JP2013230142A - Method for producing organic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for removing fermentation inhibitors and efficiently producing an organic acid by a safe and simple method.SOLUTION: A method for producing an organic acid includes the following steps (a)-(d): (a) a step of crushing lignocellulose biomass containing cellulose having more than 30% of a cellulose I degree of crystallization shown by calculation formula (1) wherein the degree of crystallization (%)=[(I-I)/Ic]×100 (1), Irepresents the diffracted intensity of the lattice plane (002) (lattice spacing=22.4 nm) in x-ray diffraction and Irepresents the diffracted intensity of the amorphous portion (lattice spacing=27.3 nm), and decreasing the cellulose I degree of crystallization to 0-30%; (b) a step of washing a crushed product obtained in the step (a) with water; (c) a step of obtaining a saccharified product from the solid obtained in the step (b) using a hydrolase; and (d) a step of fermenting the saccharified product obtained in the step (c) by filamentous fungi and obtaining an organic acid.

Description

  The present invention relates to a method for producing an organic acid from biomass.

  Research and development of biorefinery to produce fuel and chemical raw materials from biomass attracts attention. However, industrialized biorefinery uses starch or sugar contained in grains such as corn and sugarcane as raw materials, and there is concern about the impact on food supply. Therefore, lignocellulosic biomass contained in non-edible parts of plants such as sugarcane bagasse and rice straw has begun to attract attention as a raw material.

Lignocellulosic biomass is mainly contained in the leaves and stems of plants, and has a structure in which lignin and hemicellulose are firmly bound to cellulose. Therefore, the efficiency of the hydrolysis reaction is extremely low, and the hydrolysis must be performed chemically using a strong alkali or a strong acid.
As a method for avoiding the use of a strong acid or strong alkali having a large environmental load, a method is known in which lignocellulosic biomass is pulverized and then hydrolyzed with a hydrolase. However, in the case of producing a useful product by fermentation using the obtained sugar solution as a raw material, a decrease in the conversion rate and inhibition of the occurrence of lag time and the like are desired to avoid the problem. As one of the causes, an organic acid such as acetic acid forming an ester with a polysaccharide is known (see, for example, Patent Document 1).

  Patent Document 1 discloses a method for producing a biomass product such as ethanol, in which biomass is brought into contact with an aqueous solution containing ammonia, solid-liquid separation is performed, an inhibitory factor such as acetic acid is separated, and the remaining solid is used in a subsequent process. Is disclosed. Patent Document 2 discloses a method of removing a low-molecular fermentation inhibitor by membrane filtration after saccharification of biomass. Patent Document 3 discloses a method for producing lactic acid by culturing filamentous fungi as a pellet of cell aggregates. This method describes that it is easy to separate the bacterial cells and the medium after fermentation.

Special table 2010-536376 International Publication No. 2010/067785 Pamphlet Japanese Patent Laid-Open No. 6-253871

  However, in the method of Patent Document 1, not only dangerous ammonia is required, but when an organic acid is intended, the yield of the organic acid after fermentation is not sufficient. In Patent Document 2, membrane filtration is required for separation of the fermentation inhibitor, and productivity is low. Furthermore, in patent document 3, there is no description about removing a fermentation inhibitor.

In order to industrially mass-produce organic acids as biomass products, it is necessary to remove fermentation inhibitors by a safe and simple method.
Accordingly, the present invention relates to providing a method for removing an fermentation inhibitor and efficiently producing an organic acid by a safe and simple means.

Therefore, the present inventor has studied a selective organic acid production method in which cellulosic biomass is hydrolyzed with an enzyme and then fermented. Cellulose crystals are subjected to a pulverization step before subjecting the cellulosic biomass to enzymatic hydrolysis. It has been found that if cellulose is pulverized strongly until the degree of conversion is reduced to 30% or less, the subsequent hydrolysis of cellulose proceeds efficiently. However, when the saccharified product after hydrolysis was fermented to produce an organic acid, it was found that the selectivity of the organic acid was low and a large amount of alcohol was by-produced.
Therefore, as a result of various studies to improve the selectivity of the organic acid in the fermentation process, the efficiency of the saccharification reaction is improved when the crystallinity of cellulose is reduced in the pulverization process, but the organic acid selectivity in the fermentation process is high. However, it was found that the selectivity of the organic acid was drastically improved by adding a step of washing the pulverized product with water after this special pulverization step.

That is, this invention provides the manufacturing method of the organic acid containing following process (a)-(d).
(A): Lignocellulosic biomass containing cellulose having a cellulose I-type crystallinity of more than 30% represented by the following formula (1) is pulverized, and the cellulose I-type crystallinity is reduced to 0-30%. Step of Crystallinity (%) = [(I _c −I _a ) / I _c ] × 100 (1)
[I _c is the diffraction intensity of the lattice plane (002) (lattice plane spacing = 22.4 nm) in X-ray diffraction, and I _a is the diffraction intensity of the amorphous portion (lattice plane spacing = 27.3 nm)]
(B): Step of washing the pulverized product obtained in step (a) with water (c): Step of obtaining a saccharified product by hydrolyzing the solid obtained in step (b) with a hydrolase (d): (C) A step of fermenting the saccharified product obtained in the step with a filamentous fungus to obtain an organic acid

  According to the present invention, an organic acid can be obtained safely and simply at a high conversion rate and conversion rate using filamentous fungi from a lignocellulosic biomass-derived sugar solution.

The present invention is a method for producing an organic acid using lignocellulosic biomass as a raw material, and includes the following steps (a) to (d).
(A): Lignocellulosic biomass containing cellulose having a cellulose I-type crystallinity of more than 30% represented by the following formula (1) is pulverized, and the cellulose I-type crystallinity is reduced to 0-30%. Step of Crystallinity (%) = [(I _c −I _a ) / I _c ] × 100 (1)
[I _c is the diffraction intensity of the lattice plane (002) (lattice plane spacing = 22.4 nm) in X-ray diffraction, and I _a is the diffraction intensity of the amorphous portion (lattice plane spacing = 27.3 nm)]
(B): A step of washing the pulverized product obtained in step (a) with water (c): a step of hydrolyzing the solid obtained in step (b) with a hydrolase to obtain a saccharified product (d ): A step of fermenting the saccharified product obtained in step (c) with a filamentous fungus to obtain an organic acid.

<Lignocellulosic biomass as raw material>
In the present invention, “lignocellulose-based biomass” means biomass mainly composed of cellulose, hemicellulose, and lignin. Any lignocellulosic biomass can be used without particular limitation as long as it contains the above-mentioned components as main components. Specific examples include palm residues such as rice straw, rice husk, straw, bagasse, coconut husk, corn cob, weed, wood, empty fruit bunch, pulp produced from them, and paper. It can also include food industry, building industry, household waste, etc.
The lignocellulosic biomass used in the present invention has a cellulose content of 20% by mass or more, preferably 40% by mass or more, more preferably 60% by mass or more. The cellulose content in the present invention means the total amount of cellulose and hemicellulose in the remaining components obtained by subtracting water from lignocellulosic biomass.
In the case of commercially available pulp, the cellulose content is generally 75 to 99% by mass, and other components include lignin and the like. Moreover, the cellulose I type crystallinity degree of a commercially available sheet-like pulp is 60% or more normally.
Cellulose I-type crystallinity of cellulose contained in these lignocellulosic biomass exceeds 30%.

  Moreover, the water content of the lignocellulosic biomass used in the present invention can easily reduce the cellulose I type crystallinity of cellulose by the following (a) pulverization step, and the subsequent production of saccharified products and organic acids Is preferably 20% by mass or less, more preferably 15% by mass or less, and still more preferably 10% by mass or less.

<(A) Grinding step>
In the present invention, the lignocellulosic biomass is pulverized, and the cellulose type I crystallinity (also referred to as “crystallinity”) of the cellulose contained in the lignocellulosic biomass is reduced to 0 to 30%.

[Cellulose type I crystallinity]
The non-crystallized cellulose prepared in the present invention has a cellulose I type crystallinity reduced to 30% or less. Cellulose type I crystallinity is calculated by the Segal method from the diffraction intensity value by the X-ray diffraction method, and is defined by the following calculation formula (1).
Cellulose type I crystallinity (%) = [(I _c −I _a ) / I _c ] × 100 (1)
[I _c is the diffraction intensity of the lattice plane (002 plane) (lattice plane spacing = 22.4 nm) in X-ray diffraction, and I _a is the diffraction intensity of the amorphous portion (lattice plane spacing = 27.3 nm)]
If the crystallinity is 30% or less, the chemical reactivity of cellulose is improved, and hydrolysis in the step (c) is likely to proceed. From this viewpoint, the crystallization degree of the pulverized product is preferably 25% or less, more preferably 20% or less, more preferably 10% or less, and further preferably 0% in which cellulose I-type crystals are not detected by analysis. The cellulose I type crystallinity defined by the calculation formula (1) may be a negative value in calculation, but the cellulose I type crystallinity in the case of a negative value is 0%.
Here, the cellulose I type crystallinity is the ratio of the amount of crystal region of cellulose to the total amount. Cellulose type I is a crystalline form of natural cellulose. Cellulose type I crystallinity is also related to the physical and chemical properties of cellulose. The smaller the value, the lower the crystallinity of cellulose and the more non-crystalline parts. Therefore, according to the inventor's consideration, decreasing the cellulose I-type crystallinity increases the elongation, flexibility, solubility in water and solvent, and chemical reactivity, and the next step (b). It is considered that the fermentation inhibitor is easily removed from lignocellulosic biomass.

