JP5688501B2 - Method for enzymatic saccharification of woody biomass and method for producing fermented ethanol - Google Patents

Description

  The present invention relates to a method for enzymatic saccharification of woody biomass and a method for producing fermented ethanol.

  Conventionally, as a method for efficiently saccharifying cellulose contained in biomass with hydrolase, saccharification is performed by adding wheat bran and a surfactant and then reacting with hydrolase in a wood chip crushing process or enzymatic saccharification process. A method (Patent Document 1) and a saccharification method in which a substance having high affinity with lignin (skim milk) is added in advance and then a reaction with a hydrolase is started (Patent Document 2) are known.

JP 2007-97585 A JP 2009-72144 A

However, cellulose contained in woody biomass is strongly hydrogen-bonded between molecular chains to form cellulose crystals, and conventional saccharification methods do not have sufficient reaction efficiency between hydrolase and the above-mentioned crystalline cellulose. There is a problem that a sufficient saccharification rate cannot be obtained.
That is, an object of the present invention is to provide a saccharification method capable of saccharifying at an excellent saccharification rate in a reaction between a hydrolase and cellulose contained in woody biomass.

The feature of the enzymatic saccharification method of the woody biomass of the present invention is characterized by a pulverization step (1) for pulverizing at least one woody biomass selected from wood chips and waste building materials to obtain a woody biomass powder; and a polymeric activator (1), at least one anion selected from the group consisting of a polymeric activator (2), a polymeric activator (3), and a sulfosuccinic acid activator (S) represented by the general formula (1) The present invention includes a saccharification treatment step (2) in which a woody biomass powder is hydrolyzed in the presence of a bioactive agent and a hydrolase to obtain a sugar.

However, the polymeric activator (1) is a monomer represented by R ¹ OCOCH═CHCOOR ² {R ¹ and R ² are a hydrogen atom (H), ammonium (NH ₄ ), sodium atom (Na) or Formula: R ³ O— (A ¹ O) n ¹ —O— (R ³ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, A ¹ O is an oxyalkylene group having 2 to 4 carbon atoms, and n ¹ is 1. Or at least one of R ¹ and R ² is represented by the formula: R ³ O— (A ¹ O) n ¹ —O—. }, Maleic acid (salt) {in the case of salt, ammonium salt and / or sodium salt} and styrene as essential constituent monomers,
The polymer type activator (2) is composed of (meth) acrylic acid ester of ethylene oxide and / or 2-90 mol adduct of alkanol having 1 to 4 carbon atoms and (meth) acrylic acid (salt) {salt of The ammonium salt or sodium salt} as an essential constituent monomer,
The polymer type activator (3) comprises (meth) propenylene ether of 9 to 33 mol adduct of alkanol having 1 to 4 carbon atoms and / or propylene oxide and maleic anhydride as essential constituent monomers. Become.

R ⁴ and R ⁵ are each an alkyl group having 6 to 8 carbon atoms, M ¹ is an alkali metal atom or ammonium (NH ₄ ), C is a carbon atom, H is a hydrogen atom, O is an oxygen atom, and S is a sulfur atom. .

  The feature of the method for producing fermented ethanol of the present invention is that after the saccharification treatment step (2) of the enzymatic saccharification method for woody biomass or simultaneously with this saccharification treatment step (2), fermented ethanol is obtained by fermenting sugar. This is the gist.

  The enzymatic saccharification method for woody biomass of the present invention is remarkably excellent in the reaction efficiency between hydrolase and cellulose, and can be saccharified at an excellent saccharification rate. Therefore, the enzymatic saccharification method of the present invention is extremely useful for producing fermented ethanol (bioethanol) using woody biomass as a raw material.

<Powdering step (1)>
The woody biomass can be used without limitation as long as it is at least one selected from wood and waste building materials.
Wood includes conifers (pine, fir, tsuga, spruce, larch, raditapine, etc.) and broadleaf trees (eucalyptus, poplar, beech, maple, hippopotamus, etc.), kenaf, mitsumata, kouzo, ganpi, mulberry, Manila hemp, reeds , Bamboo and bagasse and the like. These timbers may be thinned wood, sawn timber, driftwood and pruned wood, and may include wood branches, roots and leaves. Waste building materials include waste wood building materials, waste wood pallets, waste wood packaging materials, and the like. As woody biomass, any of these may be used alone, or two or more kinds may be used in combination.

The wood biomass is pulverized by physical treatment {known cutters (scissors, slitters, band cutters, disc cutters, push cutters, etc.) or known cutters (hammer mill, roller mill, impact mill, A method using a grinder, a refiner, a ball mill, a jet mill, a freeze pulverizer, a wood chopper, etc.), a freeze pulverization method, etc.} and a chemical treatment {pulverization method, steaming method, etc.) can be applied.
The woody biomass powder may be used as it is after pulverization or may be used after being classified by a known method such as sieving.

  Although there is no restriction | limiting in the shape of the woody biomass powder obtained by the grinding | pulverization process, From a viewpoint of saccharification efficiency, an amorphous form is preferable.

  The woody biomass powder may be subjected to the saccharification treatment step (2) as it is, or pretreatment {cooking treatment, explosion treatment, hydrothermal treatment, acid treatment (sulfuric acid treatment, dilute sulfuric acid treatment, etc.) or alkali treatment, etc. In addition, it may be subjected to a saccharification treatment step (2) after performing a pretreatment or the like mixed with a peroxide in an alkaline aqueous solution {in the present invention, those that have not been pretreated, those that have been pretreated, Called woody biomass powder. }.

  The size of the woody biomass powder is preferably a circumscribed circle equivalent diameter of 0.5 to 1 mm, more preferably 0.05 to 0.5 mm, from the viewpoints of hydrolyzability and ethanol fermentation efficiency. Note that the circumscribed circle equivalent diameter is the diameter of a circle circumscribing the projected image of particles obtained by JIS Z-8827-1: 2008 Particle size analysis-Image analysis method-Part 1: Static image analysis method.

The loss on drying (% by weight) of the woody biomass powder is preferably from 5 to 40, particularly preferably from 10 to 30, from the viewpoint of hydrolyzability. When the loss on drying is larger than this range, it can be used after the loss on drying is within a preferred range by a known drying method {method of drying by heating with a forward air dryer, etc., method of air drying outdoors, etc.}.
In addition, loss on drying of woody biomass powder is JIS K0067-1992 Chemical product loss and residue test method 4.1 Drying loss test, 4.1.4 (2) “Method 3 Heat drying under reduced pressure” In accordance with the above, it is measured at a drying temperature of 50 ± 3 ° C. and a pressure of 1 to 10 kPa.

<Saccharification process (2)>
Among the anionic activities used in the saccharification treatment step (2), the polymer type active agent (1) will be described.

