Pure Chemical Industries) and 0.1% H ₃ PO ₃ (Wako Pure Chemical Industries). (Made by company). The column temperature was set to 35 ° C. and the flow rate was set to 1.0 mL / min. Detection was performed with ultraviolet rays, and the detection wavelength was 210 nm.

(Measurement of bacterial cell concentration)
The cell concentration was determined by calculating the cell mass using the dry cell weight method (Dry Cell Weight) and then converting this to a concentration. Specifically, the cell mass was measured by the following method. First, filter paper (manufactured by Advantech Toyo Co., Ltd.) was placed in a petri dish, dried in an oven at 105 ° C. for 2 hours, and then allowed to cool in a desiccator to quantify the filter paper. Using this filter paper, 10 mL of the culture broth was suction filtered, washed thoroughly with distilled water, and then dried in an oven at 105 ° C. for 2 hours. After standing to cool in a desiccator, the cells were weighed to determine the cell mass.

  As shown in Table 14, the fumaric acid yield after 96 hours was 29% by mass with respect to the total sugar and 17% by mass with respect to the pulverized rice straw.

<Manufacture of malic acid using rice straw saccharified solution>
[Example 33]
Add 1.0 L of tap water to a 3 L jar fermenter, add 350 g of rice straw pulverized product obtained in the same manner as in Production Example 2 while stirring, and suspend it, and add 1 N sulfuric acid to the suspension to adjust the pH. Adjusted to 4.5. To this, 1% by mass of mecerase was added to the pulverized rice straw and saccharified at 50 ° C. for 24 hours. The obtained saccharified solution was diluted so that the total sugar concentration was 120 g / L, ammonium sulfate 1.2 g / L, potassium dihydrogen phosphate 0.035 g / L, and dipotassium hydrogen phosphate 0.035 g. / L, Magnesium sulfate 0.1g / L, Iron sulfate 0.06g / L, Calcium chloride 0.1g / L, Sodium chloride 0.005g / L, Calcium carbonate 90g / L did. 2.5 L of this saccharified solution was placed in a jar fermenter (5MDL, Maruhishi Bioengineering Co., Ltd.) and sterilized in an autoclave at 121 ° C. for 25 minutes to obtain a malic acid production medium. Separately, in a sterilized 500 mL Erlenmeyer flask, preculture medium d (glucose 120 g / L, ammonium sulfate 4 g / L, potassium dihydrogen phosphate 0.75 g / L, dipotassium hydrogen phosphate 0.75 g / L 90 mg of magnesium sulfate 0.1 g / L, iron sulfate 0.005 g / L, calcium chloride 0.1 g / L, sodium chloride 0.005 g / L). In a slant for culturing Aspergillus flavus ATCC 13697, 10 mL of sterile 0.85% physiological saline was added to suspend spores. Then, the suspension was inoculated into the preculture medium, and reciprocal shaking culture was performed on a rotary shaker at 220 rpm and 32 ° C. for 18 hours. A pre-culture medium in an amount of 10% by volume with respect to the malic acid production medium was inoculated into the malic acid production medium, and cultured at a temperature of 32 ° C., a rotation speed of 300 rpm, and an aeration rate of 0.5 vvm for 264 hours. The malic acid concentration in the culture broth was determined according to the measurement of fumaric acid concentration in Example 32. The bacterial cell concentration was measured in the same manner as in Example 32. The time course of the culture is shown in Table 15.

  As shown in Table 15, the yield of L-malic acid after 264 hours was 72% by mass with respect to the total sugar and 29% by mass with respect to the pulverized rice straw.

<Production of pyruvic acid using rice straw saccharified solution>
[Example 34]
Add 1.0 L of tap water to a 3 L jar fermenter, add 300 g of rice straw pulverized product obtained in the same manner as in Production Example 2 with stirring, suspend the mixture, and add 1 N sulfuric acid to the suspension to adjust the pH. Adjusted to 4.5. To this, 1% by mass of mecerase was added to the pulverized rice straw and saccharified at 50 ° C. for 24 hours. The obtained saccharified solution was diluted so that the total sugar concentration was 50 g / L, corn steep liquor was 15 g / L, ammonium sulfate was 6 g / L, dipotassium hydrogen phosphate was 1 g / L, and magnesium sulfate was 0. 5 g / L, nicotinic acid (142-0123323, manufactured by Wako Pure Chemical Industries, Ltd., the same below) 0.4 mg / L, pyridoxine hydrochloride (168-20031, manufactured by Wako Pure Chemical Industries, Ltd., the same below) 0 0.2 g / L, 0.02 g / L of thiamine hydrochloride (203-00851, manufactured by Wako Pure Chemical Industries, Ltd., the same below), and 0 for biotin (023-08711, manufactured by Wako Pure Chemical Industries, Ltd., the same below) It added so that it might become 0.005 mg / L. And 1N sulfuric acid was added and pH was adjusted to 5.0. The medium was placed in a 3 L jar fermenter and sterilized in an autoclave at 121 ° C. for 25 minutes to obtain a pyruvic acid production medium. Separately, preculture medium e (glucose 50 g / L, corn steep liquor 15 g / L, ammonium sulfate 6 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, nicotinic acid 0.4 mg / L L, pyridoxine hydrochloride 0.2 g / L, thiamine hydrochloride 0.02 g / L, biotin 0.005 mg / L), precultured solution of Torulopsis glabrata NBRC 0005 (IFO 0005), which was previously shake-cultured at 30 ° C. for 2 days. 100 mL was added to the pyruvic acid production medium, and aeration stirring culture was performed at a culture temperature of 30 ° C. while maintaining the dissolved oxygen concentration at 20%. The pyruvic acid concentration in the culture broth was determined according to the measurement of the fumaric acid concentration in Example 32. The bacterial cell concentration was measured in the same manner as in Example 32. Table 16 shows the time course of the culture.