Thus, in order to reduce the cellulose I type crystallinity degree of the cellulose contained in lignocellulosic biomass, it can carry out using a well-known grinder. There is no restriction | limiting in particular in the grinder used, What is necessary is just an apparatus which can make a particle size of a cellulose raw material small.
Specific examples of the pulverizer include a high pressure compression roll mill, a roll mill such as a roll rotating mill, a vertical roller mill such as a ring roller mill, a roller race mill or a ball race mill, a rolling ball mill, a vibration ball mill, a vibration rod mill, and a vibration tube. Container driven media mills such as mills, planetary ball mills or centrifugal fluidization mills, tower crushers, stirring tank mills, medium stirring mills such as flow tank mills or annular mills, compaction such as high-speed centrifugal roller mills and angling mills Examples include a shear mill, a mortar, a stone mortar, a mass collider, a fret mill, an edge runner mill, a knife mill, a pin mill, and a cutter mill. Among these, from the viewpoint of improving the grinding efficiency and productivity of lignocellulosic biomass, a container-driven medium mill or a medium stirring mill is preferable, a container-driven medium mill is more preferable, a vibration ball mill, a vibration rod mill, or a vibration A vibration mill such as a tube mill is more preferable, and a vibration rod mill is more preferable.

  A preferable vibrating rod mill of the present invention has a cylindrical space, is arranged so that the central axis of the cylindrical space is substantially horizontal, and can vibrate in an in-plane direction substantially perpendicular to the central axis. And a rod (B) which is one or more rod-shaped mediums arranged so as to be able to vibrate so as to be substantially parallel to the central axis of the cylindrical space. The vibrating ball mill includes one or more balls (C) that are spherical media.

There is no restriction | limiting in particular as a material of a rod (B) or a ball | bowl (C), For example, iron, stainless steel, an alumina, a zirconia, silicon carbide, silicon nitride, glass etc. are mentioned.
The outer diameter of the rod (B) or ball (C) is preferably in the range of 0.5 to 200 mm, more preferably 1 to 100 mm, and still more preferably 5 to 50 mm.
Further, as the rod (B), those having a cross section of a quadrangle, a polygon such as a hexagon, a circle, an ellipse or the like can be used.
The length of the rod (B) is not particularly limited as long as it is shorter than the length of the pulverizer container. If the size of the rod (B) is within the above range, the desired pulverizing force can be obtained, and the cellulose can be efficiently made amorphous without contaminating the powdered cellulose by mixing fragments of the rod (B). It can be made.

  The filling ratio of the rod (B) or the ball (C) is preferably in a range of 10 to 97% by volume, more preferably 15 to 95% by volume, although a suitable range varies depending on the type of vibration mill. When the filling rate is within this range, the contact frequency between the cellulose and the rod or ball is improved, and the grinding efficiency can be improved without hindering the movement of the medium. Here, the filling rate refers to the apparent volume of the rod (B) or ball (C) relative to the volume of the cylindrical space inside the container (A).

The vibration rod mill used in the present invention includes a vibration mill manufactured by Chuo Kako Co., Ltd., a small vibration rod mill model 1045 manufactured by Yoshida Manufacturing Co., Ltd., a vibration cup mill P-9 model manufactured by Fritsch, Germany, and Nichito Kagaku Co., Ltd. A small vibration mill NB-O type or the like made from can be used.
In addition, as the vibrating ball mill, a device similar to the vibrating rod mill, for example, a vibrating mill manufactured by Chuo Kakoki Co., Ltd. can be used. The processing method may be either a batch type or a continuous type.
When the pulverization process is a batch process, from the viewpoint of smoothly vibrating the medium, the volume of the material to be crushed filled in the cylindrical space inside the container (A) is changed from the volume of the space to the rod (B) or ball ( It is preferably 99% by volume or less, more preferably 95% by volume or less, and still more preferably 90% by volume or less of the volume excluding the volume of C) (hereinafter referred to as the actual volume in the grinding container). 80% by volume or less is more preferable.
On the other hand, when the raw material to be crushed is small, the collision between the container (A) and the rod (B) or ball (C), and the rod (B) or ball (C) that are not related to pulverization increases, and the pulverization efficiency decreases. . Therefore, from the viewpoint of improving the grinding efficiency, the volume of the filled raw material to be ground is preferably 1% by volume or more of the actual volume in the container, more preferably 2% by volume or more, and 3% by volume or more. More preferably.
Here, the volume of the material to be crushed filled in the cylindrical space inside the container (A) means the volume obtained by dividing the weight of the material to be crushed by the apparent specific gravity of the material. To do.
In the case where the pulverization process is a continuous process, a preferable aspect of the retention amount of the cylindrical space inside the container (A) of the material to be pulverized is “filling amount of material to be pulverized” when the pulverization process is a batch process. Is stored in the cylindrical space inside the container (A) of the raw material to be crushed, and the volume of the raw material to be crushed is retained in the cylindrical space inside the container (A). It is the same except that it reads as “volume of raw material to be crushed”.
The frequency and amplitude of the container (A) during the pulverization process are not particularly limited, but the acceleration given to the container (A) and the rod (B) or ball (C) is increased by increasing the frequency and amplitude. And the pulverization speed of the material to be pulverized can be increased.
From the viewpoint of increasing the grinding speed, the frequency of the container (A) is preferably 8 Hz or more, more preferably 10 Hz or more, and further preferably 12 Hz or more. Further, the amplitude of the container (A) is preferably 5 mm or more, more preferably 6 mm or more, and further preferably 7 mm or more.
On the other hand, from the viewpoint of apparatus load, the frequency of the container (A) is preferably 40 Hz or less, more preferably 35 Hz or less, and further preferably 30 Hz or less. Further, the amplitude of the container (A) is preferably 25 mm or less, more preferably 20 mm or less, and still more preferably 18 mm or less.

  The processing time of the vibrating rod mill or the vibrating ball mill cannot be determined unconditionally depending on the type of the vibrating rod mill or the vibrating ball mill, the type, size and filling rate of the rod or ball, but is preferably 0 from the viewpoint of reducing the crystallinity. 0.01 to 50 hr, more preferably 0.05 to 20 hr, still more preferably 0.1 to 10 hr. Although processing temperature does not have a restriction | limiting in particular, From a viewpoint of preventing deterioration by a heat | fever, Preferably it is 5-250 degreeC, More preferably, it is 10-200 degreeC.

Moreover, in this process, it is preferable to add an alkali from a viewpoint which can reduce a cellulose I type crystallinity and can perform subsequent saccharification thing and organic acid production efficiently. Preferable alkali includes sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, and calcium hydroxide, more preferably sodium hydroxide and potassium hydroxide, and still more preferably sodium hydroxide.
Examples of the alkali added in this step include powder, granules, lumps, solutions dissolved in a solvent, or dispersions dispersed in a solvent, preferably powders, granules, or lumps.
The amount of alkali used is preferably 1 to 100 parts by mass of lignocellulosic biomass from the viewpoint of reducing cellulose I-type crystallinity and efficiently producing saccharified products and organic acids thereafter. It is 50 mass parts, More preferably, it is 3-30 mass parts, More preferably, it is 5-10 mass parts.

By the above pulverization method, a pulverized product of amorphous cellulose having a cellulose I-type crystallinity of 30% or less can be efficiently obtained from the raw lignocellulosic biomass. The pulverized material does not adhere to the surface, and can be processed by a dry method.
The average particle size of the pulverized product obtained is preferably 20 to 150 μm, more preferably 25 to 150 μm, more preferably from the viewpoint of chemical reactivity and handleability when the pulverized product is used as a raw material in step (c). Is 30-100 μm. In particular, when the average particle size is 25 μm or more, it is possible to suppress the occurrence of “maco” when the non-crystalline cellulose is brought into contact with a liquid such as water.
The average particle size of the pulverized product refers to an average value obtained by the laser diffraction scattering method according to the volume standard.

Further, in the vibration mill treatment in the present invention, lignocellulosic biomass having a bulk density of 100 kg / m ³ or more is used from the viewpoint of more efficiently performing pulverization and amorphousization with a vibration mill filled with rods or balls. It is preferably 120 kg / m ³ or more, more preferably 150 kg / m ³ or more. When the bulk density is 100 kg / m ³ or more, the raw material lignocellulosic biomass has an appropriate volume, so that handleability is improved. Moreover, since the raw material preparation amount to the vibration mill can be increased, the processing capacity is improved. On the other hand, the upper limit of the bulk density is preferably 500 kg / m ³ or less, more preferably 400 kg / m ³ or less, and still more preferably 350 kg / m ³ or less from the viewpoint of handleability and productivity. From these viewpoints, the bulk density is preferably 100 to 500 kg / m ³ , more preferably 120 to 400 kg / m ³ , and still more preferably 150 to 350 kg / m ³ .

In the present invention, it may be preferable to pretreat lignocellulosic biomass to be supplied to the vibration mill. In such a case, for example, by processing lignocellulosic biomass with an extruder, the bulk density of the lignocellulosic biomass can be brought into the preferred range described above.
It is preferable to coarsely pulverize the lignocellulosic biomass before feeding it into the extruder. The size of the coarsely pulverized product is preferably 1 to 50 mm, more preferably 1 to 30 mm. By roughly pulverizing to 1 to 50 mm, the extruder treatment can be performed efficiently and easily, and the load required for pulverization can be reduced.