The polymeric activator (1) is a monomer represented by R ¹ OCOCH═CHCOOR ² {R ¹ and R ² are each a hydrogen atom (H), ammonium (NH ₄ ), a sodium atom (Na) or a formula: R ³ O— (A ¹ O) n ¹ —O— (R ³ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, A ¹ O is an oxyalkylene group having 2 to 4 carbon atoms, and n ¹ is 1 or 2 And at least one of R ¹ and R ² is represented by the formula: R ³ O— (A ¹ O) n ¹ —O—. }, Maleic acid (salt) {in the case of salt, ammonium salt and / or sodium salt} and styrene as essential constituent monomers.

  Maleic acid (salt) means maleic acid and / or maleate.

A monomer represented by R ¹ OCOCH═CHCOOR ² is obtained by esterifying (anhydrous) maleic acid and an alcohol represented by R ³ O— (A ¹ O) n ¹ —O—H by a known method. Maleic acid (mono / di) esters and / or maleic acid monoester salts {in the case of salts, ammonium salts and / or sodium salts}. Among these, a maleic acid monoester salt is preferable, an ammonium salt or a sodium salt of maleic acid monoester is more preferable, and an ammonium salt of maleic acid monoester is particularly preferable. Maleic acid (mono / di) ester means maleic acid monoester and / or maleic acid diester.

Among the hydrogen atom or the alkyl group having 1 to 4 carbon atoms (R ³ ), the alkyl group having 1 to 4 carbon atoms includes a linear alkyl group having 1 to 4 carbon atoms and a branched alkyl group having 3 or 4 carbon atoms. Is included. Examples of the linear alkyl group having 1 to 4 carbon atoms include methyl, ethyl, n-propyl, and n-butyl. Examples of the branched alkyl group having 3 or 4 carbon atoms include iso-propyl, tert-butyl, iso-butyl, and sec-butyl. Among these, a C1-C4 linear alkyl group is preferable, More preferably, it is n-butyl.

Examples of the oxyalkylene group having 2 to 4 carbon atoms (A ¹ O) include oxyethylene, oxypropylene, and oxybutylene. Of these, oxyethylene and oxypropylene are preferred.

When n ¹ is 2, the two oxyalkylene groups may be composed of one kind of oxyalkylene group or may be composed of two kinds of oxyalkylene groups. When it is composed of two types of oxyalkylene groups, there is no limitation on the order of bonding.

Examples of the alcohol represented by R ³ O— (A ¹ O) n ¹ —O—H include ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, and diethylene glycol monobutyl ether.

The content of the monomer unit represented by R ¹ OCOCH = CHCOOR ^{2 (mol%),} based on the total moles of R 1 OCOCH = CHCOOR monomer unit represented by ^2, maleic acid (salt) units and styrene units 10-30 are preferable.

The content (mole%) of the maleic acid (salt) unit is preferably 15 to 35 based on the total number of moles of the monomer unit represented by R ¹ OCOCH═CHCOOR ² , the maleic acid (salt) unit and the styrene unit. .

The content (mol%) of the styrene unit is preferably 40 to 60 based on the total number of moles of the monomer unit represented by R ¹ OCOCH = CHCOOR ² , the maleic acid (salt) unit, and the styrene unit.

When the polymeric activator (1) contains an ammonium salt and / or a sodium salt, the ammonium salt unit contained in the monomer unit represented by R ¹ OCOCH = CHCOOR ² contained in the polymeric activator (1) and The content (mol%) of the sodium salt unit and the maleate unit is 90 to 90 based on the total number of moles of the monomer unit represented by R ¹ OCOCH = CHCOOR ² and the maleic acid (salt) unit. 100 is preferred.

The polymeric activator (1) contains other ethylenically unsaturated monomers as constituent monomers in addition to the monomer represented by R ¹ OCOCH = CHCOOR ² , maleic acid (salt) and styrene. Can do.

Other ethylenically unsaturated monomers are not particularly limited as long as they can be copolymerized with a monomer represented by R ¹ OCOCH═CHCOOR ² , maleic acid (salt), and styrene, and include aromatic vinyl monomers, olefins, ethylene Unsaturated nitriles and ethylenically unsaturated amides can be used.

  Examples of the aromatic vinyl monomer include α-methylstyrene, vinyltoluene, α-chlorostyrene, o-, m- or p-chlorostyrene, p-ethylstyrene, divinylbenzene, and the like.

  Examples of olefins include α-olefins having 2 to 20 carbon atoms (ethylene, propylene, butylene, isobutylene, hexylene, octylene, undecylene, tetradecylene, and nonadecylene), diisobutylene, butadiene, piperylene, chloroprene, pinene, limonene, isodene, cyclone. Pentadiene, bicyclopentadiene, ethylidene norbornene and the like are included.

  Examples of the ethylenically unsaturated nitrile include (meth) acrylonitrile, cyanopropene and cyanopentene.

  Examples of the ethylenically unsaturated amide include (meth) acrylamide, N-methyl (meth) acrylamide, N-2-hydroxymethyl (meth) acrylamide, N- (2-aminoethyl) (meth) acrylamide, N-vinylpyrrolidone and N- (2-dimethylaminoethyl) (meth) acrylamide and the like are included.

  The styrene-maleic anhydride copolymer can be easily obtained from the market as “SMA series” (manufactured by Sartomer) or the like, and these can also be used.

  The weight average molecular weight (Mw) of the polymeric activator (PA1) is preferably 3,000 to 100,000, more preferably 5,000 to 15,000, from the viewpoint of hydrolyzability.

  The weight average molecular weight (Mw) is measured by gel permeation chromatography (GPC) method using polyethylene glycol of known molecular weight as a standard substance (column temperature 40 ° C., eluent: 0.1-MPB hydrogen phosphate Sodium aqueous solution: 0.1-MPB sodium dihydrogen phosphate aqueous solution = 1: 1 (molar ratio), flow rate 0.8 ml / min, sample concentration: 0.4 wt% eluent solution.

  The polymer type activator (1) can be produced by a known method. For example, (maleic anhydride) and styrene and, if necessary, other ethylenically unsaturated monomers are used as constituent monomers, and the following ( It can be obtained by the method described in Method 11) or (Method 12) (hereinafter, description of other ethylenically unsaturated monomers is omitted, but can be copolymerized with styrene or the like if necessary).

(Method 11) A styrene-maleic anhydride copolymer obtained from maleic anhydride and styrene by a known method is used in the presence of an alcohol represented by R ³ O— (A ¹ O) n ¹ —O—H. To 80-90 ° C., partially esterified to produce a styrene-maleic acid-maleic acid monoester copolymer, followed by subsequent neutralization with sodium hydroxide or ammonia if necessary, and styrene-maleic acid (salt ) -Method for producing maleic acid monoester (salt) copolymer.