  As shown in Table 16, the concentration of pyruvic acid reached 44.6 g / L 36 hours after the start of the culture, and then gradually decreased. The yield of pyruvic acid after 84 hours was 31% by mass with respect to the total sugar, and 19% by mass with respect to the pulverized rice straw.

<Manufacture of xylitol using rice straw saccharified solution>
[Example 35]
Add 1.0 L of tap water to a 3 L jar fermenter, add and suspend 220 g of rice straw pulverized material obtained in the same manner as in Production Example 2 while stirring, and add 1 N sulfuric acid to the suspension to adjust the pH. Adjusted to 4.5. To this, 1% by mass of mecerase was added to the pulverized rice straw and saccharified at 50 ° C. for 24 hours. The resulting saccharified solution was diluted so that the D-xylose concentration was 6%, yeast extract 1%, dipotassium hydrogen phosphate 1.5%, ammonium sulfate 0.3%, magnesium sulfate heptahydrate Was added to 0.3%. And 1N sulfuric acid was added and pH was adjusted to 5.0. The medium was placed in a 3 L jar fermenter and sterilized in an autoclave at 121 ° C. for 25 minutes to obtain a xylitol production medium. Separately, pre-culture medium f (D-xylose 6%, yeast extract 1%, dipotassium hydrogen phosphate 1.5%, ammonium sulfate 0.3%, magnesium sulfate heptahydrate 0.3%) in advance. 100 mL of a preculture solution of Candida tropicalis NBRC 0618 (IFO 0618) that had been reciprocally shake-cultured at 30 ° C. for 2 days was added to the xylitol production medium, while maintaining the dissolved oxygen concentration at 20%, the culture temperature was 30 ° C., and the rotation speed was Aeration stirring culture was performed at 300 rpm and aeration dormitory 0.5 vvm. The xylitol concentration in the culture solution was analyzed by HPLC (LC-10A, manufactured by Shimadzu Corporation) equipped with a Shodex Asahipak NH2p-50 4E column. Measurement was performed by using acetonitrile / water = 70/30 (v / v) as a mobile phase and eluting at a rate of 1 mL / min and detecting at 257 nm. The bacterial cell concentration was measured in the same manner as in Example 32. Table 17 shows the time course of the culture.

  As shown in Table 17, the yield of xylitol 72 hours after the start of the culture was 56.8% by mass with respect to the total sugar, and 39% by mass with respect to the pulverized rice straw.

Claims (9)

  A method for producing a saccharified solution, comprising a step of pulverizing lignocellulosic biomass and a step of hydrolyzing the obtained pulverized product using a hydrolase.   The manufacturing method of the saccharified liquid of Claim 1 whose particle size of 50 mass% or more of pulverized products is less than 1.0 mm with respect to the total mass (dry mass conversion) of the said pulverized products.   The method for producing a saccharified solution according to claim 1 or 2, wherein the pulverization is dry pulverization.   The lignocellulosic biomass includes at least one selected from the group consisting of rice straw, rice husk, wheat straw, bagasse, coconut husk, corn cob, weed, wood, pulp, and paper. 2. A method for producing a saccharified solution according to item 1.   The method for producing a saccharified solution according to any one of claims 1 to 4, wherein the hydrolase contains at least one selected from the group consisting of cellulase, hemicellulase, and amylase.   The manufacturing method of a microbial metabolite including the process of culture | cultivating microorganisms in the culture medium containing the saccharified solution manufactured by the method of any one of Claims 1-5.   The method for producing a microbial metabolite according to claim 6, wherein the microbial metabolite is at least one selected from the group consisting of lactic acid, ethanol, itaconic acid, fumaric acid, malic acid, pyruvic acid, and xylitol.   The microorganism is selected from the group consisting of a microorganism belonging to the genus Rhizopus, Pachysolen, Aspergillus, Torulopsis, Candida, and Acremonium. The method for producing a microbial metabolite according to claim 6 or 7, which belongs to at least one species.   A method for producing a microbial metabolite, comprising a step of culturing a hydrolase-producing microorganism and a microorganism in a medium containing a pulverized product of lignocellulosic biomass.