JP2010279305A - Method for producing sugar and ethanol from vegetable biomass - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for treating vegetable biomass and a method for producing sugars and ethanol from the vegetable biomass. <P>SOLUTION: This method for treating the vegetable biomass is provided by comprising a process of subjecting the vegetable biomass to a hydrothermal treatment by immersing the vegetable biomass in a solution containing a water soluble organic solvent and an inorganic acid. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a process for producing useful sugars and ethanol from, for example, plant biomass.

  In recent years, the release of global warming substances such as carbon dioxide due to mass consumption of oil has become a problem. As an alternative to petroleum, waste biomass such as waste wood is attracting attention as a chemical raw material resource.

  For example, cellulose, which is one of the main constituents of wood, has glucose molecules as constituent units. Sugars such as glucose can be obtained by hydrolyzing the cellulose. By using the obtained saccharide as a raw material and performing a reaction using various catalysts, a polymer raw material produced in the petrochemical industry can be prepared. Moreover, ethanol can be manufactured by performing fermentation using the obtained saccharides.

  In the production of sugar from plant biomass, as a pretreatment method for plant biomass, for example, a method including a hydrothermal treatment process using a pressurized hot water and a mechanical pulverization process is known (Patent Document 1). ). However, this method requires treatment under high pressure conditions.

  Further, Patent Document 2 discloses a method including a steaming step in which biomass is immersed in a chemical solution containing water, a water-soluble organic solvent and an organic acid, and the plant biomass is cooked as a pretreatment method for plant biomass. . However, this method uses a large amount of a water-soluble organic solvent; the cost is high because an organic acid is used as an acid; the cost is high because it requires a recovery of the organic acid; the organic solvent is present in a high concentration Therefore, a solid-liquid separation process is required before the saccharification process; and since a solid-liquid separation process is necessary, xylose dissolved (extracted) on the liquid side cannot be used, or concentrated to use, There is a problem that the cost is high because collection of xylose or the like is necessary.

JP 2006-136263 A JP 2008-271962

  As described above, conventionally, various methods have been studied as a pretreatment method for plant biomass in the production of sugar from plant biomass. However, there is a pretreatment method that can be efficiently performed at low cost. It was not known.

  Therefore, in view of the above-described circumstances, the present invention provides a plant biomass for obtaining a raw material that can be efficiently produced at low cost and improved in the saccharification rate in the production of sugar from plant biomass. A treatment method and a method for producing sugar and ethanol from plant biomass including the pretreatment are provided.

  As a result of diligent research to solve the above problems, when plant biomass is immersed in a solution containing a water-soluble organic solvent and an inorganic acid and subjected to hydrothermal treatment, a processed product obtained by saccharification treatment is used. The inventors have found that the saccharification rate is improved and have completed the present invention.

  That is, this invention is a plant biomass processing method including the process of immersing plant biomass in the solution containing a water-soluble organic solvent and an inorganic acid, and using for hydrothermal treatment.

  Examples of the water-soluble organic solvent include acetone and ethanol. Examples of the content ratio of the water-soluble organic solvent in the mixture containing the water-soluble organic solvent and the inorganic acid include 1 to 5% by weight.

Examples of the inorganic acid include sulfuric acid and phosphoric acid. Examples of the content ratio of the inorganic acid in the mixture containing the water-soluble organic solvent and the inorganic acid include 0.1 to 0.3% by weight.
The conditions for the hydrothermal treatment include conditions of a temperature of 140 to 240 ° C. and a pressure of 0.1 to 4 MPa.

  The plant biomass treatment method can include a step of recovering the water-soluble organic solvent as a vapor simultaneously with or after the hydrothermal treatment step, and a step of neutralizing the inorganic acid with an alkaline solution after the hydrothermal treatment step. Examples of the alkaline solution used for neutralization include sodium hydroxide, ammonia and potassium hydroxide.

  The present invention also includes a step of immersing plant biomass in a solution containing a water-soluble organic solvent and an inorganic acid in accordance with the above-described plant biomass treatment method, subjecting the biomass to hydrothermal treatment, contacting the hydrothermally treated product with an enzyme, and saccharification. A method for producing sugar, comprising a step of subjecting to a treatment. An example of the enzyme used for the saccharification treatment is cellulase.