(Method 12) (Male anhydride) is partially esterified with an alcohol represented by R ³ O— (A ¹ O) n ¹ —O—H to obtain a maleic acid monoester, Anhydrous) maleic acid and styrene are copolymerized to produce a styrene-maleic acid-maleic acid monoester copolymer, followed by neutralization with sodium hydroxide or ammonia if necessary, styrene-maleic acid (salt) -maleic acid A method for producing a monoester (salt) copolymer.

(Method 13) A styrene-maleic anhydride copolymer obtained from maleic anhydride and styrene by a known method is converted into an acid catalyst (sulfuric acid, p-toluenesulfonic acid, etc.) and R ³ O— (A ¹ O) n. Heating at 100 to 120 ° C. in the presence of an alcohol represented by ^1- O—H to effect dehydration esterification to produce a styrene-maleic acid-maleic acid diester copolymer. A method for producing a styrene-maleic acid (salt) -maleic acid diester copolymer by neutralization with ammonia.

  A polymerization catalyst can be used for the copolymerization. As the polymerization catalyst, an ordinary polymerization catalyst or the like is used, and azo compounds, persulfates, inorganic peroxides, redox catalysts, organic peroxides, and the like are included. Examples of the azo compound include 2,2′-azobisisobutyl, tolyl, 4,4′-azobis-4-cyanovaleric acid, 2,2′-azobis (4-methoxy-2,4-dimethylvaleronitrile, 2,2 '-Azobis (2-methylbutyronitrile), 1,1'-azobis (cyclohexane-1-albonitrile), 2,2'-azobis (2,4,4-trimes (cyclohexane-1-albonitrile), 2,2′-azobis (2,4,4-trimethylpentane), dimethyl 2,2-azobis (2-methylpropionate), 2,2′-azobis [2- (hydroxymethyl) propionitrile] and 1,1′-azobis (1-acetoxy-1-phenylethane), etc. Examples of persulfates include ammonium persulfate, potassium persulfate, and sodium persulfate. Examples of inorganic peroxides include perborate and hydrogen peroxide, etc. Examples of redox catalysts include ascorbic acid-hydrogen peroxide, etc. Examples of organic peroxides include peroxidation. These polymerization catalysts may be used alone or in combination, among which persulfates and azo compounds are preferred, more preferably ammonium persulfate, potassium persulfate, sodium persulfate and An azo compound, particularly preferably 2,2′-azobisisobutyronitrile.

  When a polymerization catalyst is used, the amount (% by weight) of the polymerization catalyst used is preferably from 1 to 100, more preferably from 2 to 80, particularly preferably from 3 to 60, based on the weight of the constituent monomer.

  At the time of copolymerization, a continuous transfer agent for radical polymerization may be used. Examples of the chain transfer agent include thiocarboxylic acids (n-lauryl mercaptan, 2-mercaptoethanol, thiomalic acid, etc.), secondary alcohols (isopropanol, etc.), amines (dibutylamine, etc.), hypophosphites (sodium hypophosphite, etc.). ).

  A solvent may be used in the copolymerization. Solvents include water (tap water, ion exchange water, industrial water, etc.), alcohols having 1 to 4 carbon atoms (such as methanol, ethanol and isopropyl alcohol), aromatic solvents (such as toluene and xylene), and 1 to 6 carbon atoms. Ketones such as methyl isobutyl ketone and acetone can be used. Among these, water, a mixed solvent of water and an alcohol solvent, and a ketone having 1 to 6 carbon atoms are preferable, and a ketone having 2 to 6 carbon atoms is more preferable.

  When a solvent is used, the amount (% by weight) used is preferably 50 to 900, more preferably 60 to 800, and particularly preferably 100 to 600, based on the total weight of the constituent monomers.

  The copolymerization reaction temperature is preferably about 40 to 130 ° C., and the copolymerization reaction time is preferably about 1 to 15 hours.

  In addition, you may superpose | polymerize, dripping the whole quantity or one part of a structural monomer. Moreover, you may superpose | polymerize, dripping the whole quantity or one part of a polymerization catalyst. Moreover, you may superpose | polymerize, dripping the whole quantity or one part of a solvent with a structural monomer or a polymerization catalyst. On the other hand, the polymerization may be carried out while charging the solvent in the polymerization tank and removing the solvent. Among these, from the viewpoint of productivity and the like, the method of dropping the whole amount of the constituent monomer and the polymerization catalyst and the method of dropping a part of the solvent together with the constituent monomer or the polymerization catalyst are preferable, and more preferably the constituent unit. In this method, the entire amount of the monomer and the polymerization catalyst is dropped together with a part of the solvent.

  Next, among the anionic activities used in the saccharification treatment step (2), the polymer type active agent (2) will be described.

  The polymeric activator (2) is a (meth) acrylic acid ester (hereinafter referred to as (meth) acrylic acid ester) of an adduct of 2 to 90 mol of ethylene oxide and / or propylene oxide having 1 to 4 carbon atoms. , (Meth) acrylic acid (salt) {in the case of salt, ammonium salt or sodium salt} is an essential constituent monomer. In addition, (meth) acryl ... means methacryl ... and / or acrylic ..., and acrylic acid (salt) means acrylic acid and / or acrylate.

  Examples of the alkanol having 1 to 4 carbon atoms include linear alkanol having 1 to 4 carbon atoms and branched alkanol having 3 or 4 carbon atoms. Examples of the linear alkanol having 1 to 4 carbon atoms include methanol, ethanol, n-propyl alcohol, and n-butyl alcohol. Examples of the branched alkanol having 3 or 4 carbon atoms include iso-propyl alcohol, tert-butyl alcohol, iso-butyl alcohol and sec-butyl alcohol. Among these, a linear alcohol having 1 to 4 carbon atoms is preferable, and methyl alcohol is more preferable.

  When the (meth) acrylic acid ester is an ester with an adduct of ethylene oxide and propylene oxide, the bonding mode of ethylene oxide and propylene oxide may be any of block, random, and a mixture thereof.

  The content (mol%) of the (meth) acrylic acid ester unit is preferably 10 to 90 based on the total number of moles of the (meth) acrylic acid ester unit and the (meth) acrylic acid (salt) unit.

  The content (mol%) of the (meth) acrylic acid (salt) unit is preferably 10 to 90 based on the total number of moles of the (meth) acrylic acid ester unit and the (meth) acrylic acid (salt) unit.

  When the polymer type activator (2) includes a (meth) acrylate unit, the content (mol%) of the (meth) acrylate unit is the total number of moles of the (meth) acrylic acid (salt) unit. Based on 0.1 to 90. As the (meth) acrylate, an ammonium salt is preferable.

  The polymeric activator (2) can contain other ethylenically unsaturated monomers as constituent monomers in addition to (meth) acrylic acid ester and (meth) acrylic acid (salt). As the other ethylenically unsaturated monomers, the same monomers as the other ethylenically unsaturated monomers can be used.

