JP7297232B2 - Method for saccharification of lignocellulosic biomass - Google Patents

Description

TECHNICAL FIELD The present invention relates to an enzyme stabilizer containing a hydrophilic lignin derivative, a method for producing an enzyme stabilizer, a method for stabilizing an enzyme, a method for saccharifying lignocellulosic biomass, and an apparatus for producing an enzyme stabilizer.

BACKGROUND ART In recent years, the use of biomass made from plant resources has attracted attention from the viewpoint of global warming countermeasures and the effective use of waste. Generally, carbohydrates such as sugar cane and starches such as corn are often used as raw materials for producing compounds such as ethanol from biomass. However, these raw materials are originally used as food or feed, and there is a risk that their long-term use as industrial resources will cause competition with food or feed use, leading to a rise in raw material prices.

Therefore, technology development for utilizing non-edible biomass as an energy resource is underway. Cellulose is the most abundant non-edible biomass on earth, and most of it exists as lignocellulose, which is a complex with aromatic polymers such as lignin and hemicellulose.

When using this lignocellulosic biomass, a method of decomposing cellulose in the biomass into monosaccharides with concentrated sulfuric acid and then fermenting the monosaccharides has long been studied. However, this method has not been widely used because it is difficult to establish and manage an acid-resistant device for handling concentrated sulfuric acid and an efficient recovery technique for sulfuric acid.

On the other hand, a method (enzymatic saccharification/fermentation method) that uses enzymes such as cellulase to monosaccharify (saccharify) biomass polysaccharide components without using acids such as sulfuric acid and then performs ethanol fermentation with yeast is being studied. However, when this method is applied to lignocellulosic biomass such as wood, some kind of treatment (pretreatment) is required before the enzymatic saccharification treatment.

The pretreatment for enzymatic saccharification is a step of making cellulose or the like in biomass into a state in which enzymes can effectively act, and physical or chemical treatment is performed. As a physical treatment, there is a method of grinding with a ball mill or the like. As chemical treatment, there is a method of obtaining cellulose by removing lignin with a chemical agent, such as the chemical process of paper pulping. For example, in Patent Document 1, bioethanol can be efficiently produced by enzymatic saccharification/fermentation after pretreatment of biomass by alkali digestion or kraft digestion to remove most of the lignin in the biomass. disclosed.

On the other hand, lignin is one of the three main components of biomass and the second most accumulated organic compound on earth. It is separated in the chemical pulping process and bioethanol pretreatment process, and is a by-product in paper pulp production and bioethanol production, but there are few effective ways to use it other than as a heat source, and effective ways to use it are being sought. Non-Patent Document 1 discloses that a hydrophobic lignin derivative can be a functional raw material, and research is underway.

In Patent Document 2, an enzyme stabilizer containing a lignin derivative produced by the reaction of lignin and a hydrophilic compound improves the activity of the saccharifying enzyme and prevents non-specific adsorption of the saccharifying enzyme to the substrate. It is disclosed that saccharification of lignocellulosic biomass can be efficiently performed by doing so.
Patent Document 3 discloses a method for producing a lignin derivative by reacting lignin with a hydrophilic compound.

Japanese Unexamined Patent Application Publication No. 2008-92910 WO2011/111664 JP 2017-197517 A

Applied Clay Science, Volumes 132-133, November 2016, Pages 425-429

However, although Patent Document 2 discloses a method of using a lignin derivative produced by the reaction of lignin and a hydrophilic compound, the use of lignin is limited to lignin derivatives, and the lignin derivative is further used as a hydrophilic component. There is no disclosure regarding the effective utilization of each of the lignin derivatives separated and purified into a liquid fraction and a hydrophobic solid fraction.

Patent Document 3 discloses a method for producing a lignin derivative by reacting lignin with a hydrophilic compound, but the target product is a solid fraction that can be obtained by mixing an acid with the reactant and precipitating it, The use of a separated liquid fraction is not disclosed.

Further, Patent Documents 2 and 3 do not disclose the influence of the molecular weight of the hydrophilic compound to be reacted or the molecular weight of the lignin derivative on the enzyme stabilization performance of the lignin derivative to be produced.

The present invention has been made in view of the above circumstances, and a liquid fraction containing a hydrophilic lignin derivative and a solid fraction containing a hydrophobic lignin derivative are separated from a lignin derivative obtained from lignocellulosic biomass. Improve the profitability of the entire process using cellulosic biomass by using the liquid fraction containing the hydrophobic lignin derivative as a saccharifying enzyme stabilizer and using the solid fraction containing the hydrophobic lignin derivative for the production of functional raw materials. It is intended to

The present inventors added a second acid to a lignin derivative obtained by reacting a first acid with a lignocellulosic biomass and a polyalkylene glycol, followed by solid-liquid separation to obtain a liquid fraction. The hydrophilic lignin derivative obtained by stabilizes the saccharifying enzyme, so that the saccharification of cellulosic biomass by the saccharifying enzyme can be performed efficiently, and the solid fraction obtained by the solid-liquid separation The inventors have found that a functional raw material can be produced from the hydrophobic lignin derivative contained in , and the profitability of the entire process using cellulosic biomass can be improved, and the present invention has been completed.