  Furthermore, the present invention comprises a step of immersing plant biomass in a solution containing a water-soluble organic solvent and sulfuric acid and / or phosphoric acid in accordance with the above-described plant biomass treatment method and sugar production method, and subjecting the biomass to hydrothermal treatment, An ethanol production method comprising a step of neutralizing a heat-treated product with ammonia and / or potassium hydroxide, a step of bringing a hydrothermally-treated product into contact with an enzyme, subjecting it to a saccharification treatment, and subjecting a yeast to fermentation.

  According to the plant biomass treatment method of the present invention, a raw material used for saccharification treatment can be obtained at low cost. Moreover, according to the sugar production method of the present invention, the saccharification rate is improved, and sugar can be produced with excellent productivity. Furthermore, according to the method for producing ethanol according to the present invention, yeast fermentation can be performed without adding potassium phosphate and / or ammonium sulfate necessary for yeast fermentation to the medium, and the ethanol production by yeast fermentation is reduced. Cost can be reduced.

Hereinafter, the present invention will be described in detail.
This invention is a plant biomass processing method including the process of immersing plant biomass in the solution containing a water-soluble organic solvent and an inorganic acid, and using for a hydrothermal treatment. According to the plant biomass treatment method of the present invention, saccharification is performed in a saccharification treatment by cutting lignin, cellulose, hemicellulose, etc. of plant biomass with an inorganic acid and dissolving lignin, cellulose, hemicellulose, etc. with a water-soluble organic solvent. A raw material with an improved rate can be obtained.

  Here, examples of plant biomass include biomass mainly composed of lignin, cellulose, lignocellulose, and hemicellulose. Examples of the biomass mainly composed of lignocellulose include woody and herbaceous materials (wood, eucalyptus, bagasse, rice straw, cedar, wheat straw, bamboo, etc.), pulp, and wastes thereof (for example, waste paper). In the plant biomass treatment method according to the present invention, a pulverized product obtained by subjecting plant biomass to a pulverization process using a vibration mill, a cutter mill, or the like in advance can be used as the plant biomass. In addition, after pulverization, the pulverized product may be appropriately subjected to a sieve (for example, an opening mesh of 150 μm to 4 mm), and the pulverized product that has passed may be used as plant biomass. Or the said grinding | pulverization process process can also be included as a process of the plant biomass processing method which concerns on this invention. The content ratio of the plant biomass in the hydrothermal treatment is, for example, 5 to 40% by weight, preferably 20 to 40% by weight, particularly preferably 30 to 40% with respect to the mixture containing the plant biomass, the water-soluble organic solvent, and the inorganic acid. % By weight.

  Examples of the water-soluble organic solvent include acetone, ethanol, methanol, isopropanol, butanol and the like, and acetone or ethanol is preferable. The content ratio of the water-soluble organic solvent in the hydrothermal treatment is, for example, 1 to 5% by weight with respect to the mixture containing the water-soluble organic solvent and the inorganic acid.

  Examples of the inorganic acid include sulfuric acid, phosphoric acid, nitric acid, hydrochloric acid, boric acid and the like, and sulfuric acid or phosphoric acid is preferable. The content ratio of the inorganic acid in the hydrothermal treatment is, for example, 0.1 to 0.3% by weight with respect to the mixture containing the water-soluble organic solvent and the inorganic acid.

  In the plant biomass processing method according to the present invention, plant biomass is immersed in a solution containing a water-soluble organic solvent and an inorganic acid and subjected to hydrothermal treatment in a high-temperature and high-pressure vessel or the like. Examples of hydrothermal treatment conditions include a temperature of 140 to 240 ° C. and a pressure of 0.1 to 4 MPa, and more preferably a temperature of 140 to 180 ° C. and a pressure of 0.5 to 1 MPa. The treatment time may be a time sufficient for the lignin, cellulose and hemicellulose to be untangled and become fibrous, for example, 0.5 to 3 hours.