<(B) Cleaning step>
(B) In the washing step, the pulverized product obtained in the step (a) is washed with water. The water may be an aqueous solution containing a water-soluble solvent, an acidic aqueous solution, or an alkaline aqueous solution.
Examples of the water-soluble solvent include alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; polyethers such as polyethylene glycol; Examples include critical carbon dioxide. These may be used alone or in combination of two or more, and may be repeated by changing the solvent.
Among these, water is preferable from the viewpoint of removing an inhibitory factor that inhibits the growth and metabolism of filamentous fungi in step (d) and from the viewpoint of removing other inhibitory factors.

The pH of the water used in this step is preferably 4 or more, more preferably 6 or more, from the viewpoint of removing an inhibitory factor that inhibits the growth and metabolism of filamentous fungi in the step (d). On the other hand, from the viewpoint of safety, it is preferably 14 or less, more preferably 9 or less, and still more preferably 8 or less.
The pH of the water used in this step is preferably 4 to 14, more preferably 4 to 9, and further preferably 6 to 8.
For pH adjustment, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali carbonates such as sodium carbonate and sodium bicarbonate; alkali hydrogen carbonates; alkaline earth metal hydroxides such as calcium hydroxide; Inorganic alkalis such as alkaline earth metal carbonates such as calcium; inorganic acids such as hydrochloric acid, phosphoric acid, nitric acid and sulfuric acid, and organic acids such as lactic acid are preferred.

The degree of the washing treatment in the step (b) is preferably managed by the water-soluble component remaining rate. The water-soluble component remaining rate is a value obtained by dividing the mass of the water-soluble component in the pulverized product after the washing step by the mass of the water-soluble component in the pulverized product before the washing step. The pulverized product obtained in the step (a) is preferably washed until the water-soluble component remaining rate is sufficiently small.
For example, in the method of washing the pulverized product obtained in step (a) with a batch-type agitation tank or the like, the pulverized product is dispersed in water placed in a batch-type agitation tank or the like, and filtered or centrifuged from the dispersion. The liquid or supernatant is separated to obtain a solid content. Preferably, the solid content is dispersed again in water using a batch-type stirring tank or the like, and the above-described solid-liquid separation is repeated 1 to n times. From the water added each time and the mass of the filtrate or supernatant, the water-soluble component residual ratio can be calculated by the following formula (2).
Water-soluble component residual ratio = (v ₁ / w ₁ ) × (v ₂ / (v ₁ + w ₂ )) ×... × (v _n / (v _n−1 + w _n )) (2)
Here, w _n is the mass of water added to the washed n-th, v _n is the mass of the liquid present in the precipitated fraction after solid-liquid separation of washed n-th. v _n is calculated as v _n = v _n−1 + w _n −m _n , _where m _n is the mass of the liquid fraction removed after the nth solid-liquid separation operation (where v ₀ = 0). .
Formula (2) represents the washing efficiency when assuming an ideal state in which the water-soluble components in the pulverized product are all dissolved in the aqueous phase without being distributed or adsorbed on the precipitate. Not all of the inhibitors in the present invention are true, but are generally controlled by this value.
In the method of continuously washing the pulverized material obtained in step (a) with a filter press or the like, the pulverized material is dispersed in water, the dispersion is filtered, and water is supplied to the solid content remaining in the filter. Repeat the operation. In the method of continuous washing, since it is difficult to calculate with the formula (2), the water-soluble component remaining rate can be calculated from the following formula (2 ′) using the water-soluble component contained in the pulverized product as a tracer substance. In this case, it is inappropriate if the aqueous component is not completely dissolved due to saturation solubility, adsorption, chemical bonding, etc., so even if a soluble tracer substance is added separately, the residual ratio of the water-soluble component can be calculated. Good. The water-soluble component analysis method may be any method as long as it is not affected by the coexisting substance and is suitable for the water-soluble component analysis method, and examples thereof include absorbance, liquid chromatography, gas chromatography, and electrical conductivity. .
Water-soluble component residual ratio (when continuously washed) = Tracer substance mass after washing / Tracer substance mass after washing (2 ')

In the present invention, the water-soluble component residual rate is preferably 0.5 or less from the viewpoint of removing substances that inhibit the growth and metabolism of filamentous fungi in step (d) and increasing the productivity of organic acids, Preferably it is 0.4 or less, More preferably, it is 0.2 or less, More preferably, it is 0.1 or less. On the other hand, the water-soluble component residual ratio is preferably 0.001 or more from the environmental load and economical viewpoint by suppressing the amount of water used.
Further, the water-soluble component residual ratio is preferably 0.001 to 0.5, more preferably 0.001 to 0.4, still more preferably 0.001 to 0.2, and still more preferably. 0.001 to 0.1.

In order to obtain a more preferable residual ratio of water-soluble components, it is preferable to wash with 1 part by mass or more of water in total with respect to 1 part by mass of the solid content of the pulverized product obtained in step (a), and further 3 parts by mass or more Further, it is preferable to wash with 5 parts by mass or more and further 10 parts by mass or more of water. On the other hand, from the environmental load and economical viewpoint, it is preferable to wash with 100 parts by mass or less of water in total with respect to 1 part by mass of the solid content of the pulverized product obtained in the step (a), and further 50 parts by mass or less. Furthermore, it is preferable to wash with 40 parts by mass or less, and further with 30 parts by mass or less of water.
Moreover, in (b) washing | cleaning process, 1-100 mass parts in total with respect to 1 mass part of solid content of the ground material obtained at the (a) process, Furthermore, 3-50 mass parts, Furthermore, 5-40 mass parts, Furthermore, It is preferable to wash with 10 to 30 parts by mass of water.

In this step, from the viewpoint of removing substances that inhibit the growth and metabolism of filamentous fungi in step (d) and increasing the productivity of organic acid, a dispersion comprising pulverized material and water obtained in step (a) The temperature is preferably 0 ° C. or higher, more preferably 10 ° C. or higher, and still more preferably 20 ° C. or higher. Moreover, from a viewpoint of energy efficiency, Preferably it is 100 degrees C or less, More preferably, it is 90 degrees C or less, More preferably, it is 80 degrees C or less.
Moreover, the temperature of the dispersion liquid consisting of the pulverized product and water obtained in the step (a) is preferably 0 to 100 ° C, more preferably 10 to 90 ° C, and further preferably 20 to 80 ° C. .

<(C) Step of obtaining a saccharification product using a hydrolase>
In step (c), the solid obtained in step (b) is hydrolyzed with a hydrolase to obtain a saccharified product.
In the present invention, the hydrolase used in step (c) is not particularly limited as long as it is an enzyme having hydrolytic activity with respect to cellulose or hemicellulose. For example, commercially available cellulase preparations and hemicellulase preparations, cellulases and hemicellulases derived from animals, plants and microorganisms can be used.
Cellulases include, for example, Trichoderma reease (Trichoderma luses luciferase) from Cellic Ctec, Cellic Ctec2, Celluclast 1.5L (Novozymes), Accelerase 1000, Accelerase 1500, Accelerase DUET (Genencore), etc. .) Cellulase from KSM-N145 (FERM P-19727) strain, Bacillus sp. Cellulase from KSM-N252 (FERM P-17474) strain, Bacillus sp. KSM-N115 (FERM P -19726) strain-derived cellulase, Bacillus SP s sp.) Cellulase derived from KSM-N440 (FERM P-19728) strain, cellulase derived from Bacillus sp. KSM-N659 (FERM P-19730) strain, Trichoderma viride, Aspergillus sp. acletus, Clostridium thermocellum, Clostridium stercoralium, Clostridium josui, Cellulomonas flocculus Iticus), Irupekkusu Rakuteusu (Irpex lacteus), Aspergillus niger (Aspergillus niger), Humicola insolens (Humicola insolens) derived cellulase mixture, Pyrococcus horikoshii (Pyrococcus horikoshii) heat resistance cellulase derived thereof.
Among these, cellulase derived from Trichoderma reesei, cellulase derived from Trichoderma vilide, cellulase derived from Humicola insolens, and cellulase derived from Humicola insolens are preferable. Cellulase derived from (Trichoderma reesei) is more preferable, and Cellic Ctec, Cellic Ctec2 (Novozymes), Accelerase DUET (Genencore), TP-60 (Meiji Seika Co., Ltd.), or Ultraflo L (Novozymes) is more preferable.

Examples of the hemicellulase include, for example, a xylanase derived from the Bacillus sp. KSM-N546 (FERM P-19729) strain, Aspergillus niger, Trichoderma violol, H. Or xylanase from Bacillus alcalophilus, Thermomyces, Aureobasidium, Streptomyces, Clostridium thermother, T ASCUS), Karudoseramu (Caldocellum), or Thermo mono Supora (Thermomonospora) genus xylanase like from like.
Moreover, the enzyme which has hemicellulase activity contained in said cellulase mixture can also be utilized.

  Hydrolytic enzymes can be used alone, but it is effective to use these enzymes in combination for more efficient sugar production. Moreover, the efficiency of sugar production can be improved by further adding a specific cellulase component such as β-glucosidase to these enzymes. Examples of β-glucosidase to be added include enzymes derived from Aspergillus niger (for example, Novozymes 188 and Megazyme β-glucosidase), enzymes derived from Trichoderma reesei, Penicillium eri ) Derived enzymes and the like.

  In the step (c), first, the solid obtained in the step (b) 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 reaction conditions for step (c) can be selected according to the characteristics of the enzyme used. From the viewpoint of the productivity of saccharified products and organic acids, the substrate concentration is 5 to 200 (g / L). It is preferable to add hydrolase to the substrate suspension so that it preferably corresponds to 0.01 to 10% by volume, more preferably 0.1 to 2% by volume.
The pH is preferably 2 to 10, more preferably 3 to 7, and further preferably 4 to 6.
Moreover, reaction temperature becomes like this. Preferably it is 10-90 degreeC, More preferably, it is 20-70 degreeC, More preferably, it is 40-60 degreeC. The reaction time is 30 minutes to 7 days, preferably 0.5 to 5 days.