That is, the present invention relates to the following [1] to [19].
[1] By adding a first acid to lignocellulosic biomass and polyalkylene glycol and reacting them, adding a second acid to the resulting lignin derivative, and then performing solid-liquid separation to obtain a liquid fraction. An enzyme stabilizer comprising the resulting hydrophilic lignin derivative.
[2] The enzyme stabilizer of [1], wherein the hydrophilic lignin derivative has a polyalkylene glycol group at the α-position carbon of the coniferyl alcohol skeleton of lignin, and the terminal of the polyalkylene glycol is a hydroxyl group.
[3] The enzyme stabilizer of [1] or [2], wherein the lignocellulosic biomass is wood flour.
[4] The enzyme stabilizer according to any one of [1] to [3], wherein the polyalkylene glycol is polyethylene glycol.
[5] The enzyme stabilizer of [4], wherein the polyethylene glycol has an average molecular weight of 200-600.
[6] The enzyme stabilizer according to any one of [1] to [5], wherein the hydrophilic lignin derivative has an average molecular weight of 1,000 to 3,200.
[7] The enzyme stabilizer according to any one of [1] to [6], wherein the first acid or second acid is sulfuric acid.
[8] Adding a first acid to lignocellulosic biomass and polyalkylene glycol for reaction, adding a second acid to the resulting lignin derivative, and then performing solid-liquid separation to obtain a liquid fraction. A method for producing an enzyme stabilizer comprising a hydrophilic lignin derivative characterized by:
[9] A method for producing an enzyme stabilizer containing a hydrophilic lignin derivative, comprising the following steps.
(1) adding a first acid to lignocellulosic biomass and polyalkylene glycol to react them;
(2) adding alkali to the reaction solution obtained in step (1) to neutralize;
(3) removing solids from the neutralized alkaline solution obtained in step (2);
(4) adding a second acid to the liquid fraction obtained in step (3); and
(5) Perform solid-liquid separation on the precipitate precipitated in step (4) to obtain a liquid fraction containing a hydrophilic lignin derivative [10] A hydrophilic lignin derivative is at the α-position of the coniferyl alcohol skeleton of lignin having a polyalkylene glycol group at the carbon of [8] or [9], wherein the end of the polyalkylene glycol is a hydroxyl group.
[11] The method for producing an enzyme stabilizer according to any one of [8] to [10], wherein the lignocellulosic biomass is wood flour.
[12] The method for producing an enzyme stabilizer according to any one of [8] to [11], wherein the polyalkylene glycol is polyethylene glycol.
[13] The method for producing an enzyme stabilizer according to [12], wherein the polyethylene glycol has an average molecular weight of 200-600.
[14] The method for producing an enzyme stabilizer according to any one of [8] to [13], wherein the hydrophilic lignin derivative has an average molecular weight of 1000 to 3200.
[15] The method for producing an enzyme stabilizer according to any one of [8] to [13], wherein the first acid or second acid is sulfuric acid.
[16] The enzyme stabilizer according to any one of [1] to [7], or the enzyme stabilizer obtained by the production method of any one of [8] to [15], in the reaction system of the substrate and the enzyme. A method for stabilizing an enzyme, characterized by adding
[17] In the enzymatic saccharification method of lignocellulosic biomass, the enzyme stabilizer of any one of [1] to [7] or the enzyme obtained by the production method of any one of [8] to [15] A method of saccharification of lignocellulosic biomass comprising adding a stabilizer.
[18] The saccharification of [17], wherein the hydrophilic lignin derivative in the enzyme stabilizer is added so as to be 0.1 to 1.0% by mass relative to the dry weight of the lignocellulosic biomass to be enzymatically saccharified. Method.
[19] A first reaction tank having an alkali supply means, for reacting the lignocellulosic biomass and polyethylene glycol with the first acid;
a filtering device;
a second reaction tank for reacting a liquid fraction obtained by solid-liquid separation using the filtration device with a second acid;
a solid-liquid separator;
An apparatus for producing an enzyme stabilizer, comprising:

According to the present invention, a liquid fraction containing a hydrophilic lignin derivative useful as an enzyme stabilizer and a solid fraction containing a hydrophobic lignin derivative useful as a functional raw material can be obtained from lignocellulosic biomass. can improve the profitability of the overall process using lignocellulosic biomass.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic configuration diagram schematically showing a method for producing an enzyme stabilizer and an apparatus for producing the enzyme stabilizer according to an embodiment of the present invention; 1 is a diagram showing the results of saccharification reactions using the liquid fraction and solid fraction obtained in Example 1. FIG. FIG. 3 is a diagram showing the relationship between the addition rate of lignin derivatives and the C6 saccharification rate when polyethylene glycol (PEG) 200, PEG 400, and PEG 600 are used as polyethylene glycol. FIG. 2 is a diagram showing the relationship between the PEG molecular weight to be reacted and the sugar yield ratio when polyethylene glycol (PEG) 200, PEG600, and PEG1000 are used as polyethylene glycol. It is a figure which shows the relationship between the addition rate of the lignin derivative of Example 2 prepared using polyethylene glycol (PEG) 400 as polyethylene glycol, and the lignin derivative prepared in Comparative Example 1, and C6 saccharification rate.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail. In addition, in this specification and claims, the meanings of various terms are defined as follows.

(1) Enzyme stabilization As used herein, "enzyme stabilization" refers to the presence of an enzyme stabilizer in the reaction between a substrate and an enzyme to prevent deactivation of the enzyme. , means that the enzyme activity is maintained at a high value. Specifically, for example, under the enzymatic saccharification reaction conditions of Test Example 1 described later, the residual enzyme activity is 10% or more, preferably 20% or more, compared to the case where the enzyme stabilizer is not used, More preferably, it means 50% or higher. Enzyme activity measurement methods can be appropriately selected by those skilled in the art from among the methods described in this specification, methods described in catalogs for commercially available products, methods described in literature, and the like.

(2) Enzyme As used herein, the term “enzyme” refers to a high-molecular compound mainly composed of protein that catalyzes chemical reactions, and particularly refers to saccharifying enzymes. Saccharifying enzymes include cellulases that degrade cellulose, hemicellulase that decomposes hemicellulose, glucoxidase (β-glucoxidase), amylase that decomposes starch, and the like, preferably cellulase.

(3) Hydrophilic lignin derivative As used herein, the term "hydrophilic lignin derivative" refers to a polyalkylene glycol at any of the α-position carbons of the coniferyl alcohol skeleton that constitutes the hydrophilic lignin derivative. Among lignin derivatives obtained by adding sulfuric acid to lignocellulosic biomass and polyalkylene glycol having a terminal hydroxyl group, it refers to a lignin derivative that does not precipitate when the pH is adjusted to 2 to 3 with sulfuric acid. As the hydrophilic lignin derivative, those having a molecular weight of 1,000 to 3,200 are preferred. This is because if the molecular weight is less than 1000, the hydrophilicity tends to be insufficient, while if the molecular weight is greater than 3200, the function as an enzyme stabilizer tends to decrease due to the high molecular weight.