  In this way, a treated product obtained after hydrothermal treatment in a solution containing an inorganic acid and a water-soluble organic solvent (hereinafter referred to as “hydrothermal treated product”) can be used as a raw material used for saccharification treatment. . At the same time or after the hydrothermal treatment step, the high-temperature and high-pressure state can be utilized to recover the water-soluble organic solvent as a vapor and reuse it.

  Further, a hydrothermally treated product may be neutralized with an alkali solution and adjusted to pH 5 to 6, for example, as a hydrothermally treated product and used for saccharification treatment. When the water-soluble organic solvent recovery is performed, the neutralization step can be performed before or after the water-soluble organic solvent recovery or at the time of the recovery. Here, examples of the alkaline solution include sodium hydroxide, ammonia, and potassium hydroxide.

  Furthermore, the present invention provides a pretreatment step in which plant biomass is immersed in a solution containing a water-soluble organic solvent and an inorganic acid in accordance with the above-described plant biomass treatment method according to the present invention, and subjected to hydrothermal treatment. A method for producing sugar, comprising a step of bringing a product into contact with an enzyme and subjecting the product to a saccharification treatment. The resulting saccharification product (sugar) can be converted to ethanol by fermentation using yeast.

  Here, examples of the enzyme include cellulase, hemicellulase (xylanase, xylobiase), mannanase and the like. For example, glucose (sugar) can be obtained by using cellulase as an enzyme. Moreover, xylose (sugar) can be obtained by using hemicellulase as an enzyme. Furthermore, mannose (sugar) can be obtained by using mannanase as an enzyme. As these enzymes, microorganisms that produce the enzymes themselves, culture solutions or culture supernatants of the microorganisms, immobilized enzymes, and the like can be used as the enzymes. For example, when cellulase is used, for example, 4 to 40 FPU (Filter Paper Unit) cellulase is used per 1 g of the hydrothermally treated product.

For example, when cellulase is used, the saccharification step uses a reaction product containing cellulase to which a buffer solution (for example, sodium acetate buffer (pH 5)) is added and a hydrothermal treatment, for example, at a temperature of 35 to 45 ° C. Preferably, it is carried out by subjecting to a reaction at 38 to 42 ° C. for 12 to 48 hours. In addition, you may perform the said reaction under shaking. The obtained processed product (hereinafter referred to as “saccharified product”) may be used as a saccharide as it is, or a product that has been subjected to purification or extraction treatment and purified or extracted may be used as a saccharide.
Examples of the sugar to be obtained include glucose, xylose, mannose, galactose and the like.

によって算出される糖化率(％)を指標に判断することができる。 Whether or not saccharification was significantly performed by saccharification treatment is determined by quantifying the amount of sugar by HPLC or the following formula:

The saccharification rate (%) calculated by the above can be used as an index.

  Further, the present invention provides a hydrothermal treatment by immersing plant biomass in a solution containing a water-soluble organic solvent and sulfuric acid and / or phosphoric acid according to the plant biomass treatment method and sugar production method according to the present invention described above. A step of pre-treating the hydrothermally treated product with ammonia and / or potassium hydroxide; and contacting the hydrothermally treated product with an enzyme, subjecting the hydrothermally treated product to saccharification, and subjecting the yeast to fermentation. It is a manufacturing method. In this method, the neutralization step and the saccharification step are usually performed after the neutralization step, but may be performed after the saccharification step. Furthermore, the saccharification / fermentation step is usually performed after the saccharification step, but may be performed simultaneously.

  The yeast used here may be any yeast that can perform ethanol fermentation. Examples include yeasts belonging to the genus Pichia and Candida.