  As for the saccharified product after completion of the hydrolysis reaction, insolubles in the saccharified product may be appropriately filtered, or may be subjected to the next step (d) as it is.

The mass of the monosaccharide contained in the saccharified product produced in the step (c) (hereinafter also referred to as “total sugar amount”) is the ligno after washing subjected to the hydrolysis reaction from the viewpoint of improving the yield of the organic acid. Preferably it is 10-90 mass% with respect to the total mass of the ground material of a cellulose biomass, More preferably, it is 20-80 mass%, More preferably, it is 30-70 mass%.
Examples of monosaccharides include glucose, fructose, mannose, galactose, xylose, and arabinose.

<(D) Step of fermenting a saccharified product to obtain an organic acid>
In step (d), the saccharified product obtained in step (c) is fermented with filamentous fungi.
The medium used in step (d) includes the saccharified product produced in step (c).
The medium generally contains a carbon source, a nitrogen source, and an inorganic salt, but when the saccharified product produced in step (c) sufficiently contains these nutrient sources necessary for culture. Can use only the above-mentioned saccharified product, the concentration of which is appropriately adjusted, and if necessary, a medium may be prepared by adding a nutrient source in addition to the saccharified product.

In this step, an organic acid is produced by adding and culturing a filamentous fungus capable of producing an organic acid to the medium. The organic acid according to the present invention is a compound having a carboxyl group as an acidic group. 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, ascorbic acid, citric acid, Examples include kojic acid, dipicolinic acid, and aconitic acid. Of these, lactic acid and fumaric acid are preferred.
The organic acid can be produced by culturing filamentous fungi capable of producing them. Examples of these filamentous fungi include microorganisms belonging to the genus Rhizopus. Specifically, Rhizopus oryzae and Rhizopus delemar are preferable from the viewpoint of producing a large amount of L-lactic acid and fumaric acid.

  The culture temperature is preferably 20 to 40 ° C, more preferably 30 to 37 ° C. In addition, the pH of the medium is preferably 2 to 7, more preferably 4 to 6, from the viewpoint of bacterial cell growth and organic acid productivity. The pH control is preferably performed using calcium hydroxide, sodium hydroxide, calcium carbonate, ammonia, sulfuric acid, hydrochloric acid, or the like.

As the culture method, any one of anaerobic conditions and aerobic conditions can be appropriately employed. A conventionally well-known thing can be employ | adopted suitably for the culture tank used for culture | cultivation. Specific examples include an aeration and stirring type culture tank, a bubble column type culture tank, a fluidized bed culture tank, and a packed bed culture tank. In order to improve the production rate of the organic acid, an aeration stirring type culture tank, a bubble column type culture tank, and a fluidized bed culture tank are preferable.
Further, from the viewpoint that the organic acid can be easily separated from the culture medium after fermentation and continuous production is possible, the filamentous fungus is preferably cultivated by forming a pellet (mycelium mass).
As a method for culturing pellet-like filamentous fungi, known methods can be employed. For example, after inoculating filamentous fungal spores in a liquid medium, the spores are germinated to form hyphae, and the cells are formed from the hyphae. And pelletized. After culturing, the filamentous fungal pellet can be extracted from the culture tank together with the culture solution, separated and recovered from the medium by operations such as filtration and centrifugation, and used in step (d). It is also possible to leave the filamentous fungus pellet in the culture tank and perform the step (d) in the same culture tank.

  According to the culture, alcohol is usually by-produced in addition to the organic acid. However, according to the present invention, by combining the steps (a) and (b), an organic acid is selectively obtained, and the amount of by-product alcohol is reduced.

  After step (d), an ester may be produced using the product obtained in step (d) as a raw material, and the resulting ester may be distilled. By the distillation step, substances other than the organic acid can be removed from the product obtained in the step (d).

  The mass ratio (organic acid / alcohol) of the organic acid and alcohol obtained in the step (d) is preferably 1.3 or more, more preferably 2.0 or more, from the viewpoint of the yield of the organic acid. Preferably it is 3.0 or more, More preferably, it is 4.0 or more.

  In relation to the above-described embodiment, the present invention further discloses the following manufacturing method.

<1> A method for producing an organic acid comprising the following steps (a) to (d).
(A): Lignocellulosic biomass containing cellulose having a cellulose I-type crystallinity of more than 30% represented by the following formula (1) is pulverized, and the cellulose I-type crystallinity is reduced to 0-30%. Step of Crystallinity (%) = [(I _c −I _a ) / I _c ] × 100 (1)
[I _c is the diffraction intensity of the lattice plane (002) (lattice plane spacing = 22.4 nm) in X-ray diffraction, and Ia is the diffraction intensity of the amorphous portion (lattice plane spacing = 27.3 nm)]
(B): Step of washing the pulverized product obtained in step (a) with water (c): Step (d) of obtaining a saccharified product by hydrolyzing the solid obtained in step (b) with a hydrolase : (C) Step of fermenting the saccharified product obtained in the step with a filamentous fungus to obtain an organic acid

<2> The method for producing an organic acid according to <1>, wherein the cellulose content of the lignocellulosic biomass is preferably 20% by mass or more, more preferably 40% by mass or more, and still more preferably 60% by mass or more.
<3> Step (a) is preferably a pulverizer selected from a vertical roller mill, a container drive medium mill, a medium agitation mill and a compaction shear mill, more preferably a container drive medium mill or a medium agitation mill, and more preferably Is a method for producing an organic acid according to <1> or <2>, which is performed using a container drive medium mill, more preferably a vibration mill filled with rods or balls.
<4> The method for producing an organic acid according to any one of <1> to <3>, wherein an alkali is added in the step (a).
<5> The above <4>, wherein the amount of alkali used is preferably 1 to 50 parts by mass, more preferably 3 to 30 parts by mass, and even more preferably 5 to 10 parts by mass with respect to 100 parts by mass of lignocellulosic biomass. The manufacturing method of the organic acid as described in any one of.
<6> The above <4>, wherein the alkali is preferably sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate or calcium hydroxide, more preferably sodium hydroxide or potassium hydroxide, still more preferably sodium hydroxide. Or the manufacturing method of the organic acid as described in <5>.
<1> The cellulose I type crystallinity after pulverization in the step <7> (a) is preferably 25% or less, more preferably 20% or less, still more preferably 10% or less, and further preferably 0%. <1> The manufacturing method of the organic acid in any one of <6>.
<8> In the step (b), the water-soluble component residual ratio of the pulverized product is preferably 0.5 or less, more preferably 0.4 or less, still more preferably 0.2 or less, and further preferably 0.1 or less. The method for producing an organic acid according to any one of <1> to <7>, wherein the organic acid is washed with water until it becomes.
<9> The method for producing an organic acid according to any one of <1> to <8>, wherein in the step (b), the pulverized product is washed with water until the water-soluble component remaining ratio is preferably 0.001 or more.
<10> In the step (b), the water-soluble component residual ratio of the pulverized product is preferably 0.001 to 0.5, more preferably 0.001 to 0.4, and still more preferably 0.001 to 0.2. More preferably, the method for producing an organic acid according to any one of <1> to <7>, wherein the organic acid is washed with water until 0.001 to 0.1.
<11> In the step (b), the total amount is preferably 1 part by mass, more preferably 3 parts by mass or more, still more preferably 5 parts by mass with respect to 1 part by mass of the solid content of the pulverized product obtained in the step (a). As mentioned above, The manufacturing method of the organic acid in any one of said <1>-<10> wash | cleaned with 10 mass parts or more of water more preferably.
<12> In the step (b), the total amount is preferably 100 parts by mass or less, more preferably 50 parts by mass or less, still more preferably 40 parts by mass with respect to 1 part by mass of the solid content of the pulverized product obtained in the step (a). Hereinafter, the method for producing an organic acid according to any one of <1> to <11>, wherein the organic acid is more preferably washed with 30 parts by mass or less of water.
<13> In the washing treatment in step (b), the total amount is preferably 1 to 100 parts by weight, more preferably 3 to 50 parts by weight, more preferably 1 part by weight of the solid content of the pulverized product obtained in step (a) The method for producing an organic acid according to any one of <1> to <10>, wherein the organic acid is preferably washed with 5 to 40 parts by mass, more preferably 10 to 30 parts by mass of water.
<14> In the step (b), the temperature of the dispersion comprising the pulverized product and water obtained in the step (a) is preferably 0 ° C. or higher, more preferably 10 ° C. or higher, and further preferably 20 ° C. The method for producing an organic acid according to any one of the above items <1> to <13>.
<15> In the step (b), the temperature of the dispersion obtained from the pulverized product and water obtained in the step (a) is preferably 100 ° C. or lower, more preferably 90 ° C. or lower, and further preferably 80 ° C. or lower. The method for producing an organic acid according to any one of <1> to <14>.
<16> In the step (b), the temperature of the dispersion comprising the pulverized product and water obtained in the step (a) is preferably 0 to 100 ° C., more preferably 10 to 90 ° C., and still more preferably 20 The manufacturing method of the organic acid in any one of said <1>-<13> which is -80 degreeC.
<17> The water is preferably water, an aqueous solution containing a water-soluble solvent, an acidic aqueous solution or an alkaline aqueous solution, more preferably water or an aqueous solution containing a water-soluble solvent, and further preferably water. The manufacturing method of the organic acid in any one of <16>.
<18> The method for producing an organic acid according to any one of <1> to <17>, wherein the hydrolase in step (c) is cellulase.
<19> The cellulase in step (c) is preferably a cellulase derived from Trichoderma reesei, a cellulase derived from a strain of Bacillus sp. KSM-N145 (FERM P-19727), or a Bacillus sp. ) Cellulase derived from KSM-N252 (FERM P-17474) strain, Bacillus sp. Cellulase derived from KSM-N115 (FERM P-19726) strain, Bacillus sp. KSM-N440 (FERM P-) 19728) strain-derived cellulase, Bacillus sp. KSM-N659 (FERM P-19730) strain-derived cellulase, Trichoderma bili (Trichoderma viride), Aspergillus Akureatasu (Aspergillus acleatus), Clostridium thermocellum (Clostridium thermocellum), Clostridium stercorarium (Clostridium stercorarium), Clostridium josui (Clostridium josui), Cellulomonas Fimi (Cellulomonas fimi), Acremonium cell Lori caustics (Acremonium celluloriticus ), Irpex lacteus, Aspergillus niger, Humicola insolens A heat-resistant cellulase derived from Pyrococcus horikoshii, more preferably a cellulase derived from Trichoderma reesei, a cellulase derived from Trichoderma viride, or an insulase derived from Trichoderma viride. The method for producing an organic acid according to <18>, which is a cellulase derived from Trichoderma reesei.
<20> The organic acid is preferably lactic acid, fumaric acid, itaconic acid, malic acid, pyruvic acid, tartaric acid, succinic acid, maleic acid, glutaric acid, levulinic acid, propionic acid, gluconic acid, ascorbic acid, citric acid, koj The method for producing an organic acid according to any one of <1> to <19>, which is an acid, dipicolinic acid or aconitic acid, more preferably lactic acid or fumaric acid.
<21> The filamentous fungus is preferably a microorganism belonging to the genus Rhizopus, more preferably Rhizopus oryzae or Rhizopus delemar, and more preferably Rhizopus oryzae (Rhizpus). The method for producing an organic acid according to any one of <1> to <20>.