(4) Hydrophobic Lignin Derivative As used herein, the term “hydrophobic lignin derivative” refers to a polyalkylene glycol at any of the α-position carbons of the coniferyl alcohol skeleton that constitutes the hydrophobic lignin derivative. Among lignin derivatives obtained by adding sulfuric acid to lignocellulosic biomass and polyalkylene glycol, it refers to a lignin derivative that precipitates when the pH is adjusted to 2 to 3 with sulfuric acid. Hydrophobic lignin derivatives having a molecular weight of 4,000 to 20,000 are preferred.

[Embodiment 1: Enzyme stabilizer and method for producing the enzyme stabilizer]
The enzyme stabilizer according to the present embodiment is obtained by adding a first acid to lignocellulose biomass and polyalkylene glycol to react, adding a second acid to the obtained lignin derivative, and then performing solid-liquid separation. It is an enzyme stabilizer containing a hydrophilic lignin derivative obtained by obtaining a liquid fraction with Here, a hydrophilic lignin derivative obtained by adding a second acid to the lignin derivative obtained by adding a first acid to lignocellulose-based biomass and polyalkylene glycol to react with each other and performing solid-liquid separation is obtained. It has been demonstrated in the examples described later that the liquid fraction containing the enzyme can stabilize the enzyme activity in the reaction between the substrate and the enzyme. Therefore, the enzyme stabilizer according to this embodiment can prevent enzyme deactivation and stabilize enzyme activity.

(lignocellulosic biomass)
In the present invention, the lignocellulosic biomass is of any type as long as it is at least one selected from the group consisting of woody plants, herbaceous plants, processed products thereof, and waste products thereof. preferable.

Woody plants in the present invention include cedar, cypress, larch, pine, rice pine, rice cedar, rice hemlock, poplar, white birch, willow, eucalyptus, sawtooth oak, konara oak, oak, seaweed, beech, acacia, bamboo, bamboo, Oil palm, sago palm and the like can be exemplified, but cedar is preferable from the viewpoint of stability of properties. In addition, bark, branches, fruit clusters, fruit shells, etc. can also be used. In addition, processed materials such as plywood, fiberboard and laminated lumber using these materials can also be used. In addition, members dismantled after being used in buildings can also be used. In addition, processed products of lignocellulose biomass such as paper and used paper can also be used.

Examples of herbaceous plants in the present invention include rice, wheat, sugar cane, reeds, pampas grass, and corn.

(polyalkylene glycol)
In the present invention, the polyalkylene glycol is not particularly limited as long as it is polymerized alkylene glycol, but preferably has a terminal hydroxyl group. Examples thereof include polyethylene glycol, polypropylene glycol, etc., and polyethylene glycol is preferred. The molecular weight of the polyalkylene glycol is not particularly limited as long as it can be used for the production of the enzyme stabilizer of the present invention.

These polyalkylene glycols may be commercially available or may be prepared by methods known in the art.

(lignin derivative)
A lignin derivative can be obtained in a liquid fraction from lignin in the lignocellulosic biomass by adding a first acid to the lignocellulosic biomass and polyalkylene glycol and reacting them. The liquid fraction containing the lignin derivative thus obtained contains the hydrophilic lignin derivative and the hydrophobic lignin derivative. FIG. 1 shows an outline of a method for producing a liquid fraction containing a lignin derivative according to the present invention.
Hereinafter, a method for producing a liquid fraction containing the lignin derivative will be specifically described with reference to FIG.

A liquid fraction containing the lignin derivative according to the present invention can be produced by the following steps.
(1) a step of adding a first acid to lignocellulosic biomass and polyalkylene glycol to react (11 in the figure);
(2) a step of adding an alkali to the reaction solution obtained in step (1) to neutralize it (12 in the figure);
(3) a step of removing the solid fraction from the neutralized alkaline solution obtained in step (2) (13 in the figure);

In the above step (1), the first acid used when reacting the lignocellulose biomass and the polyalkylene glycol is not particularly limited as long as it can produce a lignin derivative from the lignocellulose biomass and the polyalkylene glycol. Hydrochloric acid, sulfuric acid, or the like is used, and sulfuric acid is preferably used. The amount added is usually 0.1 to 3.0 parts by weight based on the polyalkylene glycol.

In the reaction between lignocellulosic biomass and polyalkylene glycol, the amount of polyalkylene glycol to be reacted with lignocellulosic biomass depends on the type of lignin and polyalkylene glycol in the lignocellulosic biomass used and the desired amount. It can be determined depending on the performance of the enzyme stabilizer. For example, the amount of polyalkylene glycol to be added is calculated based on the amount of hydroxyl groups in lignin present in the lignocellulosic biomass to be used and the amount of hydroxyl groups in the polyalkylene glycol to be added. Polyalkylene glycol reacts with the hydroxyl groups attached to the α carbon atoms of lignin. The amount of polyalkylene glycol is usually 5 to 100 parts by weight, preferably 10 to 60 parts by weight, more preferably 20 to 50 parts by weight based on 10 parts by dry weight of lignocellulosic biomass.

Although the reaction temperature is not particularly limited, it is usually 100°C to 200°C, preferably 120°C to 160°C.

Although the reaction time is not particularly limited, it is usually 30 to 180 minutes, preferably 60 to 120 minutes.

In the above step (2), the alkali to be added is not particularly limited as long as it can neutralize the reaction solution obtained in step (1). Calcium, magnesium hydroxide, lithium hydroxide and the like can be mentioned.
The pH of the reaction solution after addition of the alkali is preferably 6 or more and 12 or less, more preferably 6 or more and 10 or less.
In the above step (3), the method for removing the solid content from the neutralized alkaline solution is not particularly limited as long as the solid content can be removed from the neutralized alkaline solution. membranes and the like. The liquid fraction obtained by removing the solid content in step (3) contains hydrophilic and hydrophobic lignin derivatives, and the hydrophilic lignin derivative can be used as the enzyme stabilizer of the present invention.

(Hydrophilic lignin derivative)
After adding a second acid to the liquid fraction containing the lignin derivative obtained by adding the first acid to the lignocellulosic biomass and polyalkylene glycol and reacting them, solid-liquid separation is performed to obtain a solid can be separated to obtain a liquid fraction containing the hydrophilic lignin derivative. Hereinafter, a method for producing a liquid fraction containing the hydrophilic lignin derivative will be specifically described with reference to FIG.