The basic medium used for yeast culture is generally yeast extract, glucose, KH ₂ PO ₄ , MgSO ₄ .7H ₂ O, NaCl, CaCl ₂ , (NH ₄ ) ₂ SO ₄ , H ₃ BO ₄ at a predetermined concentration. CuSO ₄ · 5H ₂ O, KI, FeCl ₃ · 6H ₂ O, MnSO ₄ · 5H ₂ O, ZnSO ₄ · 7H ₂ O and Na ₂ MoO ₄ · 2H ₂ O. In the method for producing ethanol according to the present invention, the hydrothermally treated product containing sulfuric acid and / or phosphoric acid is neutralized with ammonia and / or potassium hydroxide, so that each is a component of the above-mentioned basal medium (NH ₄ ) _2. SO ₄ and KH ₂ PO ₄ are produced. Therefore, in the fermentation process, it is not necessary to add (NH ₄ ) ₂ SO ₄ and / or KH ₂ PO ₄ together with glucose to the medium containing the saccharified product.

In the fermentation process, yeast is cultured in a medium containing the saccharified product and the above-mentioned medium composition (excluding either or both of (NH ₄ ) ₂ SO ₄ and KH ₂ PO ₄ together with glucose). The culture conditions may be any conditions as long as ethanol fermentation is sufficiently performed and the yeast grows.For example, the temperature is 25 to 45 ° C. (preferably 30 to 37 ° C.), pH 3.0 to 7.0 (preferably pH 4.0). -6.0) for 12-72 hours (preferably 24-48 hours). The culture may be shaking culture. The obtained processed product (hereinafter referred to as “fermented processed product”) may be used as ethanol as it is, or may be subjected to purification or extraction treatment and purified or extracted to be used as ethanol.

によって算出されるエタノール変換率(％)を指標に判断することができる。 Whether ethanol was significantly obtained by fermentation is determined by quantifying the amount of ethanol by HPLC or the like, or the following formula:

The ethanol conversion rate (%) calculated by the above can be used as an index.

  Furthermore, the ethanol production method according to the present invention can also be applied as a yeast culture method.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
1. Production of sugar from plant biomass In Examples 1 to 8 and Comparative Examples 1 to 9 below, production of sugar from plant biomass was evaluated by a saccharification rate using glucose as an index.

[Example 1]
Eucalyptus ground product (50 g) (vibration mill ground 150 μm mesh passing product) was collected in a beaker as plant biomass (hereinafter referred to as “BM”). Next, the reaction solution (450 g) was added to adjust the total weight to 500 g. The reaction solution was 0.1% by weight sulfuric acid (98% sulfuric acid (special grade, manufactured by Wako Pure Chemical Industries): equivalent to “inorganic acid” in the present invention) and 5% by weight acetone (99%) with respect to the reaction solution (450 g). Acetone (special grade, manufactured by Wako Pure Chemical Industries, Ltd.): equivalent to “water-soluble organic solvent” in the present invention.

Further, the mixture was placed in a high-temperature and high-pressure vessel (made of SUS, 1 L), heated to 180 ° C. with stirring (heated up in about 30 minutes), and maintained at a pressure of about 1 MPa (saturated water vapor pressure) for 15 minutes (previous processing). Immediately after the heat treatment, the mixture was cooled to room temperature with water.
After cooling with water, the pH of the mixture was measured, neutralized with 1N NaOH (special grade, manufactured by Nacalai Tesque), and adjusted to pH = 5.

  Next, the obtained pre-treated solid-liquid mixture (20 g) (corresponding to `` hydrothermally treated product '' in the present invention) was collected in a falcon tube, and cellulase (CBH, EG) (1 g of BM by dry weight) (CBH, EG) ( 1 ml of NS50013 (Novozyme) and 0.2 ml of cellulase (BGL) (NS50010, Novozyme) were added. The total unit of these cellulases was 40 FPU.

Further, sodium acetate buffer (pH 5.0) was used as a buffer, and added to the above mixture so that the final concentration was 50 mM.
The mixture was reacted in a shaker at 45 ° C. for 48 hours with shaking at a speed of 120 revolutions per minute.

により算出された。 Next, the amount of glucose in the mixture after the reaction (corresponding to the “saccharified product” in the present invention) was quantified by HPLC, and the saccharification rate was calculated. Saccharification rate (%) is calculated using the formula:

It was calculated by.

[Example 2]
Saccharide was produced in the same manner as in Example 1 except that the reaction solution contained 1% by weight of acetone instead of 5% by weight of acetone with respect to the reaction solution (450 g), and the saccharification rate was calculated. did.