  The preferred range of the weight average molecular weight (Mw) of the polymeric activator (2) is preferably from 3,000 to 100,000, more preferably from 10,000 to 30,000, from the viewpoint of hydrolyzability.

  The polymer type activator (2) can be produced by a known method. For example, using (meth) acrylic acid (salt) and other ethylenically unsaturated monomers as necessary, the following (Method 21) to It can be obtained by the method described in (Method 25) (hereinafter, description of other ethylenically unsaturated monomers is omitted, but it can be copolymerized with (meth) acrylic acid (salt) or the like if necessary. ).

(Method 21) Poly (meth) acrylic acid obtained by a known polymerization method is partially esterified with an adduct of 2 to 90 mol of ethylene oxide and / or propylene oxide of an alkanol having 1 to 4 carbon atoms to obtain (meth) acrylic acid ester A method for obtaining a (meth) acrylic acid copolymer.

(Method 22) Poly (meth) acrylic acid obtained by a known polymerization method is partially esterified with 2-90 mol adduct of ethylene oxide and / or propylene oxide having 1 to 4 carbon atoms, and then sodium hydroxide or ammonia To obtain a (meth) acrylic acid ester- (meth) acrylic acid (salt) copolymer.

(Method 23) Known method {Method of heating (meth) acrylic acid with an ethylene oxide and / or propylene oxide 2-90 mol adduct of alkanol having 1 to 4 carbon atoms in the presence of an acid catalyst. } To obtain a (meth) acrylic acid ester- (meth) acrylic acid copolymer by copolymerizing the (meth) acrylic acid ester obtained in (1) and (meth) acrylic acid by a known method.

(Method 24) A (meth) acrylic acid ester obtained by a known method and (meth) acrylic acid are copolymerized by a known method, and then neutralized with sodium hydroxide or ammonia to obtain a (meth) acrylic acid ester ( A method for obtaining a (meth) acrylic acid (salt) copolymer.

(Method 25) A (meth) acrylic acid ester obtained by a known method and (meth) acrylic acid (salt) are copolymerized by a known method, and (meth) acrylic acid ester- (meth) acrylic acid (salt) A method for obtaining a polymer.

  The (meth) acrylic acid ester can be easily obtained from the market in addition to a known method, and as a trade name, for example, “Blemmer PME series, PSE series, PP series, AE series, etc.” (NOF) “Blemmer” is a registered trademark of NOF Corporation.), “Light acrylate BO-A, EC-A, MTG-A, EHDG-A, 130A, DMP-A” (Kyoeisha Chemical Co., Ltd.) Manufactured), "light ester BO, BC, MTG, 130MA, 041MA" (manufactured by Kyoeisha Chemical Co., Ltd.), "NK ester M-20G, M-40G, M-90G, M-230G, AMP-10G, AMP-20G" AMP-60G, AM-90G, LA, etc. "(manufactured by Shin-Nakamura Chemical Co., Ltd.).

  The copolymerization in (Method 21) to (Method 24) can be carried out under the same conditions and method as the copolymerization in (Method 11) and (Method 12) in the polymeric activator (1).

  Next, among the anionic activities used in the saccharification treatment step (2), the polymer type active agent (3) will be described.

  The polymer type activator (3) is a (meth) propenylene ether of an alkanol having 1 to 4 carbon atoms and / or an adduct of 9 to 33 mol of propylene oxide (hereinafter referred to as (meth) propenyl ether). } And maleic anhydride as essential constituent monomers. In addition, (meth) propenyl ... means propenyl ... and / or 2-methyl 2-propenyl ....

  As the alkanol having 1 to 4 carbon atoms, the same alkanol as the alkanol having 1 to 4 carbon atoms can be used, and preferable ones are also the same. In the case of an adduct of ethylene oxide and propylene oxide, the bonding mode of ethylene oxide and propylene oxide may be block, random, or a mixture thereof.

  The content (mol%) of the (meth) propenyl ether unit is preferably 40 to 60 based on the total number of moles of the (meth) propenyl ether unit and the maleic anhydride unit.

  As for content (mol%) of a maleic anhydride unit, 40-60 are preferable based on the total number of moles of a (meth) propenyl ether unit and a maleic anhydride unit.

  The polymeric activator (3) can contain other ethylenically unsaturated monomers as constituent monomers in addition to (meth) propenyl ether and maleic anhydride. As the other ethylenically unsaturated monomers, the same monomers as the other ethylenically unsaturated monomers can be used.

  The preferred range of the weight average molecular weight (Mw) of the polymeric active agent (3) is the same as that of the polymeric active agent (2).

  The polymeric activator (3) can be produced by a known method, for example, a known method {for example, addition of 9 to 33 mol of ethylene oxide and / or propylene oxide of (meth) propenyl halide and alkanol having 1 to 4 carbon atoms. Can be obtained by copolymerizing (meth) propenyl ether, maleic anhydride and, if necessary, other ethylenically unsaturated monomers obtained by a known method by a known method (hereinafter referred to as “Williamson synthesis method with a product”) The description about other ethylenically unsaturated monomers is omitted, but if necessary, it can be copolymerized with maleic anhydride and the like.)

  (Meth) propenyl ether can be easily obtained from the market in addition to a known method. For example, “Uniox PKA series” (manufactured by NOF Corporation, “Uniox” Are registered trademarks of NOF Corporation), "Allyl glycol, allyl glycol H (manufactured by Nippon Emulsifier Co., Ltd.), and the like.

  For the copolymerization, the same polymerization catalyst, radical chain transfer agent and solvent as those described in the above (Method 11) and the like can be used, and preferred polymerization catalysts, radical chain transfer agents and solvents are also the same. The same temperature and method can be used for the polymerization reaction temperature and the polymerization method, and the preferred temperature and method are also the same.

  Next, among the anionic activities used in the saccharification treatment step (2), the sulfosuccinic acid type activator (S) will be described.

The sulfosuccinic acid type activator (S) is represented by the general formula (1).

R ⁴ and R ⁵ are each an alkyl group having 6 to 8 carbon atoms, M ¹ is an alkali metal atom or ammonium (NH ₄ ), C is a carbon atom, H is a hydrogen atom, O is an oxygen atom, and S is a sulfur atom. .

The alkyl group having 6 to 8 carbon atoms ^{(R 4,} R ^5), a linear alkyl group having 6 to 8 carbon atoms, cyclic alkyl groups and branched-chain alkyl group. Examples of the linear alkyl group include hexyl and octyl. Examples of the cyclic alkyl group include cyclohexyl, and examples of the branched alkyl group include isohexyl and 2-ethylhexyl.