A liquid fraction containing the hydrophilic lignin derivative according to the present invention can be produced by the following steps.
(4) a step of adding a second acid to the liquid fraction obtained in step (3) above (14 in the figure);
(5) A step of performing solid-liquid separation on the solid precipitated in step (4) to obtain a liquid fraction containing a lignin derivative (15 in the figure).

The second acid added to the liquid fraction containing the lignin derivative is not particularly limited as long as it can acidify the liquid fraction, but an acid capable of adjusting the pH to 1 to 4 is preferable, and sulfuric acid is particularly preferred. preferable. The amount added is usually 0.01 to 3.0 parts by weight based on the liquid fraction.
By adding a second acid to the liquid fraction to make it acidic, unreacted lignin and hydrophobic lignin derivatives are precipitated, solid-liquid separation is performed, and the solids are separated to obtain hydrophilic lignin derivatives. A liquid fraction containing Since the liquid fraction contains unreacted polyalkylene glycol in addition to the hydrophilic lignin derivative, the unreacted polyalkylene glycol in the liquid fraction can be reused in step (1). is.
The method for solid-liquid separation is not particularly limited as long as it is a method capable of separating the solids produced by adding the second acid, but centrifugation is preferred. Centrifugation is usually from 2000g to 20000g, preferably from 5000g to 15000g.

The liquid fraction containing the hydrophilic lignin derivative obtained by the reaction can be used as it is as an enzyme stabilizer. It may be subjected to ultrafiltration, ion exchange resin, synthetic adsorbent, activated carbon, or the like.

If necessary, the liquid fraction containing the hydrophilic lignin derivative obtained by the reaction may be completely dried by a conventionally used drying method such as a freeze dryer.

(enzyme stabilizer)
When the liquid fraction containing the hydrophilic lignin derivative of the present invention is used as an enzyme stabilizer, it may be used in the form of an aqueous solution, or it may be dried and powdered. .

Any additive may be added to the enzyme stabilizer of the present invention as long as it does not impair its performance. Examples of such additives include pH adjusters, antioxidants, water-soluble or water-insoluble carriers, dispersants, water-soluble inorganic or organic acid salts of metals, and the like.

Examples of the pH adjuster include citric acid, sodium citrate, potassium citrate, and the like. These may be used individually by 1 type, and may use 2 or more types together.

Examples of the antioxidant include butylhydroxytoluene, distyrenated cresol, sodium sulfite and sodium hydrogensulfite. These may be used individually by 1 type, and may use 2 or more types together.

Examples of the water-soluble or water-insoluble carrier include water, alcohols (ethanol, isopropyl alcohol, etc.), diols (eg, ethylene glycol, 1,2-propanediol, 1,6-hexanediol), other polyols ( polyethylene glycol, polypropylene glycol, etc.), ethers (e.g., dipropylene glycol monomethyl ether, etc.), ketones (e.g., cyclohexanone, etc.), esters (e.g., dimethyl succinate, dimethyl adipate, etc.), nitrogen-containing compounds Water-soluble carriers such as (e.g., N-methyl-2-pyrrolidone, N,N-dimethylformamide, acetonitrile, etc.); aliphatic and aromatic hydrocarbon solvents (e.g., pentane, hexane, heptane, xylene, toluene, water-insoluble carriers such as dodecylbenzene, mineral spirits, aromatic naphtha, etc.), oils and fats (eg, coconut oil, soybean oil, rapeseed oil, castor oil, linseed oil, etc.). These may be used individually by 1 type, and may use 2 or more types together.

Examples of the dispersant include acrylate homopolymers, acrylate copolymers, mixtures thereof, and the like. These may be used individually by 1 type, and may use 2 or more types together.

Examples of the inorganic or organic acid salts of water-soluble metals include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium, and alkanolamine salts such as mono-, di-, and triethanolamine salts. be done. These may be used individually by 1 type, and may use 2 or more types together.

[Embodiment 2: Enzyme stabilization method]
The method for stabilizing an enzyme according to the present embodiment is a method for stabilizing an enzyme, which comprises adding the enzyme stabilizer to a reaction system between a substrate and an enzyme. Here, it will be described later that the liquid fraction containing the hydrophilic lignin derivative produced by the reaction between the lignocellulosic biomass and the polyalkylene glycol can stabilize the enzymatic activity in the reaction between the substrate and the enzyme. Examples demonstrate this. Therefore, the enzyme stabilization method according to the present embodiment uses the liquid fraction containing the hydrophilic lignin derivative, so that the enzyme activity can be stabilized.

The hydrophilic lignin derivative used in the enzyme stabilization method according to the present embodiment is basically the same as the hydrophilic lignin derivative used for the enzyme stabilizer specifically described in Embodiment 1. It has a configuration and operational effects. Therefore, descriptions of the same contents as in the first embodiment will be omitted as appropriate.

As the enzyme for the enzymatic reaction, a saccharifying enzyme is preferred, and cellulase is particularly preferred. As the substrate for the enzymatic reaction, cellulose is preferred, and lignocellulose derived from wood or the like is particularly preferred.

The amount of the enzyme stabilizer used in the method for stabilizing enzymes according to the present embodiment is arbitrary. The weight of the lignin derivative is preferably 0.1 to 3.0% by mass, more preferably 0.2 to 1.0% by mass, and particularly preferably 0.3 to 0.7% by mass. With such a preferable addition amount, deactivation of the enzyme can be further prevented, and the enzyme activity can be further stabilized.
The concentration of the hydrophilic lignin derivative added to the reaction solution can be calculated by the following formula.

[Embodiment 3: Enzymatic saccharification method]
The enzymatic saccharification method according to the present embodiment is a method for saccharifying lignocellulosic biomass by adding the enzyme stabilizer according to Embodiment 1 to the enzymatic saccharification method for lignocellulosic biomass. As described above, the enzyme stabilizer used in this embodiment can prevent enzyme deactivation and stabilize enzyme activity. Therefore, according to the saccharification method using the enzyme stabilizer, it is possible to reuse the used enzyme or reduce the amount of enzyme used.