<Analysis method>
[Calculation of crystallinity]
It measured on condition of the following using "Rigaku RINT 2500VC X-RAY diffractometer" made from Rigaku Corporation, and calculated cellulose I type crystallinity based on the above-mentioned calculation formula (1).
The measurement conditions were X-ray source: Cu / Kα-radiation (wavelength λ = 0.154056 nm), tube voltage: 40 kv, tube current: 120 mA, measurement range: 2θ = 5-45 °. The measurement sample was prepared by compressing a pellet having an area of 320 mm ² × thickness of 1 mm. The X-ray scan speed was measured at 10 ° / min.
As is I _c diffraction intensity at 2θ = 22.6 °, as I _a using the diffraction intensity at 2θ = 18.5 °.

[Measurement of various components by high-performance liquid chromatograph (HPLC)]
The fermentation broth was appropriately diluted with a 0.0085N sulfuric acid aqueous solution and filtered using a cellulose acetate membrane filter (ADVANTEC) having a pore size of 0.22 μm to obtain a sample for HPLC analysis. The HPLC analysis conditions were as follows: Column: ICSep ICE-ION-300, eluent: 0.0085N sulfuric acid, 0.4 mL / min, detection method: RI (HITACHI, L-2490), column temperature: 40 ° C., injection volume : 20 μL, retention time: 40 minutes. The retention time of each component in this analytical system was glucose: 16 minutes, xylose: 17 minutes, lactic acid: 23 minutes, fumaric acid: 27 minutes, ethanol: 34 minutes.

[Measurement of water content]
The moisture content was measured using an electronic moisture meter (MOISTUREBALANCE MOC-120H (manufactured by Shimadzu Corporation)). A sample was placed on the moisture meter and measurement was started. The temperature was raised to 120 ° C., which was a preset temperature, and then held at 120 ° C. The time when the change in the mass of the sample in 30 seconds was less than 0.05% was regarded as the end of measurement. The mass reduction rate from the start of measurement to the end of measurement was taken as the amount of water.

[Calculation of residual ratio of water-soluble components]
The pulverized product was washed 1 to 3 times, and the residual ratio of water-soluble components was calculated based on the above formula (2).

<Preparation of pelletized filamentous fungus>
[Preparation of spore suspension]
The strain is a filamentous fungus R. cerevisiae obtained from the National Institute of Technology and Evaluation (NITE). oryzae NBRC5384 was used. Filamentous fungi are streaked / coated on slanted agar medium (Difco Potato Dextrose Agar, Becton, Dickinson and Company) formed in a test tube, and statically cultured at room temperature, and periodically passaged. Went. When using bacterial cells, add 10 mL of sterilized distilled water to the test tube in which the cells have grown, then collect the spores by stirring for 4 minutes with a touch mixer, and then add and dilute by adding sterile distilled water. A solution adjusted to 10 ⁶ spores / mL was used as a spore suspension.

[Pelletization of filamentous fungi]
The pelleted filamentous fungus was prepared by the following two-stage culture.
In the first stage culture, a 200 mL baffled Erlenmeyer flask charged with 60 mL of PDB medium (Difco Potato Dextrose Broth, Becton, Dickinson and Company) was sterilized, and the spore suspension prepared by the above method was 1 × 10 ^The cells were inoculated so as to have ⁴ spores / mL, and cultured for 3 days under a culture condition of 27 ° C. and 100 r / m (PRECI, PRXYg-98R).
In the second stage of culture, pellet formation medium (glucose (reagent) 10% by mass, magnesium sulfate heptahydrate 0.025% by mass, zinc sulfate heptahydrate 0.009% by mass, ammonium sulfate 0.1% by mass, Sterilize a 500 mL Erlenmeyer flask charged with 100 mL of potassium dihydrogen phosphate (0.06% by mass), inoculate 5.0 g of calcium carbonate and 4 mL of the first stage culture solution at 27 ° C., 170 r / m ( (PRECI, PRXYg-98R) for 1.5 days.

[Collecting pellets]
The filamentous fungus pellet culture obtained above was filtered with gauze for about 1 minute until the filtrate drip settled to obtain a wet filamentous fungus pellet. The obtained pellet was immediately subjected to fermentability evaluation.

<Evaluation of fermentability>
[Culture method]
Enzymatic saccharified solution of lignocellulosic biomass 100 mL and various minor components (magnesium sulfate heptahydrate 0.025% by mass, zinc sulfate heptahydrate 0.009% by mass, ammonium sulfate 0.1% by mass, dihydrogen phosphate 0.06% by mass of potassium and 5.0% by mass of calcium carbonate (both as final concentrations) were added to a sterilized 500 mL Erlenmeyer flask, followed by filamentous fungal pellet 12 prepared in <Preparation of pelleted filamentous fungus>. .5 g (wet state) was added. Immediately after that, sampling was performed at 0 hour of culture, and then cultured under the culture conditions of 35 ° C. and 170 r / m (PRECI, PRXYg-98R). Sampling was performed again at 48 hours.

[Evaluation method]
From the analysis values at 0 hour and 48 hours, (1) conversion rate from sugar to lactic acid (P [%]), (2) conversion rate from sugar to ethanol (Q [%]), (3) The four items of the ratio of lactic acid and ethanol produced (R [%]) and (4) conversion rate from sugar to fumaric acid (P ′ [%]) were used as evaluation axes. The calculation formula of each item is shown in Table 1 and formulas (3) to (6).

Conversion rate from sugar to lactic acid
P [%] = (L ₄₈ −L ₀ ) / (G ₀ + X ₀ ) × 100 (3)
Conversion rate from sugar to ethanol
_{Q [%] = (E 48} -E 0) / (G 0 + X 0) × 100 (4)
Ratio of lactic acid and ethanol produced from sugar
_{R [%] = (L 48} -L 0) / (E 48 -E 0) × 100 (5)
Conversion rate from sugar to fumaric acid P ′ [%] = (F ₄₈ −F ₀ ) / (G ₀ + X ₀ ) × 100 (6)

(Examples 1-5) Effect of water-washing of dry-pulverized sugarcane bagasse [drying treatment of biomass]
Sugar cane bagasse was dried at 80 ° C. overnight to obtain dry sugar cane bagasse having a moisture content of 6% by mass. The cellulose content of sugarcane bagasse was 38% and the crystallinity was 37%.

[(A) Step of grinding lignocellulosic biomass]
(Dry grinding process)
50 g of the dried sugarcane bagasse obtained in the drying step was put into a vibration mill (Chuo Kako Co., Ltd., “MB-1”, total vessel capacity 3.58 L), and the rod had an outer diameter of 30 mm, a length of 211 mm, A stainless steel material, 13 rods having a circular cross-sectional shape, were filled in a vibration mill (filling rate: 58% by volume) and processed for 0.5 hours under conditions of an amplitude of 8 mm and a circular rotation of 1200 r / m. The temperature of the obtained pulverized product was 65 ° C. due to heat generated by the treatment.
After completion of the treatment, no pulp sticking matter was found on the wall or bottom of the vibration mill. The obtained pulverized product was taken out from the vibration mill. The crystallinity of the pulverized product was 6%. The average particle size based on volume was 25 μm.