  Examples of the sulfosuccinic acid type activator (S) include sulfosuccinic acid dicyclohexyl sodium salt, sulfosuccinic acid di-2-ethylhexyl sodium salt, sulfosuccinic acid di-2-ethylhexyl ammonium salt, and sulfosuccinic acid dioctyl ester sodium salt. Of these, from the viewpoint of saccharification rate, di-2-ethylhexyl sulfosuccinate sodium salt and di-2-ethylhexyl ammonium sulfosuccinate are preferable.

  The sulfosuccinic acid type activator (S) can be obtained by a known method (Japanese Patent Laid-Open No. 08-337567). In addition, the sulfosuccinic acid type activator (S) can be easily obtained from the market, and as a trade name, for example, “Viewlight ESS, LSS, SSS, A-5000” (manufactured by Sanyo Chemical Industries, Ltd., “Viewlight”). Is a registered trademark of Sanyo Kasei Kogyo Co., Ltd.), “Rika Surf Series” (manufactured by Nippon Nippon Chemical Co., Ltd., “Rika Surf” is a registered trademark of Shin Nippon Rika Co., Ltd.), “Perex Series” (Kao "Perex" is a registered trademark of Kao Corporation.), "Koha Cool Series" (manufactured by Toho Chemical Industry Co., Ltd., "Koha Cool" is a registered trademark of Toho Chemical Industry Co., Ltd.), " "Rapidol A Series" (manufactured by NOF Corporation, "RAPISOL" is a registered trademark of NOF Corporation) and "Air Roll" (manufactured by Toho Chemical Industry Co., Ltd., "Air Roll" Is a registered trademark of Toho Chemical Industry Co., Ltd.), And the like.

In the saccharification treatment step (2), the ratio (% by weight) of the anionic active agent is preferably 0.05 to 10 and particularly preferably 0.1 to 5 with respect to the dry weight of the woody biomass powder. Within this range, the saccharification rate is good, which is preferable.
The dry weight of the woody biomass powder is JIS K0067-1992 Chemical product weight loss and residue test method 4.1 Drying weight loss test 4.1.4 (2) “Method 3 Method of heat drying under reduced pressure” , And the weight after drying at a drying temperature of 50 ± 3 ° C. and a pressure of 1 to 10 kPa.

As the hydrolase, any enzyme capable of hydrolyzing cellulose can be used without limitation.
Examples of cellulose include polysaccharides containing a β-1,4-glucoside bond, and cellulose and hemicellulose (such as xylan) are included.

  Enzymes include cellulose-degrading enzymes and xylan-degrading enzymes (for example, “Cellulase”, Kodansha Co., Ltd., Murao Sawao, published in 1987, pp. 16-18) and the like. Of these, cellulose-degrading enzymes are preferred, and cellulase (EC number 3.2.1.4), exo-1,4-β-D-glucosidase (EC number 3.2.74) and exo are more preferred. It is a complex enzyme consisting of at least two selected from the group consisting of cellobiohydrase (EC number 2.2.1.91). Cellulose-degrading enzymes include enzymes derived from animals (snail, termites, etc.), plants, microorganisms (eg, “Cellulase” (publisher Kodansha Co., Ltd., copyright Murao Sawao, published in 1987, page 24, etc.)). Commercially available cellulase preparations can be used, such as “Cellulase“ Onozuka ”3S, Cellulase Y-NC etc.” (manufactured by Yakult Yakuhin Co., Ltd.), “Sucrase C etc.” (manufactured by Mitsubishi Chemical Foods Corporation) "Cellulase A" Amano "3, Cellulase T" Amano "4 etc." (Amano Enzyme Co., Ltd.), "Mecerase" (Meiji Pharmaceutical Co., Ltd.), "Cellulosin AC40, Cellulosin AL, Cellulosin T3, etc." Company-made), "Entilon CM, MCH, Bio-Hit, Bio-Star, etc." Company) and “Phenzyme, Viscozyme, etc.” (manufactured by Novozymes Japan), “Spezyme CP, GC220, Multifect CX, Prima First, Accel Race, Husband Petitmash, Optimase CX, Puratax HA, etc.” (Genencor Kyowa Co., Ltd.).

There is no restriction | limiting in particular as usage-amount of an enzyme, Although it can select suitably according to the objective, About 0.1-30FPU is preferable with respect to 1g of dry weights of woody biomass powder, and 3-15FPU is especially preferable. .
The dry weight of the woody biomass powder is measured in the same manner as described above. FPU is a standard enzyme unit and means an enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute from filter paper (Pure & Appl. Chem., Vol. 59, No. 2, pp. 257-268, 1987). .).

  Although there is no restriction | limiting in particular as a hydrolysis temperature in a saccharification process process, 10-60 degreeC is preferable, More preferably, it is 20-55 degreeC. There is no restriction | limiting in particular as pH of a hydrolysis, 3-7 are preferable, More preferably, it is 4-6.

  In the saccharification treatment step, a known buffer solution may be included in addition to the anionic active agent, hydrolase, and woody biomass powder. Examples of the buffer solution include an acetate buffer solution, a phosphate buffer solution, a carbonate buffer solution, and a citrate buffer solution.

  The saccharification treatment step is preferably performed in a reaction solvent. As the reaction solvent, water and an organic solvent can be used. As water, tap water, industrial water, distilled water, ion exchange water, distilled water, ground water, seawater, hot spring water, etc. can be used. Of these, one type may be used, or two or more types may be mixed and used. Among these, purified water such as distilled water and ion exchange water is preferable from the viewpoint of hydrolyzability and the like.

  Organic solvents include hydrocarbon solvents (benzene, toluene, hexane, etc.), ether solvents (ethyl ether, isopropyl ether, etc.), chlorine solvents (carbon chloride, methylene chloride, etc.), ester solvents (methyl acetate, ethyl acetate, etc.), Examples include alcohols (methanol, ethanol, isobutanol, etc.), ketone solvents (acetone, methyl ethyl ketone, etc.), dioxane, tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF), pyridine, and the like. Among organic solvents, alcohol, ketone solvent, dioxane, tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF), and pyridine.

  The saccharification treatment step can be performed by mixing woody biomass powder, hydrolase and an anionic active agent. Woody biomass powder can be used as a solid and dispersion. The enzyme may be used in an enzyme solution or an enzyme dispersion, and the anionic active agent may be used in a solution, a solid or a dispersion.

  In the saccharification treatment step, the order of addition of the woody biomass powder, the enzyme, and the anionic active agent is not limited, and can be performed by the following method, for example.

(Reaction method 1) A method of uniformly mixing a woody biomass powder and an anionic active agent, adding an enzyme, and subsequently performing a hydrolysis reaction under predetermined conditions.

(Reaction method 2) A method in which an anionic active agent and an enzyme are uniformly mixed in a reaction solvent, and then a woody biomass powder is added, followed by a hydrolysis reaction under predetermined conditions.