As used herein, the “enzymatic saccharification method for lignocellulosic biomass” may be any method as long as it enzymatically saccharifies lignocellulosic biomass. A saccharification method (method for producing ethanol) described in a publication may be used. The contents of Japanese Patent Application Laid-Open No. 2008-92910 are incorporated herein by reference.

The enzymatic saccharification method according to this embodiment includes:
(a) a step of mixing acid or alkali with lignocellulosic biomass and performing hydrothermal treatment (pretreatment);
(b) An enzyme, an enzyme stabilizer of the present invention, and water are added to the pretreated lignocellulosic biomass, and cellulose and hemicellulose in the biomass are hydrolyzed (saccharified) into monosaccharides of glucose and xylose, respectively. and (c) a step of adding yeast to the obtained sugar solution and fermenting it to produce ethanol.

In the enzymatic saccharification method of the present invention, the saccharification efficiency can be examined by calculating the C6 saccharification rate. Here, the C6 saccharification rate indicates the ratio of the amount of glucose produced through saccharification from cellulose (in terms of glucose) in the pretreated biomass. Originally, xylose (C5 sugar) is also produced through pretreatment and saccharification, but since a high proportion of xylose is produced by pretreatment with dilute sulfuric acid, the enzymatic saccharification efficiency should be examined by the C6 saccharification rate.

The enzyme stabilizer used in the saccharification method according to this embodiment has the same configuration and effects as the enzyme stabilizer specifically described in the first embodiment. Therefore, descriptions of the same contents as in the first embodiment will be omitted as appropriate.

The amount of the enzyme stabilizer used in the saccharification method according to the present embodiment is arbitrary. It is preferably 0.1 to 3.0% by mass, more preferably 0.2 to 1.0% by mass, and particularly preferably 0.3 to 0.70% by mass based on the dry weight. With such a preferable addition amount, the adsorption of the enzyme to the biomass component can be further suppressed, and the activity of the enzyme can be further improved.

The weight of the hydrophilic lignin derivative can be calculated by the method described in the section of the enzyme stabilization method.

Any enzyme having cellulase, hemicellulase or β-glucoxidase activity can be used as the saccharifying enzyme used in the present invention. Examples of these saccharifying enzyme-producing bacteria include aerobic Trichoderma, Aspergillus, Humicola, Irpex, and Acremonium.

The amount of the saccharifying enzyme used in the saccharification reaction is adjusted so that 5 to 50 units of cellulase activity are included with respect to 1 g of cellulose of the lignocellulosic biomass serving as the raw material substrate.

The saccharified liquid obtained by the saccharification method of the present invention can be used as a raw material for ethanol fermentation, lactic acid fermentation, amino acid fermentation, butanol fermentation, isobutanol fermentation, and the like. Below, the case where it uses for ethanol fermentation is demonstrated.

In ethanol fermentation, the saccharified liquid obtained above is fermented. Since the optimum temperature for the saccharification reaction differs from the optimum temperature for fermentation, the saccharification reaction is carried out at a temperature suitable for the saccharification reaction, preferably 40 to 60°C. The pH of the reaction solution is also suitable for the saccharification reaction, preferably 4-7. After completion of the saccharification reaction, the saccharification reaction solution is taken out and supplied to the fermenter. The ethanol-fermenting bacteria in the fermenter may or may not be immobilized, but are preferably immobilized. Fermentation conditions are carried out under conditions suitable for ethanol fermentation. The pH is preferably 4-8, and the temperature is preferably 20-40°C. Ethanol produced during ethanol fermentation can also be separated and recovered.

By adding ethanol-fermenting bacteria to the obtained saccharified liquid, the saccharified liquid can be fermented to produce ethanol. Examples of such ethanol-fermenting bacteria include Saccharomyces, Zymomonas, and Pichia. Genetically modified ones can also be used if alcoholic fermentation is possible. These ethanol-fermenting bacteria are desirably pre-cultured in a liquid medium before ethanol fermentation to increase the amount of bacteria. The greater the amount of ethanol-fermenting bacteria input, the better the fermentation efficiency.

The saccharification reaction and ethanol fermentation can also be performed simultaneously by adding an ethanol-fermenting bacterium together with the saccharifying enzyme. In the simultaneous saccharification and fermentation, a method of performing saccharification reaction and fermentation in the same reactor or a method of performing saccharification reaction and fermentation in separate reactors may be used.

When the saccharification reaction and the fermentation are performed in the same reactor, the pH and temperature of the reaction solution are set so that both the saccharification reaction and the fermentation can work. As conditions, priority is given to fermentation conditions for ethanol-fermenting bacteria, preferably pH of 4 to 7 and temperature of 20 to 40°C. In addition, by performing simultaneous saccharification and fermentation under anaerobic conditions, it is possible to suppress the growth of saccharifying enzyme-producing bacteria, which are aerobic bacteria, and to suppress the consumption of sugar accompanying the growth of saccharifying enzyme-producing bacteria. In addition, stirring the simultaneous saccharification and fermentation facilitates the progress of the saccharification reaction, resulting in improved ethanol productivity. Simultaneous saccharification and fermentation can also be performed while separating and recovering the produced ethanol. This method can simplify the manufacturing process because all saccharification reactions and ethanol fermentation can be performed in one reactor.

In the method in which the saccharification reaction and fermentation are simultaneously performed in separate reactors, the saccharification reaction is performed at a temperature suitable for the saccharification reaction, preferably at 40-60°C. The pH of the reaction solution is the same as the fermentation conditions, preferably 4-6. The saccharification reaction liquid is continuously taken out and supplied to the fermenter. The ethanol-fermenting bacteria in the fermenter may or may not be immobilized, but are preferably immobilized. Fermentation conditions are preferably pH 4 to 7 and temperature 20 to 40°C. The ethanol fermentation liquid is returned to the saccharification reaction tank again, and saccharification reaction and fermentation are performed simultaneously. At that time, the produced ethanol can also be separated and recovered.

As the saccharification reaction progresses, the lignocellulosic biomass in the reactor is decomposed and reduced, so it is desirable to aseptically charge the lignocellulosic biomass into the reactor as needed to continue the reaction.