[(B) Step of washing the pulverized product]
For each example, the pulverized product was washed 1 to 3 times.
First wash: Distilled water (W ₁ unit: gram) was added to 50 g of the pulverized powder, and mixed and stirred at room temperature for 10 minutes. The temperature of the dispersion consisting of the pulverized product and distilled water was room temperature. Subsequently, the solid was precipitated by centrifugation (centrifuge: HITACHI HIMAC CR22GIII, rotor: R9A, 7000 r / m, 5 to 10 minutes), and then the supernatant (W ₂ ) was discarded.
Second wash: After adding the same weight (W ₂ ) of distilled water as the supernatant discarded in the first wash to the precipitate fraction obtained in the first wash, mixing and stirring again, the same conditions as in the first wash Centrifugation was performed and the supernatant (W ₃ ) was discarded.
Wash 3rd: Add distilled water of the same weight (W ₃ ) as the supernatant discarded in the 2nd wash to the precipitate fraction obtained in the 2nd wash and mix and stir again. Centrifugation was performed and the supernatant (W ₄ ) was discarded.
Table 2 shows values of W _{1 to} W ₄ in Examples 1 to 5. In addition, Table 3 shows the residual ratio of water-soluble components calculated from these values.

[(C) Step of obtaining a saccharified product by hydrolyzing the solid obtained in the washing step with a hydrolase]
Distilled water was added to the precipitate fraction (solid matter) obtained in the washing step until the total weight reached 250 g, and then saccharifying enzyme (Cellic Ctec2, Novozyme, 2.5 mL) was added, and the temperature was 50 ° C. and 150 r. / M (PRECI, PRXYg-98R) for 4.5 days for saccharification reaction. The saccharification reaction completion liquid was filtered with a filter paper (no.2, ADVANTEC) and a membrane filter system (IWAKI, Cat no. 8030-001) having a pore diameter of 0.22 μm. The obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.

[(D) Fermentation process of saccharified product by filamentous fungi]
Fermentability was evaluated by the method shown in <Evaluation of fermentability>. The results are shown in Table 3. By changing the degree of washing, the production ratio of lactic acid and ethanol changed greatly, and an increase in lactic acid production was recognized as the degree of washing increased.

(Comparative Example 1) (b) Fermentation of sugar-containing liquid obtained by enzymatic saccharification of dry-milled sugarcane bagasse without washing step (b) The same operation as in Example 1 was performed except that step (b) was not carried out. It was.
(C) After the step, the obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.
Table 3 shows the results obtained after the step (d). Compared with Example 1, the total of the conversion rate to lactic acid and the conversion rate to ethanol was low, and R was small.

(Comparative Example 2)
The shredder treatment of sugarcane bagasse was performed as described below, and the step of washing the obtained crushed sugarcane bagasse and the step of drying the sugarcane bagasse obtained by washing with water were performed.
Subsequently, (a) process, (c) process, and (d) process were performed with respect to sugarcane bagasse similarly to Examples 1-5. In addition, the (b) process was not performed after the (a) process.

[Shredder treatment process]
Sugar cane bagasse was crushed by a shredder (COMIX, cross-cut shredder S330).
[Washing process]
A total of three washing operations were performed on the crushed sugarcane bagasse obtained in the shredder treatment step.
First wash: 500 g of distilled water was added to 50 g of sugarcane bagasse, and mixed and stirred at room temperature for 10 minutes. Subsequently, the slurry was suction filtered on a Nutsche with a gauze drawn, and 285 g of liquid was separated and discarded by pressing with a spoon.
Second wash: 500 g distilled water was added to the cake fraction obtained in the first wash and mixed and stirred again, followed by filtration under the same conditions as the first, and 480 g of liquid was separated and discarded.
3rd washing: 500 g of distilled water was added to the cake fraction obtained in the 2nd washing and mixed and stirred again, followed by filtration and separation under the same conditions as the first, and 480 g of liquid was separated and discarded.
Table 3 shows the calculated water-soluble component residual ratio.
[Drying process]
The sugar cane bagasse obtained in the water washing step was dried at 80 ° C. overnight to obtain a dry sugar cane bagasse having a moisture content of 6% by mass.
Table 3 shows the results obtained in steps (c) and (d). Compared with Examples 1-5, the sum total of the conversion rate to lactic acid and the conversion rate to ethanol was low, ethanol production was high, lactic acid production was low, and R was small.

(Example 6) Effect of water washing of dry alkali mixed pulverized sugarcane bagasse (a) The same operation as in Examples 1 to 5 was performed except that the step (b) and the step (c) were performed as follows.
[(A) Step of grinding lignocellulosic biomass]
[Dry alkali mixed pulverization]
50 g of dried sugarcane bagasse and 4.4 g of sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) are put into a vibration mill (manufactured by Chuo Kako Co., Ltd., “MB-1”, total capacity 3.58 L) as a rod. , 30 mm outer diameter, 211 mm length, stainless steel, 13 rods with a circular cross-sectional shape are filled into a vibration mill (filling rate 58% by volume) and processed for 1 hour under conditions of amplitude 8 mm and circular rotation 1200 r / m. Went. After completion of the treatment, the obtained pulverized product was taken out from the vibration mill. The crystallinity of the pulverized product was 2%. The volume-based average particle size was 36 μm.

[(B) Step of washing the pulverized product]
The obtained pulverized product was washed three times.
First wash: 540 g of distilled water was added to 60 g of the pulverized powder, and mixed and stirred at room temperature for 10 minutes. The temperature of the dispersion consisting of the pulverized product and distilled water was room temperature. Subsequently, the solid was settled by centrifugation (centrifuge: HITACHI HIMAC CR22GIII, rotor: R9A, 7000 r / m, 5 to 10 minutes), and then 360 g of the supernatant was discarded.
Second wash: 500 g of distilled water was added to the precipitate fraction obtained in the first wash and the mixture was stirred again, then centrifuged under the same conditions as the first, and 502 g of the supernatant was discarded.
3rd washing: 500 g of distilled water was added to the precipitate fraction obtained in the 2nd washing and the mixture was stirred again, then centrifuged under the same conditions as in the first, and 498 g of the supernatant was discarded. Table 3 shows the calculated water-soluble component residual ratio.

[(C) Step of obtaining a saccharified product using a hydrolase on the solid obtained in the washing step]
Distilled water was added to the cake obtained in the washing step to make it a little less than 250 g, and after adjusting the pH to 5.0 with hydrochloric acid, distilled water was added so that the total weight was 250 g. Subsequently, a saccharification enzyme (Cellic Ctec2, Novozyme, 2.5 mL) was added, and a saccharification reaction was performed at 50 ° C. and 100 r / m (PRECI, PRXYg-98R) for 4 days. The saccharification reaction completion liquid was filtered with a filter paper (no.2, ADVANTEC) and a membrane filter system (IWAKI, Cat no. 8030-001) having a pore diameter of 0.22 μm to obtain an aseptic liquid. The obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.
Table 3 shows the results of fermentability evaluation performed by the method shown in <Fermentation Evaluation>.

(Comparative Example 3) Fermentation of sugar-containing liquid obtained by enzymatic saccharification of dry alkali mixed pulverized sugarcane bagasse (b) The same operation as in Example 6 was performed except that the step was not performed.
Table 3 shows the results obtained in steps (c) and (d). Compared with Example 6, the total of the conversion rate to lactic acid and the conversion rate to ethanol was low, ethanol production was high, lactic acid production was low, and R was small.

(Comparative Example 4) Fermentation of sugar-containing liquid obtained by washing sugarcane bagasse with water, followed by dry alkaline mixed pulverization and enzymatic saccharification The steps of washing sugarcane bagasse with water and drying the sugarcane bagasse obtained with water As before (a).
Next, as in Example 6, the steps (a), (c), and (d) were performed on sugarcane bagasse. In addition, the (b) process was not performed after the (a) process.

[Washing process]
(A) A total of three washing operations were performed on sugarcane bagasse before the process.
First wash: Distilled water (540 g) was added to sugarcane bagasse (60 g), and the mixture was stirred at room temperature for about 10 minutes. The temperature of the dispersion consisting of the pulverized product and distilled water was room temperature. Subsequently, solids were settled by centrifugation (centrifuge: HITACHI HIMAC CR22GIII, rotor: R9A, 7000 r / m, 5 to 10 minutes), and then 203 g of the supernatant was discarded through gauze.
Second wash: 540 g of distilled water was added to the precipitate fraction obtained in the first wash and the solid matter on the gauze, mixed and stirred again, then centrifuged under the same conditions as the first, and passed through the gauze. 527 g of supernatant was discarded.
Third wash: 540 g of distilled water was added to the precipitate fraction obtained in the second wash and the solid matter on the gauze, mixed and stirred again, then centrifuged under the same conditions as the first, and passed through the gauze. 527 g of supernatant was discarded. Table 3 shows the calculated water-soluble component residual ratio.
[Drying process]
The sugar cane bagasse obtained in the water washing step was dried at 80 ° C. overnight to obtain a dry sugar cane bagasse having a moisture content of 6% by mass.
Table 3 shows the results obtained in steps (c) and (d). Compared with Example 6, the total of the conversion rate to lactic acid and the conversion rate to ethanol was low, ethanol production was high, lactic acid production was low, and R was small.

(Example 7) Effect of water washing on dry pulverized EFB (fruit bunch after removing palm fruit) [drying treatment of biomass]
Shredder-treated (COMIX, cross-cut shredder S330) EFB was dried at 80 ° C. overnight to obtain a dry EFB with a moisture content of 5 mass%. The cellulose content of EFB was 36%, and the crystallinity was 40%.