(Reaction method 3) A method in which a woody biomass powder, an enzyme, and an anionic active agent are added to a reaction solvent at a time, and then uniformly mixed, followed by a hydrolysis reaction under predetermined conditions.

  Of these, (Reaction Method 1) or (Reaction Method 3) is preferable, and (Reaction Method 1) is particularly preferable.

  In (Reaction Method 1), when mixing the woody biomass powder and the anionic active agent, it is preferable to uniformly mix the woody biomass powder and the anionic active agent in the following pretreatment step because the saccharification rate is further improved. This is considered to be because the penetration of the anionic active agent into the woody biomass powder proceeds, and the enzyme easily hydrolyzes the inside of the woody biomass powder.

  In the enzyme saccharification method of the present invention, a pretreatment step of woody biomass powder can be performed after the pulverization step. As a pretreatment process, for example, a method of mixing with a peroxide in an aqueous alkali solution in addition to a method such as steaming, explosion treatment, hydrothermal treatment, acid treatment (sulfuric acid treatment, dilute sulfuric acid treatment, etc.) or alkali treatment is used. be able to.

  In the enzymatic saccharification method of the present invention, a known lignocellulosic biomass can be used in the saccharification treatment step in addition to the woody biomass powder. For example, herbaceous biomass (such as crop residues (stems and leaves) of rice, wheat, corn, etc.) is used. be able to.

(Production of ethanol)
In the method for producing fermented ethanol of the present invention, a sugar solution obtained in the saccharification treatment step in the presence of an anionic active agent is obtained by a known method (for example, “Bioenergy Technology and Application Dissemination Version” (Publisher Co., Ltd.). Ethanol fermentation at CMC Publishing, published on April 22, 2009, pages 171 to 173, etc.)).

  The sugar solution includes a primary sugar solution and a secondary sugar solution, and ethanol fermentation may be performed separately for each of the primary sugar solution and the secondary sugar solution, or may be performed by mixing the two. .

The primary sugar solution is a sugar derived from hemicellulose and contains pentose sugars such as xylose and arabinose and hexose sugars such as glucose, galactose and mannose.
Among these, pentasaccharides, for example, introduce pentose metabolism genes into E. coli that does not produce ethanol, genetically modified Escherichia coli in which a gene derived from a microorganism that produces ethanol is introduced, or ethanol-fermenting Zymomonas bacteria. And a method for recovering ethanol and carbon dioxide by subjecting a pentose sugar to ethanol fermentation (for example, JP-T-5-502366 and JP-T-6-504436) Fermentation ethanol can be manufactured by well-known methods, such as (Unexamined-Japanese-Patent No. 2006-111593).
The hexose can produce fermented ethanol by a known method such as a method of fermenting with yeast or a method of using a bacterium having a gene necessary for ethanol production by gene recombination.

  The secondary sugar solution contains hexoses such as cellulase-derived glucose, galactose, and mannose, and can produce fermented ethanol in the same manner as the aforementioned hexasaccharides.

  Ethanol fermentation may be performed continuously after the saccharification treatment step of the method for saccharification of woody biomass, or may be performed simultaneously with the saccharification treatment step. Moreover, conditions, such as fermentation temperature of ethanol fermentation, pH, can be suitably set according to the kind of saccharide | sugar used as a raw material, the yeast to be used, or the kind of fermentation microbe.

  The fermented liquor is subjected to a separation process for separating the produced ethanol. As a method for separating ethanol, known methods such as a distillation method and a pervaporation membrane method can be used. The ethanol obtained by separation may be used as it is, or may be used after purification by a known method such as distillation.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this. Unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.

The weight average molecular weights of the polymer type activators (A11 to A14, A21 to A24, A31, and A32) produced in Production Examples 1 to 10 were measured using the following apparatus.
(Weight average molecular weight)
Model: Waters LCM1
Detector: Waters 410 Differential refraction detector analysis software: Waters Millenium Ver. 2.18
Dissolved solution: 0.1-MPB disodium hydrogen phosphate aqueous solution: 0.1-MPB sodium dihydrogen phosphate aqueous solution = 1: 1 (molar ratio)
Eluent flow rate: 0.8ml / min
Column temperature: 40 ° C
Sample concentration: 0.4% eluent solution column: TSKgel GuardColumn SWXL + G4000SWXL + G3000SWXL + G2000SWXL manufactured by Tosoh Corporation
Standard substance: Polyethylene glycol, weight average molecular weight (Mw) 272500, 219300, 85000, 46000, 24000, 12600, 4250, 7100, 1470

  In Production Examples 1 to 12, the dry weight was measured according to JIS K0067-1992, the weight loss and residue test method of chemical products 4.1 Drying weight loss test, 4.1.4 (1) “First method, heating under atmospheric pressure The drying was carried out at a drying temperature of 130 ± 3 ° C. according to “Method of drying”.

<Production Example 1>
500 parts of ion-exchanged water, 200 parts of isopropyl alcohol and 3 parts of 2-mercaptoethanol were put into a pressure-resistant reaction vessel equipped with a dropping line, a stirrer and a thermometer, and heated to 80 to 100 ° C. with stirring in a sealed state. Subsequently, while stirring at 80 to 100 ° C., 50 parts of acrylic acid and 1000 parts of acrylic acid methoxypolyoxyethylene (oxyethylene: 2 mol) ester [Blemmer AE-90, manufactured by NOF Corporation] The mixed monomer and 100 parts of 40% sodium persulfate aqueous solution were dropped from separate drop lines at a constant rate over 2 hours. After completion of the dropwise addition, the mixture was kept at 95 to 100 ° C. for 3 hours in a sealed state. Subsequently, isopropyl alcohol was distilled off while gradually releasing pressure while maintaining 95 to 100 ° C. After the distillation of isopropyl alcohol ceased, 150 parts of ion-exchanged water was added dropwise while maintaining 95-100 ° C. Subsequently, isopropyl alcohol was distilled off under reduced pressure while maintaining 95-100 ° C. The mixture was cooled to 30 ° C. after the isopropyl alcohol was not distilled off. The total weight of distilled isopropyl alcohol was 200 parts. Subsequently, 0.1 part of a 48% aqueous sodium hydroxide solution (special grade reagent, manufactured by Kanto Chemical Co., Inc.) was gradually added dropwise with stirring so as not to reach 40 ° C or higher. Subsequently, ion-exchanged water was added to adjust the concentration so that the dry weight was 40%, and the polymer type activator (A21) [acrylic acid (10.8 mol%-sodium acrylate salt (0.02 mol) %)-Acrylic acid methoxypolyoxyethylene (oxyethylene; 2 mol) ester (89.2 mol%) copolymer], which had a weight average molecular weight of 4,000.