In fermentation, ethanol accumulates in the reactor, and fermentation is suppressed when the ethanol concentration rises. Therefore, fermentation may be performed while separating and recovering ethanol from the fermentation broth. In that case, a pervaporation membrane may be used, or an evaporation device may be used. At that time, it is preferable to operate at 50° C. or less at which the fermenting microorganisms are not deactivated. However, if the fermentation broth after recovering ethanol is not returned to the reactor, this is not the case, and the temperature suitable for recovering ethanol can be used. In addition, since fermenting microorganisms remain in the liquid after ethanol recovery, it is preferable to aseptically return the liquid to the reactor for reuse.
Enzymes and fermenting microorganisms may be added aseptically as needed.

In addition, insoluble residue accumulates in the reactor and suppresses stirring efficiency, so it may be removed using a centrifugal separator or the like. When a large amount of cellulose remains in the residue, it may be mixed with the raw material lignocellulose biomass, or may be decomposed by adding a saccharifying enzyme-producing bacteria culture solution.
The recovered ethanol can be distilled with a distillation apparatus.

(Hydrophobic lignin derivative)
After adding a second acid to the liquid fraction containing the lignin derivative obtained by adding the first acid to the lignocellulosic biomass and polyalkylene glycol and reacting them, solid-liquid separation is performed to obtain a hydrophobic A solid fraction containing the organic lignin derivative can be obtained. Hereinafter, a method for producing a solid fraction containing the hydrophobic lignin derivative will be specifically described with reference to FIG.

A solid fraction containing the hydrophobic lignin derivative according to the present invention can be produced by the following steps.
(4) a step of adding a second acid to the liquid fraction obtained in step (3) above (14 in the figure);
(6) A step of performing solid-liquid separation on the precipitate precipitated in step (4) to obtain a solid fraction containing the lignin derivative (15 in the figure).

The second acid added to the liquid fraction containing the lignin derivative is not particularly limited as long as it can acidify the liquid fraction, but an acid capable of adjusting the pH to 1 to 4 is preferable, and sulfuric acid is particularly preferred. preferable. The amount to be added is usually 0.1 to 3.0 parts by weight based on the liquid fraction.
Hydrophobic lignin derivatives are precipitated by adding a second acid to the liquid fraction to acidify it, and a solid fraction containing the hydrophobic lignin derivative is obtained by solid-liquid separation. The solid fraction obtained by solid-liquid separation contains unreacted lignin in addition to the hydrophobic lignin derivative.
The solid-liquid separation method is not particularly limited as long as it is a method capable of separating the precipitate formed by adding the second acid, but centrifugation is preferred. Centrifugation is usually from 2000g to 20000g, preferably from 5000g to 15000g.

If necessary, the solid fraction containing the hydrophobic lignin derivative obtained by the reaction may be completely dried by a conventionally used drying method such as a freeze dryer.
The concentration of the hydrophobic lignin derivative in the solid fraction can be calculated by the following formula.

The hydrophobic lignin derivative contained in the solid fraction can be used as a functional raw material for heat-resistant films, cement dispersants, thermosetting resins, and the like.

[Embodiment 4: Apparatus for producing enzyme stabilizer]
FIG. 1 is a schematic configuration diagram schematically showing an apparatus for producing an enzyme stabilizer according to a first embodiment of the present invention. Hereinafter, each configuration of the apparatus for producing an enzyme stabilizer according to this embodiment will be described with reference to FIG.
An enzyme stabilizer manufacturing apparatus 100 according to the first embodiment of the present invention includes a first reaction tank 1 , a filtration device 2 , a second reaction layer 3 , and a solid-liquid separation device 4 . The first reaction tank 1 has an alkali supply means 1a, and reacts the lignocellulosic biomass and polyethylene glycol with the first acid. The second reaction tank 3 has a second acid supply means 3a.

According to the apparatus for producing an enzyme stabilizer of the present embodiment, an enzyme stabilizer capable of stabilizing enzyme activity can be obtained at a high yield. Furthermore, the apparatus for producing an enzyme stabilizer of the present embodiment can simultaneously obtain a hydrophilic lignin derivative that can be used as an enzyme stabilizer and a hydrophobic lignin derivative that is suitable as a functional raw material. It is possible to improve the profitability of the entire industrial production process using biomass.

[Reaction tank]
The reaction tank 1 is a tank for obtaining a lignin derivative by reacting the lignocellulosic biomass and polyethylene glycol with the first acid. The lignocellulosic biomass, polyethylene glycol, and first acid are the same as those described for the enzyme stabilizer above.
The amount of polyalkylene glycol to be added is not particularly limited, and is usually 5 to 100 parts by weight, preferably 10 to 60 parts by weight, more preferably 20 to 50 parts by weight with respect to 10 parts by dry weight of lignocellulose biomass. be.
The amount of the first acid to be added is, for example, 0.1 to 0.3 parts by weight with respect to 100 parts by weight of polyalkylene glycol.

Moreover, the reaction tank 1 is not particularly limited as long as it is acid-resistant.
The reaction tank 1 may have a stirring mechanism such as a stirring blade.
Moreover, the temperature of the reaction solution in the stirring tank is not particularly limited, and is usually 100°C to 200°C, preferably 120°C to 160°C.
In order to keep the temperature in the reaction vessel 1 within the above range, the stirring vessel 1 may be equipped with a temperature control device or a thermometer 1b.
Moreover, the reaction vessel 1 has an alkali supply means 1a. Furthermore, as shown in FIG. 1, a lignocellulose-based biomass supply means 1c, a polyalkylene glycol supply means 1d, a first acid supply means 1e, and the like may be provided.
Moreover, the reaction tank 1 may have a pH meter. A pH meter may be used to measure the pH of the reaction solution after alkali supply and to appropriately adjust the amount of alkali supply.

(Alkali supply means)
The alkali supply means 1a is arranged in the reaction tank 1 and is for supplying the alkali to the reaction tank 1. As shown in FIG. By supplying the alkali, the reaction liquid can be neutralized after the production reaction of the lignin derivative is completed in the reaction vessel 1 . The alkali is the same as those exemplified in the method for producing the enzyme stabilizer described above.
The pH of the reaction solution after supplying the alkali is preferably 6 or more and 12 or less, more preferably 6 or more and 10 or less.
The alkali supply means 1a may have a pump, a valve, etc. for adjusting the amount of alkali to be supplied.