[(A) Step of grinding lignocellulosic biomass]
(Dry grinding process)
50 g of the obtained dry EFB was put into a vibration mill (manufactured by Chuo Kako Co., Ltd., “MB-1”, container total capacity 3.58 L), and as a rod, an outer diameter of 30 mm, a length of 211 mm, a stainless steel material, a cross-sectional shape Were filled in a vibration mill (filling rate: 58% by volume) and processed for 1.0 hour under conditions of an amplitude of 8 mm and a circular rotation of 1200 r / m. The temperature of the obtained pulverized product was 70 ° C. due to heat generated by the treatment.
After completion of the treatment, no pulp sticking matter was found on the wall or bottom of the vibration mill. The obtained pulverized product was taken out from the vibration mill. The crystallinity of the pulverized product was 0%. The volume-based average particle size was 20 μm.

[(B) Step of washing the pulverized product]
The obtained pulverized product was washed three times.
First wash: 500 g of distilled water was added to 50 g of the pulverized powder, and mixed and stirred at room temperature for 10 minutes. The temperature of the dispersion consisting of the pulverized product and distilled water was room temperature. Subsequently, solids were precipitated by centrifugation (centrifuge: HITACHI HIMAC CR22GIII, rotor: R9A, 7000 r / m, 5 to 10 minutes), and then 365 g of the supernatant was discarded.
Second wash: To the precipitate fraction obtained in the first wash, distilled water of the same weight (365 g) as the supernatant discarded in the first wash was added, mixed and stirred again, and centrifuged under the same conditions as the first wash. Separation was performed and 362 g of the supernatant was discarded.
3rd washing: Add the same weight (362 g) of distilled water as the supernatant discarded in the 2nd washing to the precipitate fraction obtained in the 2nd washing, mix and stir again, then centrifuge under the same conditions as the 1st Separation was performed and 361 g of the supernatant was discarded.
Table 3 shows the calculated water-soluble component residual ratio.

[(C) Step of obtaining a saccharified product using a hydrolase to the solid obtained in the washing step]
Distilled water was added to the precipitate fraction obtained by the washing treatment until the total weight became 280 g, saccharification enzyme (Cellic Ctec2, Novozyme, 2.5 mL) was added, and 50 ° C., 150 r / m (PRECI) The saccharification reaction was carried out for 4.5 days using the company PRXYg-98R). The saccharification reaction completion liquid was filtered with a filter paper (no.2, ADVANTEC) and a membrane filter system (IWAKI, Cat no. 8030-001) having a pore diameter of 0.22 μm. The obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.

[(D) Fermentation process of saccharified product by filamentous fungi]
Fermentability was evaluated by the method shown in <Evaluation of fermentability>. The results are shown in Table 3.

(Comparative Example 5)
(B) The same operation as Example 7 was performed except not performing a process.
(C) After the step, the obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.
Table 3 shows the results obtained after the step (d). Compared with Example 7, the total of the conversion rate to lactic acid and the conversion rate to ethanol was low, and R was small.

(Example 8) Effect of water washing on dry pulverized inowara [drying treatment of biomass]
The rice bran cut into 5 cm long pieces with a scissors was dried at 80 ° C. overnight to obtain a dry rice bran having a moisture content of 6% by mass. Inowara had a cellulose content of 40% and a crystallinity of 54%.

[(A) Step of grinding lignocellulosic biomass]
(Dry grinding process)
50 g of the dried inowara obtained in the above drying process is put into a vibration mill (Chuo Kako Co., Ltd., “MB-1”, container total capacity 3.58 L), and the rod has an outer diameter of 30 mm, a length of 211 mm, and a material. Thirteen rods with a circular cross-sectional shape made of stainless steel were filled in a vibration mill (filling rate: 58% by volume), and the treatment was performed for 0.5 hour under conditions of an amplitude of 8 mm and a circular rotation of 1200 r / m. The temperature of the obtained pulverized product was 65 ° C. due to heat generated by the treatment.
After completion of the treatment, no pulp sticking matter was found on the wall or bottom of the vibration mill. The obtained pulverized product was taken out from the vibration mill. The crystallinity of the pulverized product was 7%. The average particle size based on volume was 25 μm.

[(B) Step of washing the pulverized product]
The obtained pulverized product was washed three times.
First wash: 500 g of distilled water was added to 50 g of the pulverized powder, and mixed and stirred at room temperature for 10 minutes. The temperature of the dispersion consisting of the pulverized product and distilled water was room temperature. Subsequently, the solid was settled by centrifugation (centrifuge: HITACHI HIMAC CR22GIII, rotor: R9A, 7000 r / m, 5 to 10 minutes), and then 410 g of the supernatant was discarded.
Second wash: The same amount (410 g) of distilled water as the supernatant discarded in the first wash was added to the precipitate fraction obtained in the first wash, mixed and stirred again, and then centrifuged under the same conditions as the first wash. Separation was performed and 406 g of the supernatant was discarded.
3rd washing: Add the same weight (406 g) of distilled water as the supernatant discarded in the 2nd washing to the precipitate fraction obtained in the 2nd washing, mix and stir again, then centrifuge under the same conditions as the 1st Separation was performed, and 410 g of the supernatant was discarded.
The calculated water-soluble component residual ratio is shown in Table 3.

[(C) Step of obtaining a saccharified product using a hydrolase to the solid obtained in the washing step]
The same operation as step (c) described in Examples 1 to 5 was performed. The obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.

[(D) Fermentation process of saccharified product by filamentous fungi]
Fermentability was evaluated by the method shown in <Evaluation of fermentability>. The results are shown in Table 3.

(Comparative Example 6)
(B) The same operation as in Example 8 was performed except that the step was not performed.
(C) After the step, the obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.
Table 3 shows the results obtained after the step (d). Compared with Example 8, the total of the conversion rate to lactic acid and the conversion rate to ethanol was low, and R was small.

From the results of Examples 1 to 5 and Comparative Example 1, the conversion rate of sugar to lactic acid increases when the (b) washing step is performed after the (a) step and the (c) step and (d) step are performed. I understood.
From the results of Examples 1 to 5 and Comparative Example 2, it was found that the conversion rate of sugar to lactic acid was increased when the (b) washing step was performed after the (a) step, not before the (a) step.

(Example 9) Effect of water-washing of dry pulverized sugarcane bagasse (effect of pulverization medium)
[Biomass drying treatment]
Sugar cane bagasse was dried at 80 ° C. overnight to obtain dry sugar cane bagasse having a moisture content of 5 mass%. The cellulose content of sugarcane bagasse was 38% and the crystallinity was 37%.

[(A) Step of grinding lignocellulosic biomass]
(Dry grinding process)
50 g of the dried sugarcane bagasse obtained in the above drying step is put into a vibration mill (Chuo Kako Co., Ltd., “MB-1”, container total capacity 3.58 L), and the grinding medium has an outer diameter of 10 mm and is made of stainless steel. 15.4 kg of spheres were filled in a vibration mill (filling rate: 58% by volume) and treated for 1.0 hour under conditions of an amplitude of 8 mm and a circular rotation of 1200 r / m.
After completion of the treatment, no pulp sticking matter was found on the wall or bottom of the vibration mill. The obtained pulverized product was taken out from the vibration mill. The crystallinity of the pulverized product was 0%. The volume-based average particle size was 36 μm.

[(B) Step of washing the pulverized product]
The obtained pulverized product was washed three times.
First wash: 500 g of distilled water was added to 50 g of the pulverized powder, and mixed and stirred at room temperature for 10 minutes. The temperature of the dispersion consisting of the pulverized product and distilled water was room temperature. Subsequently, the solid was precipitated by centrifugation (centrifuge: HITACHI HIMAC CR22GIII, rotor: R9A, 7000 r / m, 10 minutes), and then 373 g of the supernatant was discarded.
Second wash: The same amount (373 g) of distilled water as the supernatant discarded in the first wash was added to the precipitate fraction obtained in the first wash, mixed and stirred again, and then centrifuged under the same conditions as in the first wash. Separation was performed and 373 g of the supernatant was discarded.
3rd washing: Add the same weight (373 g) of distilled water as the supernatant discarded in the 2nd washing to the precipitate fraction obtained in the 2nd washing, mix and stir again, and then centrifuge under the same conditions as the 1st Separation was performed and 372 g of the supernatant was discarded.
Table 3 shows the calculated water-soluble component residual ratio.

[(C) Step of obtaining a saccharified product using a hydrolase to the solid obtained in the washing step]
Distilled water was added to the precipitate fraction obtained in the washing step until the total weight reached 250 g, saccharification enzyme (Cellic Ctec2, Novozyme, 2.5 mL) was added, and 50 ° C., 100 r / m (PRECI) The saccharification reaction was carried out for 4.5 days using the company PRXYg-98R). The saccharification reaction completion liquid was filtered with a filter paper (no.2, ADVANTEC) and a membrane filter system (IWAKI, Cat no. 8030-001) having a pore diameter of 0.22 μm. The obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.

[(D) Fermentation process of saccharified product by filamentous fungi]
Fermentability was evaluated by the method shown in <Evaluation of fermentability>. The results are shown in Table 3. From the results of Example 9, it was found that the conversion rate of sugar to lactic acid was increased by the introduction of the (c) washing step even when the grinding medium was a ball.

(Example 10) Effect of washing dry crushed sugarcane bagasse (influence of pulverization degree)
[Biomass drying treatment]
Sugar cane bagasse was dried at 80 ° C. overnight to obtain dry sugar cane bagasse having a moisture content of 5 mass%. The cellulose content of sugarcane bagasse was 38% and the crystallinity was 37%.