[Example 3]
Implementation was performed except that the reaction solution contained 0.1% by weight phosphoric acid (85% phosphoric acid (special grade, manufactured by Wako Pure Chemical Industries)) instead of 0.1% by weight sulfuric acid with respect to the reaction solution (450g). Sugar was produced in the same manner as in Example 1, and the saccharification rate was calculated.

[Example 4]
In the same manner as in Example 1, except that the reaction solution contains 5 wt% ethanol (special grade, manufactured by Wako Pure Chemical Industries) instead of 5 wt% acetone with respect to the reaction solution (450 g). And the saccharification rate was calculated.

[Example 5]
Sugar was produced in the same manner as in Example 1 except that a eucalyptus ground product (100 g) (vibrated mill ground 150 μm mesh-passed product) was used as BM, and the reaction solution was 400 g, and the saccharification rate was calculated.

[Example 6]
Sugar was produced in the same manner as in Example 1 except that a eucalyptus ground product (50 g) (cutter mill 4 mm mesh passed product) was used as BM, and the saccharification rate was calculated.

[Example 7]
A sugar was produced in the same manner as in Example 1 except that bagasse (50 g) (pulverized 4 mm mesh-passed product) was used as BM, and the saccharification rate was calculated.

[Example 8]
Other than using bagasse (50g) (pulverized 4mm mesh passing product) as BM and heat treatment conditions at 165 ° C (temperature rise in about 30 minutes) and pressure about 0.7MPa (saturated water vapor pressure) for 15 minutes Produced sugar in the same manner as in Example 1, and calculated the saccharification rate.

[Comparative Example 1]
A sugar was produced in the same manner as in Example 1 except that the reaction solution did not contain acetone, and the saccharification rate was calculated.

[Comparative Example 2]
A sugar is produced in the same manner as in Example 1 except that the reaction solution contains 0.1% by weight of phosphoric acid instead of 0.1% by weight of sulfuric acid and 450% of the reaction solution (450 g). The saccharification rate was calculated.

[Comparative Example 3]
A sugar was produced in the same manner as in Example 1 except that the reaction solution did not contain sulfuric acid, and the saccharification rate was calculated.

[Comparative Example 4]
A sugar is produced in the same manner as in Example 1 except that the reaction solution contains 1% by weight of acetone instead of 5% by weight of acetone with respect to the reaction solution (450 g) and does not contain sulfuric acid. The saccharification rate was calculated.

[Comparative Example 5]
A sugar is produced in the same manner as in Example 1 except that the reaction solution contains 5% by weight of ethanol instead of 5% by weight of acetone with respect to the reaction solution (450 g) and does not contain sulfuric acid. The saccharification rate was calculated.

[Comparative Example 6]
Sugar was produced in the same manner as in Example 1 except that bagasse, rice straw and cedar ground products (50 g) (passing through vibration mill 150 μm mesh) were used as BM, and the reaction solution did not contain acetone. The saccharification rate was calculated.

[Comparative Example 7]
Bagasse, rice straw and cedar pulverized product (50 g) as BM (vibrated mill pulverized 150 μm mesh product), reaction solution does not contain acetone, and heat treatment condition is 165 ° C. (heated up in about 30 minutes) The sugar was produced in the same manner as in Example 1 except that the pressure was about 0.7 MPa (saturated water vapor pressure) for 15 minutes, and the saccharification rate was calculated.

[Comparative Example 8]
Example 1 was used except that bagasse, rice straw and cedar ground products (50 g) (vibrated mill ground 150 μm mesh-passed product) were used as BM, and the reaction solution contained neither sulfuric acid nor acetone. Sugar was produced and the saccharification rate was calculated.

[Comparative Example 9]
Bagasse, rice straw and cedar ground product (50 g) (product passing through vibration mill grinding 150 μm mesh) was used as BM, the reaction solution contained neither sulfuric acid nor acetone, and the heat treatment conditions were 165 ° C. (about 30 minutes) The saccharification rate was calculated in the same manner as in Example 1 except that the temperature was increased for 15 minutes under a pressure of about 0.7 MPa (saturated water vapor pressure).
Table 1 below shows saccharification rates in Examples 1 to 8 and Comparative Examples 1 to 9.