[Filtration device]
The filtering device 2 is for removing a solid fraction from the reaction solution.
The filtering device 2 is not particularly limited as long as the solid fraction can be removed from the neutralized alkaline solution, and examples thereof include centrifugal separation, screw press, and filter press.

[Solid-liquid separator]
The second reaction tank 3 is for reacting the liquid fraction obtained by the filtering device 2 with the second acid.
The amount of the second acid to be added is, for example, 0.01 to 3.0 parts by weight with respect to 100 parts by weight of the liquid fraction.
Moreover, the second reaction tank 3 is not particularly limited as long as it is acid-resistant.
The second reaction vessel 3 may have a stirring mechanism such as a stirring blade.
The solid-liquid separation device 4 is for solid-liquid separation of the reaction liquid obtained in the second reaction tank 3 to remove solids, thereby obtaining a liquid fraction containing a hydrophilic lignin derivative. .
The solid-liquid separator 4 is not particularly limited as long as it can separate solid matter generated by adding acid, and examples thereof include a centrifugal separator.

A method for producing an enzyme stabilizer using the apparatus 100 for producing an enzyme stabilizer shown in FIG. 1 will be described below.
First, lignocellulose-based biomass, polyalkylene glycol, and a first acid are added to the reaction tank 1 by the lignocellulose-based biomass supply means 1c, the polyalkylene glycol supply means 1d, and the first acid supply means 1e. The kind, addition amount, reaction temperature and reaction time of each material are as described in the method for producing the enzyme stabilizer described above. The above materials are reacted to produce a lignin derivative.
Next, an alkali is added from the alkali supply means 1a to the reaction liquid containing the lignin derivative after the reaction to neutralize the reaction liquid. The type and amount of alkali to be added are as described in the method for producing the enzyme stabilizer described above. Next, the neutralized reaction liquid is sent to the filtration device 2 through the pipe 5 while adjusting the amount of liquid to be sent by the pump 5a. In the filtering device 2, a solid fraction is removed from the sent reaction liquid to obtain a liquid fraction. The solid content is discharged through pipe 6a.
On the other hand, the obtained liquid fraction is sent to the second reaction tank 3 via the pipe 6b, and the second acid is added. The reaction liquid is sent to the solid-liquid separation device 4 through the pipe 7, where solids containing unreacted lignin and hydrophobic lignin derivatives are separated as a solid fraction.
On the other hand, the obtained liquid fraction contains unreacted polyalkylene glycol in addition to the hydrophilic lignin derivative. Therefore, the unreacted polyalkylene glycol contained in the liquid fraction can be sent to the polyalkylene glycol supply means 1d through a pipe (not shown) and reused.

In the present invention, a hydrophilic lignin derivative useful as an enzyme stabilizer and a hydrophobic lignin derivative useful as a functional raw material can be simultaneously obtained from lignocellulose biomass. It is possible to improve the profitability of the entire process.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[Example 1: Preparation of hydrophilic lignin derivative and hydrophobic lignin derivative]
To 250 g of air-dried cedar wood powder, 1250 g of polyethylene glycol 200 (PEG200, manufactured by Lion Corporation) and 4 g of 95% sulfuric acid were added, heated to 140° C., and stirred for 90 minutes to react. After completion of the reaction, 150 ml of 2N aqueous sodium hydroxide solution was added for neutralization, and the lignin derivative was extracted. The resulting reaction solution was applied to a filtration membrane (manufactured by ADVANTEC) to remove solids.
3N sulfuric acid was added to the resulting filtrate until the pH reached 2. A liquid fraction containing a hydrophilic lignin derivative was obtained by centrifuging the sediment precipitated by sulfuric acid at 13000 g for 15 minutes.
The precipitate obtained by centrifugation was washed with 3 L of water and freeze-dried to obtain a solid fraction containing the hydrophobic lignin derivative.

[Example 2: Preparation of hydrophilic lignin derivative and hydrophobic lignin derivative]
Liquid fractions containing hydrophilic lignin derivatives were obtained in the same manner as in Example 1, except that PEG400 (manufactured by Lion Corporation) or PEG600 (manufactured by Lion Corporation) was used instead of PEG200.

[Example 3: Preparation of hydrophilic lignin derivative and hydrophobic lignin derivative]
A liquid fraction containing a hydrophilic lignin derivative was obtained in the same manner as in Example 1, except that PEG1000 (manufactured by WAKO) was used instead of PEG200.

[Comparative Example 1: Preparation of lignin derivative according to Patent Document 2]
Air-dried cedar chips were digested with alkali to obtain alkali-treated lignin. 10 g of the resulting alkali-treated lignin was dissolved in 100 ml of 1N sodium hydroxide aqueous solution, and 40 g of lauryl alcohol-polyethylene oxide-glycidyl ether was added. The resulting solution was heated to 70° C. and reacted with stirring for 3 hours. After the reaction, acetic acid was added to the obtained solution to adjust the pH to 4, and the solution was filtered with an ultrafiltration membrane that excludes particles with a molecular weight of 1,000 or less. After filtration, the residue was collected to obtain the lignin derivative.

Here, the method for producing the lignin derivatives of Examples 1 to 3 and the method for producing the lignin derivative of the comparative example are compared. As a raw material, the production methods of Examples 1 to 3 use lignocellulose biomass, whereas the production method of Comparative Example 1 uses lignin obtained by treating biomass with alkali. In addition, regarding the types of catalyst and solvent in the synthesis of the lignin derivative, in Examples 1 to 3, sulfuric acid was used as the catalyst and polyalkylene glycol was used as the solvent. The difference is that a solution is used. Further, the synthesis temperature is 100 to 200° C. in Examples 1 to 3, whereas it is 70° C. in Comparative Example 1. In the derivative separation method, in Examples 1 to 3, centrifugation was performed after precipitation with acid, to obtain a hydrophilic lignin derivative as a liquid fraction and a hydrophobic lignin derivative as a solid fraction. Comparative Example 1 is different in that a lignin derivative is obtained as a solid fraction by an ultrafiltration membrane.