<Production Example 2>
“200 parts of isopropyl alcohol, 3 parts of 2-mercaptoethanol, 50 parts of acrylic acid and 1000 parts of methoxypolyoxyethylene acrylate (oxyethylene: 2 mol) ester and 0.6 part of 48% aqueous sodium hydroxide” Alcohol 0 part, 2-mercaptoethanol 1 part, methacrylic acid 50 parts, methacrylic acid methoxypolyoxyethylene (oxyethylene: 90 mol) ester [Blenmer PME-4000, manufactured by NOF Corporation] 583 parts and 28% aqueous ammonia solution [Reagent special grade, manufactured by Nacalai Tesque Co., Ltd.] 0.5 parts "In the same manner as in Production Example 1, the polymer type activator (A22) [methacrylic acid (81.9 mol%)-methacrylic Ammonium acid (1 mol%)-Methoxypolyoxyethylene methacrylate ( Kishiechiren: to obtain a 90 mol) ester (17.2%) copolymer. The weight average molecular weight of this copolymer was 20,000.

<Production Example 3>
“200 parts of isopropyl alcohol, 3 parts of 2-mercaptoethanol, 50 parts of acrylic acid and 1000 parts of methoxypolyoxyethylene acrylate (oxyethylene: 2 mol) ester and 0.6 part of 48% aqueous sodium hydroxide” Other than changing to 0 parts alcohol, 0.1 parts 2-mercaptoethanol, 50 parts acrylic acid, 52 parts methoxypolyoxyethylene acrylate (oxyethylene: 2 mol) ester and 49 parts 48% sodium hydroxide aqueous solution, In the same manner as in Production Example 1, polymer type activator (A23) [acrylic acid (10.7 mol%)-acrylic acid sodium salt (59.2 mol%)-methoxypolyoxyethylene acrylate (oxyethylene: 2) Mol) ester (30.1 mol%)]. The weight average molecular weight of this copolymer was 100,000.

<Production Example 4>
“200 parts of isopropyl alcohol, 3 parts of 2-mercaptoethanol, 50 parts of acrylic acid and 1000 parts of methoxypolyoxyethylene acrylate (oxyethylene: 2 mol) ester and 0.6 part of 48% aqueous sodium hydroxide” 0 parts of alcohol, 1.5 parts of 2-mercaptoethanol, 50 parts of methacrylic acid, methoxypolyoxyethylene methacrylate (oxyethylene: 23 mol) ester [Blenmer PME-1000, manufactured by NOF Corporation] 380 parts and 28% Except for changing to 8.7 parts of aqueous ammonia solution, the polymer type activator (A24) [methacrylic acid (15.3 mol%)-methacrylic acid sodium salt (5 mol%)- Methoxypolyoxyethylene methacrylate (oxyethylene: 23 mol) Este Was obtained (79.6 mol%)]. The weight average molecular weight of this copolymer was 30,000.

<Production Example 5>
4 parts of ion exchange water, 50 parts of maleic anhydride and 36.1 parts of ethylene glycol monobutyl ether are charged into a reaction vessel equipped with a dropping line, a stirrer and a thermometer, and stirred at 70 to 90 ° C. for 3 hours while stirring in a sealed state. After heating, 100 parts of methyl ethyl ketone was added under stirring. Subsequently, 62.6 parts of styrene and 100 parts of 40% sodium persulfate aqueous solution were dropped from separate drop lines at a constant rate over 2 hours while maintaining the temperature of 70 to 80 ° C. After completion of dropping, the mixture was heated to 80 to 100 ° C. in a sealed state, and this temperature was maintained for 3 hours. Subsequently, methyl ethyl ketone was distilled off while gradually releasing pressure while maintaining 80 to 100 ° C., and at the same time, 100 parts of ion-exchanged water was added from the dropping line (the ion-dropping water dropping rate was not controlled). After the distillation of methyl ethyl ketone was stopped, the mixture was cooled to 30 ° C. The total weight of the distilled methyl ethyl ketone was 100 parts. Subsequently, 25.5 parts of a 28% aqueous ammonia solution was gradually added dropwise with stirring so as not to exceed 40 ° C. Subsequently, ion-exchanged water was added to adjust the concentration so that the dry weight was 40%, and the polymer type activator (A11) [maleic acid ammonium salt (18 mol%)-maleic acid monobutoxyethylene glycol ester Ammonium salt (28 mol%)-styrene (54 mol%)] was obtained. The copolymer had a weight average molecular weight of 10,000.

<Production Example 6>
Example 5 and Example 5 except that “36.1 parts of ethylene glycol monobutyl ether and 25.5 parts of 28% aqueous ammonia” were changed to “24.8 parts of ethylene glycol monobutyl ether and 52.7 parts of 28% aqueous ammonia”. Similarly, polymeric activator (A12) [maleic acid ammonium salt (32 mol%)-maleic acid monobutoxydiethylene glycol ester ammonium salt (14 mol%)-styrene (54 mol%)] was obtained. The weight average molecular weight of this copolymer was 11,000.

<Production Example 7>
125 parts of polymer type activator (A12) obtained in Production Example 6 and sulfosuccinic acid type activator (S3) {sulfosuccinic acid di-2-ethylhexyl sodium salt 71% water / isopropyl alcohol (weight ratio 18/11) mixed solution , Trade name Rikasurf P-10, manufactured by Shin Nippon Rika Co., Ltd.} 50 parts was uniformly stirred and mixed at 15 to 30 ° C. for 1 hour to obtain an anionic active agent mixture (A41).

<Production Example 8>
175 parts of polymer type activator (A12) obtained in Production Example 6 and poly (n = 23) ethylene glycol monostearate (trade name: IONET MS-1000, manufactured by Sanyo Chemical Industries, Ltd., "IONET" 30 parts of the mixture was uniformly stirred and mixed at 15 to 30 ° C. for 1 hour to obtain an anionic active agent mixture (A42).

<Production Example 9>
50 parts of a sulfosuccinic acid type activator (S3) and 70 parts of poly (n = 20) ethylene glycol sorbitan cocoate (trade name Ionette T-20C, manufactured by Sanyo Chemical Industries, Ltd.) at 15-30 ° C. for 1 hour The mixture was stirred and mixed to obtain an anionic active agent mixture (A43).

<Evaluation of enzymatic saccharification method for woody biomass>
Woody biomass is a natural product, and the evaluation data may vary greatly due to variations in its composition and the like.
Therefore, a saccharification test was performed using a reagent (crystalline cellulose, fine powder, trade name: Cellulose, Microcrystalline, manufactured by Alfa Aesar) as the woody biomass powder. Cellulose biomass contained in woody biomass is strongly hydrogen-bonded to form crystalline cellulose and is not easily hydrolyzed (not easily enzymatically saccharified). If obtained, it is expected that good results will be obtained even in woody biomass.