2. Production of ethanol from plant biomass In Example 9 and Comparative Examples 10 and 11 below, ethanol was produced from plant biomass and the amount of ethanol was quantified.

[Example 9]
(1) Preparation of yeast strain
Each of the following reagents was added to 1 L of the solution to a predetermined concentration to prepare a liquid medium [yeast extract (Extract yeast Dried (special grade: manufactured by Nacalai Tesque)): 0.2 g / L , Glucose (special grade: manufactured by Nacalai Tesque): 20 g / L, KH ₂ PO ₄ (special grade: manufactured by Nacalai Tesque): 0.2 g / L, MgSO ₄ · 7H ₂ O (special grade: Nacalai Tesque) )): 0.5 g / L, NaCl (special grade: manufactured by Nacalai Tesque): 0.1 g / L, CaCl ₂ (special grade: manufactured by Nacalai Tesque): 0.1 g / L, (NH ₄ ) ₂ SO ₄ (special grade: manufactured by Nacalai Tesque Co., Ltd.): 0.5 g / L and the following mineral liquid: 1.0 ml / L].

  Subsequently, wine yeast (Saccharomyces cerevisiae, OC-2) strain was inoculated on the liquid medium and precultured. Pre-culture was performed under the conditions of 30 ° C. and stationary culture. The wine yeast (Saccharomyces cerevisiae, OC-2) strain was obtained from National Institute of Technology and Evaluation. After preculture, the yeast was subjected to centrifugation at 25 ° C. and 4000 rpm for 5 minutes. After centrifugation, sterile water was added to prepare a yeast suspension.

(2) Preparation of mineral liquid Mineral liquid was prepared by adding the following reagents to a predetermined concentration to 1 L of solution [H ₃ BO ₄ (special grade: manufactured by Nacalai Tesque) : 0.5g / L, CuSO ₄ · 5H ₂ O (for drainage test (10w / v%): Wako Pure Chemical Industries): 0.04g / L, KI (special grade: manufactured by Nacalai Tesque): 0.1g / L , FeCl ₃ · 6H ₂ O (special grade: made by Nacalai Tesque): 0.2 g / L, MnSO ₄ · 5H ₂ O (special grade: made by Nacalai Tesque): 0.4 g / L, ZnSO ₄ · 7H ₂ O (special grade: manufactured by KANTO Chemical): 0.4 g / L and Na ₂ MoO ₄ · 2H ₂ O (special grade: manufactured by Nacalai Tesque): 0.2 g / L].

(3) Preparation of reaction solution after saccharification (corresponding to “saccharified product” in the present invention)
(i) Preparation of reaction solution 1 after saccharification
Eucalyptus ground product (50 g) (vibrated mill ground 150 μm mesh product) was collected in a beaker as BM. Next, the reaction solution (450 g) was added to adjust the total weight to 500 g. The reaction solution contains 0.1 wt% sulfuric acid and 5 wt% acetone with respect to the reaction solution (450 g).

Further, the mixture was placed in a high-temperature and high-pressure vessel (made of SUS, 1 L), heated to 180 ° C. with stirring (heated up in about 30 minutes), and maintained at a pressure of about 1 MPa (saturated water vapor pressure) for 15 minutes (previous processing). Immediately after the heat treatment, the mixture was cooled to room temperature with water.
After cooling with water, the pH of the mixture was measured, neutralized with 10% aqueous ammonia (special grade, manufactured by Wako Pure Chemical Industries), and adjusted to pH = 5.

  Next, the obtained solid-liquid mixture (20 g) after the pretreatment was collected in a falcon tube, and 0.25 ml of cellulase (CBH, EG) and 0.05 ml of cellulase (BGL) were added to 1 g of BM by dry weight. . The total unit of these cellulases was 10 FPU.

Further, sodium acetate buffer (pH 5.0) was used as a buffer, and added to the above mixture so that the final concentration was 50 mM.
The mixture was reacted in a shaker at 45 ° C. for 72 hours with shaking at a speed of 120 revolutions per minute.
The reaction solution obtained after the reaction is hereinafter referred to as “post-saccharification reaction solution 1”.