Using the obtained liquid fraction containing the lignin derivative as an enzyme stabilizer, the following tests were carried out.

[Test Example 1: Enzyme Activity Maintenance Effect Confirmation Test (1)]
The liquid fraction containing the hydrophilic lignin derivative prepared in Example 1 or the hydrophobic lignin derivative was added to 10 g of the dry weight of the pretreated bagasse obtained by pretreating 20 g of bagasse with dilute sulfuric acid and 2 g of the saccharifying enzyme solution diluted with water. The solid fraction contained was added so that the amount of lignin derivative was 0.1 to 2.2% by weight based on the dry weight of bagasse, and water was added to bring the total volume to 100 ml. This was reacted at 50° C. for 48 hours to carry out a saccharification reaction. The C6 saccharification rate after the reaction was measured, and the relationship between the lignin derivative addition rate and the C6 saccharification rate was investigated. The results are shown in FIG. Here, the addition rate of 0% by weight means that the liquid fraction or solid fraction prepared in Examples is not added. As is clear from FIG. 2, the addition of 0.25% by weight of the liquid fraction containing the hydrophilic lignin derivative prepared in Example 1 increased the C6 saccharification rate by about 5%. In contrast, when the solid fraction containing the hydrophobic lignin derivative was added, the C6 saccharification rate increased by about 5% by adding 1 to 2% by weight. As a result, it was confirmed that the liquid fraction containing the hydrophilic lignin derivative increased the reactivity between the substrate and the enzyme and improved the activity of the enzyme.

[Test Example 2: Polyethylene glycol effect confirmation test (1)]
In Test Example 1, a saccharification test was carried out in the same manner as in Test Example 1 except that the hydrophilic lignin prepared in Example 2 was used instead of the hydrophilic lignin derivative prepared in Example 1. 3 shows the relationship between the addition rate of the hydrophilic lignin derivative and the C6 saccharification rate.

As is clear from FIG. 3, in the case of the hydrophilic lignin derivative of Example 1 prepared using PEG200, the addition of 0.25% by weight increased the C6 saccharification rate by about 5%. In contrast, in the case of the hydrophilic lignin derivative of Example 2 prepared using PEG400 or PEG600, the addition of 0.25% by weight of the lignin derivative increased the C6 saccharification rate by about 10%. From this result, it was found that the saccharification efficiency was higher when using the hydrophilic lignin derivatives prepared using PEG400 and PEG600 than when using PEG200.

[Test Example 3: Polyethylene glycol effect confirmation test (2)]
A saccharification test was carried out in the same manner as in Test Example 1 except that the hydrophilic lignin derivative prepared in Example 3 was used in Test Example 1 instead of the hydrophilic lignin derivative prepared in Example 1. The addition rate of the hydrophilic lignin derivative was added so as to be 0.5% by weight based on the dry weight of the pretreated bagasse. FIG. 4 shows the relationship between the molecular weight (200, 600) of PEG used in Examples 1 and 2 and the molecular weight (1000) of PEG used in Example 3 and the sugar yield ratio. The sugar yield ratio is the ratio of the amount of glucose at the end of saccharification when no lignin derivative is added to the amount of glucose at the end of saccharification when a lignin derivative is added. It shows that the addition increased the saccharification rate. As a result, it was confirmed that the higher the molecular weight of PEG used, the higher the saccharification rate. Furthermore, the average molecular weights of the hydrophilic lignin derivatives using PEG200, PEG600 and PEG1000 were 1530, 2250 and 3060, respectively. From the above, it is estimated that the hydrophilic lignin derivative has a molecular weight of preferably about 1,000 to 3,200, more preferably about 1,500 to 2,300.

[Test Example 4: Enzyme activity maintenance effect confirmation test (3)]
In Test Example 1, instead of the hydrophilic lignin derivative prepared in Example 1, the hydrophilic lignin derivative of Example 2 prepared using PEG400 and the lignin derivative prepared in Comparative Example 1 were used. A saccharification test was performed in the same manner as in 1. The results are shown in FIG.

From FIG. 5 , the hydrophilic lignin derivative prepared in Example 2 tended to have a higher C6 saccharification rate than the lignin derivative prepared in Comparative Example 1. From this result, it was confirmed that the lignin derivative prepared in Example 2 had a higher effect of improving enzyme activity than the lignin derivative prepared in Comparative Example 1.

According to the present invention, a liquid fraction containing hydrophilic lignin derivatives and a solid fraction containing hydrophobic lignin derivatives can be obtained from lignocellulosic biomass. A liquid fraction containing a hydrophilic lignin derivative is useful as an enzyme stabilizer, and a solid fraction containing a hydrophobic lignin derivative is useful as a raw material for producing a functional raw material.

DESCRIPTION OF SYMBOLS 1... First reaction tank 1a... Alkaline supply means 1b... Thermometer 1c... Lignocellulosic biomass supply means 1d... Polyalkylene glycol supply means 2... Filtration device 3... Second reaction tank 4 ... solid-liquid separator, 5, 6a, 6b, 7 ... piping, 5a ... pump, 100 ... enzyme stabilizer manufacturing apparatus

Claims (2)

A method for saccharification of lignocellulosic biomass comprising the following steps .
(1) Lignocellulosic biomass, which is at least one selected from the group consisting of woody plants, herbaceous plants, processed products thereof, and waste products thereof, and polyethylene glycol having an average molecular weight of 400 to 600. adding an acid to react,
(2) adding alkali to the reaction solution obtained in step (1) to neutralize;
(3) removing solids from the neutralized alkaline solution obtained in step (2);
(4) adding a second acid to the liquid fraction obtained in step (3);
(5) performing solid-liquid separation on the precipitate precipitated in step (4) to obtain a liquid fraction containing a hydrophilic lignin derivative;
(6) Step of adding the liquid fraction obtained in step (5) to lignocellulosic biomass In the step (6), the liquid fraction obtained in the step (5) is subjected to enzymatic saccharification of the hydrophilic lignin derivative in the liquid fraction obtained in the step (5). The saccharification method according to claim 1 , wherein the dry weight is added so as to be 0.1 to 1.0% by mass.

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