[(A) Step of grinding lignocellulosic biomass]
(Dry grinding process)
50 g of the dried sugarcane bagasse obtained in the above drying step was put into a vibration mill (Chuo Kako Co., Ltd., “MB-1”, container total capacity 3.58 L), and the outer diameter was 30 mm and the length was 211 mm as a grinding medium. Then, 13 rods with a stainless steel material and a circular cross section were filled in a vibration mill (filling rate: 58% by volume), and the treatment was performed for 0.17 hours under the conditions of an amplitude of 8 mm and a circular rotation of 1200 r / m.
After completion of the treatment, no pulp sticking matter was found on the wall or bottom of the vibration mill. The obtained pulverized product was taken out from the vibration mill. The crystallinity of the pulverized product was 21%. The average particle size based on volume was 38 μm.

[(B) Step of washing the pulverized product]
The obtained pulverized product was washed three times.
First wash: 500 g of distilled water was added to 50 g of the pulverized powder, and mixed and stirred at room temperature for 10 minutes. The temperature of the dispersion consisting of the pulverized product and distilled water was room temperature. Subsequently, the solid was precipitated by centrifugation (centrifuge: HITACHI HIMAC CR22GIII, rotor: R9A, 7000 r / m, 10 minutes), and then 380 g of the supernatant was discarded.
Second wash: The same amount (380 g) of distilled water as the supernatant discarded in the first wash was added to the precipitate fraction obtained in the first wash, mixed and stirred again, and centrifuged under the same conditions as in the first wash. Separation was performed and 379 g of the supernatant was discarded.
3rd washing: Add the same weight (379 g) of distilled water as the supernatant discarded in the 2nd washing to the precipitate fraction obtained in the 2nd washing, mix and stir again, then centrifuge under the same conditions as the 1st Separation was performed and 380 g of the supernatant was discarded. Table 3 shows the calculated water-soluble component residual ratio.

[(C) Step of obtaining a saccharified product using a hydrolase to the solid obtained in the washing step]
Distilled water was added to the precipitate fraction obtained in the washing step until the total weight reached 250 g, saccharification enzyme (Cellic Ctec2, Novozyme, 2.5 mL) was added, and 50 ° C., 100 r / m (PRECI) The saccharification reaction was carried out for 4.5 days using the company PRXYg-98R). The saccharification reaction completion liquid was filtered with a filter paper (no.2, ADVANTEC) and a membrane filter system (IWAKI, Cat no. 8030-001) having a pore diameter of 0.22 μm. The obtained liquid was analyzed by HPLC. Table 3 shows the total concentration (G ₀ + X ₀ ) of glucose and xylose in the obtained saccharified product.

[(D) Fermentation process of saccharified product by filamentous fungi]
Fermentability was evaluated by the method shown in <Evaluation of fermentability>. The results are shown in Table 3. From the results of Example 10, it was found that if the crystallinity after pulverization was 30% or less, the conversion rate of sugar to lactic acid was high.

(Example 11) Effect of washing dry milled sugarcane bagasse with water (influence on fumaric acid production)
[Biomass drying treatment]
Sugar cane bagasse was dried at 80 ° C. overnight to obtain dry sugar cane bagasse having a moisture content of 6% by mass. The cellulose content of sugarcane bagasse was 38% and the crystallinity was 37%.

[(A) Step of grinding lignocellulosic biomass]
(Dry grinding process)
50 g of the dried sugarcane bagasse obtained in the above drying step was put into a vibration mill (Chuo Kako Co., Ltd., “MB-1”, container total capacity 3.58 L), and the outer diameter was 30 mm and the length was 211 mm as a grinding medium. In addition, 13 rods with a material of stainless steel and a circular cross section were filled in a vibration mill (filling rate: 58% by volume), and the treatment was performed for 0.5 hours under the conditions of an amplitude of 8 mm and a circular rotation of 1200 r / m.
After completion of the treatment, no pulp sticking matter was found on the wall or bottom of the vibration mill. The obtained pulverized product was taken out from the vibration mill. The crystallinity of the pulverized product was 13%. The average particle size based on volume was 25 μm.

[(B) Step of washing the pulverized product]
The obtained pulverized product was washed three times.
First wash: 500 g of distilled water was added to 50 g of the pulverized powder, and mixed and stirred at room temperature for 10 minutes. The temperature of the dispersion consisting of the pulverized product and distilled water was room temperature. Subsequently, solids were precipitated by centrifugation (centrifuge: HITACHI HIMAC CR22GIII, rotor: R9A, 7000 r / m, 10 minutes), and then 384 g of the supernatant was discarded.
Second wash: The same amount (384 g) of distilled water as the supernatant discarded in the first wash was added to the precipitate fraction obtained in the first wash, mixed and stirred again, and then centrifuged under the same conditions as in the first wash. Separation was performed and 384 g of the supernatant was discarded.
3rd washing: Add the same weight (384 g) of distilled water as the supernatant discarded in the 2nd washing to the precipitate fraction obtained in the 2nd washing, mix and stir again, then centrifuge under the same conditions as the 1st Separation was performed and 384 g of the supernatant was discarded.
Table 4 shows the calculated water-soluble component residual ratio.

[(C) Step of obtaining a saccharified product by hydrolyzing the solid obtained in the washing step with a hydrolase]
Distilled water was added to the precipitate fraction obtained in the washing step until the total weight reached 250 g, saccharification enzyme (Cellic Ctec2, Novozyme, 2.5 mL) was added, and 50 ° C., 100 r / m (PRECI) The saccharification reaction was carried out for 4.5 days using the company PRXYg-98R). The saccharification reaction completion liquid was filtered with a filter paper (no.2, ADVANTEC) and a membrane filter system (IWAKI, Cat no. 8030-001) having a pore diameter of 0.22 μm. The obtained liquid was analyzed by HPLC. Table 4 shows the total concentration of glucose and xylose (G ₀ + X ₀ ) in the obtained saccharified product.

[(D) Fermentation process of saccharified product by filamentous fungi]
R. as a filamentous fungus Oryzae NBRC4749 strain was used and a filamentous fungus pellet was prepared by the method shown in <Preparation of pelletized filamentous fungus>. Moreover, fermentability was evaluated by the method shown in <Evaluation of fermentability> except that 20 g (wet weight) of the filamentous fungal pellet was used and the amount of the enzyme saccharified solution of biomass was 80 mL. . The results are shown in Table 4.
In Example 11, the productivity of fumaric acid was higher than that in Comparative Example 7, that is, the improvement in fumaric acid productivity was recognized by carrying out washing.

(Comparative Example 7) (b) Fermentation of sugar-containing liquid obtained by enzymatic saccharification of dry-milled sugarcane bagasse without performing a washing step (effect on fumaric acid productivity)
(B) The same operation as Example 11 was performed except not performing a process.
(C) After the step, the obtained liquid was analyzed by HPLC. Table 4 shows the total concentration of glucose and xylose (G ₀ + X ₀ ) in the obtained saccharified product.
Table 4 shows the results of the evaluation of fermentability in the same manner as in Example 11.
In Comparative Example 7, fumaric acid productivity was lower than that in Example 11.

Claims (13)

The manufacturing method of the organic acid containing following process (a)-(d).
(A): Lignocellulosic biomass containing cellulose having a cellulose I-type crystallinity of more than 30% represented by the following formula (1) is pulverized, and the cellulose I-type crystallinity is reduced to 0-30%. Step of Crystallinity (%) = [(I _c −I _a ) / I _c ] × 100 (1)
[I _c is the diffraction intensity of the lattice plane (002) (lattice plane spacing = 22.4 nm) in X-ray diffraction, and Ia is the diffraction intensity of the amorphous portion (lattice plane spacing = 27.3 nm)]
(B): Step of washing the pulverized product obtained in step (a) with water (c): Step of obtaining a saccharified product by hydrolyzing the solid obtained in step (b) with a hydrolase (d): (C) A step of fermenting the saccharified product obtained in the step with a filamentous fungus to obtain an organic acid   The method for producing an organic acid according to claim 1, wherein the step (a) is performed using a pulverizer selected from a vertical roller mill, a container drive medium mill, a medium stirring mill, and a consolidation shear mill.   The method for producing an organic acid according to claim 2, wherein the container drive medium mill is a vibration mill filled with rods or balls.   The method for producing an organic acid according to any one of claims 1 to 3, wherein an alkali is added in the step (a).   The manufacturing method of the organic acid of Claim 4 whose usage-amount of an alkali is 1-50 mass parts with respect to 100 mass parts of lignocellulosic biomass.   The method for producing an organic acid according to claim 4 or 5, wherein the alkali is sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate or calcium hydroxide.   (B) The manufacturing method of the organic acid of any one of Claims 1-6 which wash | cleans with water until the water-soluble component residual rate of a ground material becomes 0.5 or less in a process.   The method for producing an organic acid according to claim 1, wherein the water further contains a water-soluble solvent.   The method for producing an organic acid according to any one of claims 1 to 8, wherein in the step (b), the temperature of the dispersion comprising the pulverized product and water obtained in the step (a) is 0 to 100 ° C.   The method for producing an organic acid according to any one of claims 1 to 9, wherein the hydrolase is cellulase.   The method for producing an organic acid according to claim 10, wherein the cellulase is a cellulase derived from Trichoderma reesei.   The method for producing an organic acid according to any one of claims 1 to 11, wherein the organic acid is lactic acid or fumaric acid.   The method for producing an organic acid according to any one of claims 1 to 12, wherein the filamentous fungus is a microorganism belonging to the genus Rhizopus.