(ii) Preparation of reaction solution 2 after saccharification
Eucalyptus ground product (50 g) (vibrated mill ground 150 μm mesh product) was collected in a beaker as BM. Next, the reaction solution (450 g) was added to adjust the total weight to 500 g. The reaction solution contains 0.1 wt% phosphoric acid and 5 wt% acetone with respect to the reaction solution (450 g).

Further, the mixture was placed in a high-temperature and high-pressure vessel (made of SUS, 1 L), heated to 180 ° C. with stirring (heated up in about 30 minutes), and maintained at a pressure of about 1 MPa (saturated water vapor pressure) for 15 minutes (previous processing). Immediately after the heat treatment, the mixture was cooled to room temperature with water.
After cooling with water, the pH of the mixture was measured, neutralized with 1N KOH (special grade, manufactured by Nacalai Tesque), and adjusted to pH = 5.

  Next, the obtained solid-liquid mixture (20 g) after the pretreatment was collected in a falcon tube, and 0.25 ml of cellulase (CBH, EG) and 0.05 ml of cellulase (BGL) were added to 1 g of BM by dry weight. . The total unit of these cellulases was 10 FPU.

Further, sodium acetate buffer (pH 5.0) was used as a buffer, and added to the above mixture so that the final concentration was 50 mM.
The mixture was reacted in a shaker at 45 ° C. for 72 hours with shaking at a speed of 120 revolutions per minute.
The reaction solution obtained after the reaction is hereinafter referred to as “post-saccharification reaction solution 2”.

(4) Preparation of fermentation broth For 1 L of the culture broth, the yeast extract was 0.2 g / L, the inorganic salt was MgSO ₄ · 7H ₂ O: 0.5 g / L, NaCl: 0.1 g / L Each reagent was added to prepare L, CaCl ₂ : 0.1 g / L, and mineral liquid: 1.0 ml / L.

(5) Fermentation The yeast suspension prepared in (1) above was added to the fermentation broth so that the turbidity of the fermentation broth was OD ₆₀₀ = 2. After mixing saccharification reaction liquids 1 and 2 (ratio of saccharification reaction liquids 1 and 2 = 1: 1), add to the fermentation broth so that the final concentration is 10% (w / v) BM. It was. The mixture was prepared to pH 5.0 with 1N NaOH.

により算出された。 The mixture was then subjected to shaking culture at 30 ° C. at a speed of 120 revolutions per minute for fermentation. After 2 days of fermentation, the amount of ethanol in the fermentation culture was quantified by HPLC, and the ethanol conversion rate was calculated. The ethanol conversion rate (%) is calculated using the formula:

It was calculated by.

[Comparative Example 10]
In this comparative example (positive control), the reaction solution after saccharification prepared in the same manner as in Example 9 except that it was neutralized with 1N NaOH instead of 10% ammonia water during the preparation in the reaction solution 1 after saccharification. used.

In addition, fermentation was performed using the fermentation broth used in (5) fermentation of Example 9 with (NH ₄ ) ₂ SO ₄ : 0.6 g / L and KH ₂ PO ₄ : 0.6 g / L. The amount of ethanol was quantified in the same manner, and the ethanol conversion rate was calculated.

[Comparative Example 11]
In this comparative example (negative control), the same procedure as in Comparative Example 10 was performed except that (NH ₄ ) ₂ SO ₄ and KH ₂ PO ₄ were not added to the fermentation broth used in the fermentation of Example 9 (5). The amount of ethanol was quantified and the ethanol conversion rate was calculated.

  As a result of Example 9 and Comparative Examples 10 and 11, the amount of ethanol produced was 14.65 g / L (Example 9), 14.25 g / L (Comparative Example 10) and 0 g / L (Comparative Example 11), respectively. . The ethanol conversion rates were 68% (Example 9), 65% (Comparative Example 10), and 0% (Comparative Example 11), respectively.

Claims (26)

  A plant biomass treatment method comprising a step of immersing a plant biomass in a solution containing a water-soluble organic solvent and an inorganic acid and subjecting the plant biomass to hydrothermal treatment.   The plant biomass treatment method according to claim 1, wherein a content ratio of the water-soluble organic solvent is 1 to 5% by weight.   The plant biomass treatment method according to claim 1 or 2, wherein the water-soluble organic solvent is acetone or ethanol.   The plant biomass treatment method according to any one of claims 1 to 3, wherein a content ratio of the inorganic acid is 0.1 to 0.3% by weight.   The plant biomass treatment method according to any one of claims 1 to 4, wherein the inorganic acid is sulfuric acid or phosphoric acid.   The plant biomass treatment method according to any one of claims 1 to 5, wherein the hydrothermal treatment is performed under conditions of a temperature of 140 to 240 ° C and a pressure of 0.1 to 4 MPa.   The plant biomass treatment method according to any one of claims 1 to 6, further comprising a step of recovering the water-soluble organic solvent as a vapor simultaneously with or after the hydrothermal treatment step.   The plant biomass treatment method according to any one of claims 1 to 7, further comprising a step of neutralizing the inorganic acid with an alkaline solution after the hydrothermal treatment step.   The plant biomass treatment method according to claim 8, wherein the alkaline solution is sodium hydroxide, ammonia, or potassium hydroxide. Immersing plant biomass in a solution containing a water-soluble organic solvent and an inorganic acid, and subjecting the biomass to hydrothermal treatment;
Contacting the hydrothermally treated product with an enzyme and subjecting it to a saccharification treatment;
A method for producing sugar, comprising:   The method for producing a saccharide according to claim 10, wherein the content of the water-soluble organic solvent is 1 to 5% by weight.   The method for producing a sugar according to claim 10 or 11, wherein the water-soluble organic solvent is acetone or ethanol.   The method for producing sugar according to any one of claims 10 to 12, wherein a content ratio of the inorganic acid is 0.1 to 0.3% by weight.   The method for producing a saccharide according to any one of claims 10 to 13, wherein the inorganic acid is sulfuric acid or phosphoric acid.   The method for producing sugar according to any one of claims 10 to 14, wherein the hydrothermal treatment is performed under conditions of a temperature of 140 to 240 ° C and a pressure of 0.1 to 4 MPa.   The method for producing sugar according to any one of claims 10 to 15, wherein the enzyme is cellulase.   The method for producing sugar according to any one of claims 10 to 16, comprising a step of recovering the water-soluble organic solvent as a vapor simultaneously with or after the hydrothermal treatment step.   The method for producing sugar according to any one of claims 10 to 17, further comprising a step of neutralizing the inorganic acid with an alkaline solution after the hydrothermal treatment step.   The method for producing sugar according to claim 18, wherein the alkaline solution is sodium hydroxide, ammonia or potassium hydroxide. Immersing plant biomass in a solution containing a water-soluble organic solvent and sulfuric acid and / or phosphoric acid, and subjecting the biomass to hydrothermal treatment;
Neutralizing the hydrothermally treated product with ammonia and / or potassium hydroxide;
Contacting the hydrothermally treated product with an enzyme, subjecting it to a saccharification treatment, and subjecting the yeast to fermentation;
A method for producing ethanol, comprising:   The method for producing ethanol according to claim 20, wherein the content of the water-soluble organic solvent is 1 to 5% by weight.   The method for producing ethanol according to claim 20 or 21, wherein the water-soluble organic solvent is acetone or ethanol.   The method for producing ethanol according to any one of claims 20 to 22, wherein a content ratio of the sulfuric acid and / or phosphoric acid is 0.1 to 0.3% by weight.   The method for producing ethanol according to any one of claims 20 to 23, wherein the hydrothermal treatment is performed under conditions of a temperature of 140 to 240 ° C and a pressure of 0.1 to 4 MPa.   The method for producing ethanol according to any one of claims 20 to 24, wherein the enzyme is a cellulase.   The method for producing ethanol according to any one of claims 20 to 25, comprising a step of recovering the water-soluble organic solvent as a vapor simultaneously with or after the hydrothermal treatment step.