  As the anionic active agent used in the saccharification test, in addition to the anionic active agents [A11, A12, A21 to A24, A41 to A43) obtained in Production Examples 1 to 9, the following polymeric active agents ( A31, A32) and sulfosuccinic acid type activators (S1-S4) were used.

  Polymer type activator (A31): Trade name Marialim AKM-0531 {maleic anhydride (50 mol%)-methoxypoly (n = 9) ethylene glycol allyl ether (50 mol%) copolymer [viscosity of 99% dry weight Liquid preparation, weight average molecular weight 18,000, manufactured by NOF Corporation, “Mariarim” is a registered trademark of the same company. }

  Polymer type activator (A32): trade name Marialim AKM-1511-60 {maleic anhydride (50 mol%)-methoxypoly (n = 33) ethylene glycol allyl ether (50 mol%) copolymer aqueous solution [dry weight 60 %, Weight average molecular weight 16,000, NOF Corporation}

  Sulfosuccinic acid type activator (S1): Trade name Aerol CT-1P {Sulfosuccinic acid dioctyl sodium salt 70% water / isopropyl alcohol (weight ratio 17/13) mixed solution, manufactured by Toho Chemical Co., Ltd.}

  Sulfosuccinic acid type activator (S2): Trade name Perex CS {Sulfosuccinic acid dicyclohexyl sodium salt 45% aqueous solution, manufactured by Kao Corporation}

  Sulfosuccinic acid type activator (S3): trade name Rikasurf P-10 {sulfosuccinic acid di-2-ethylhexyl sodium salt 71% water / isopropyl alcohol (weight ratio 18/11) mixed solution, manufactured by Shin Nippon Rika Co., Ltd.}

  Sulfosuccinic acid type activator (S4): trade name Rikasurf G-600 {sulfosuccinic acid di-2-ethylhexylammonium salt 60% water / propylene glycol (weight ratio 16/24) mixed solution, manufactured by Shin Nippon Rika Co., Ltd.}

<Saccharification test of crystalline cellulose>
In a glass container with a capacity of 200 mL, 1 g of crystalline cellulose (reagent), 100 mL of 200 mmol / L citrate buffer (pH 4.8) adjusted to pH 4.8 and the anionic activator (A11, A12, A21 to A24, A31, A32, S1 to S4, A41 to A43) or a comparative additive (any of H1 to H3) was added and mixed uniformly for 60 minutes. Subsequently, cellulase preparation [trade name: Accelerase 1000, manufactured by Genencor Kyowa Co., Ltd.] 15 FPU was added and sealed, and left in a constant temperature bath at 40 ° C. for 144 hours.
Subsequently, the filtrate obtained by taking out from the thermostatic bath and solid-liquid separation with a glass filter was introduced into a liquid chromatograph, and the monosaccharide concentration in the filtrate was quantified. The saccharification rate (%) per cellulose content was calculated from the quantified monosaccharide concentration and listed in Table 1.

Note that the sugar concentration was measured by liquid chromatography using the following apparatus.
Model: Waters Alliance 2695
Detector: RID
Analysis software: Waters Empower2
Eluent: water eluent flow rate: 0.6 mL / min
Column temperature: 80 ° C
Injection volume: 20 μL
Column: Bio-Rad Aminex HPX-87P (300 x 7.8 mm)

  In Table 1, the number in () is the amount of anionic active agent or additive added (g), and the number in <> is the concentration of the anionic active agent or additive added to crystalline cellulose (%). H1, H2, and H3 are the following comparative additives.

Additive for comparison 1 (H1): Mixture of 100 parts of wheat bran and 6 parts of polyoxyethylene sorbitan monolaurate [trade name: Tween 20, manufactured by Kanto Chemical Co., Ltd.] Additive for comparison 2 (H2): Skim milk [Morinaga Made by Dairy Co., Ltd.]
Additive for comparison 3 (H3): polyoxyethylene sorbitan monolaurate [trade name: Tween 20, manufactured by Kanto Chemical Co., Inc.]

  The enzymatic saccharification method (Examples 1 to 15) of the present invention is compared with the enzymatic saccharification method (Comparative Examples 1 to 3) using an additive for comparison and the enzymatic saccharification method (blank 1) containing no additive. Was significantly larger. That is, the enzymatic saccharification method of the present invention in which hydrolysis is performed in the presence of an anionic active agent and a hydrolase improves the reaction efficiency between crystalline cellulose and hydrolase, and the crystalline cellulose has an excellent saccharification rate. It was confirmed that saccharification was possible. From the above results with the reagent (crystalline cellulose), it is predicted that saccharification of woody biomass can naturally be performed with an excellent saccharification rate.

  Using a sulfosuccinic acid type activator (S3) as an anionic activator, a saccharification test of woody biomass and production of fermented ethanol were carried out by the following methods. Similarly, a saccharification test of woody biomass and production of fermented ethanol were conducted using a comparative additive (H3) instead of the sulfosuccinic acid type activator (S3).

<Powdering process (production of woody biomass powder)>
After pulverizing 3 g of building waste material roughly crushed to a size of 100 to 120 mm with an impact pulverizer (high-speed vibration sample pulverizer TI-100, manufactured by CMT Corporation) for 2 minutes, the opening size is 1 mm. Screening was performed using a stainless steel wire mesh, and the materials that passed through the screen were collected to obtain a crushed powder having a circumscribed circle equivalent diameter of 0.8 mm. The pulverized powder was dried under reduced pressure at a drying temperature of 50 ± 3 ° C. and a pressure of 1 to 10 kPa. As a result, the residual heating amount of the pulverized powder was 15%.

<Pretreatment of pulverized powder>
In a 100 mL glass pressure vessel, crushed powder with a dry weight of 20 g and 200 g of a 0.25M sodium hydroxide aqueous solution were placed, heated at 100 ° C. for 1 hour, and then allowed to cool to room temperature (20-30) ° C. Subsequently, a 30% aqueous hydrogen peroxide solution was added and reacted for 24 hours, and then allowed to cool to room temperature (20-30) ° C. Subsequently, after washing the filtration residue solid-liquid separated with a glass filter, water on the surface was removed with filter paper to obtain a woody biomass powder. The amount of cellulose contained in this woody biomass powder was 55%.

The amount of cellulose contained in the woody biomass powder was determined by the following method.
A 10 mL test tube was dried at a drying temperature of 50 ± 3 ° C. under reduced pressure at a pressure of 1 to 10 kPa. A dry weight of 0.3 g of woody biomass powder and 3% 72% sulfuric acid aqueous solution were added and stirred at 30 ° C. for 1 hour. Mixed. Subsequently, this mixture and 84 mL of ion-exchanged water were placed in a 100 mL glass pressure vessel and heat-treated at 121 ° C. for 1 hour. Then, it stood to cool to room temperature (20-30) degreeC, and solid-liquid separation by suction filtration was performed. Subsequently, the supernatant liquid neutralized to pH 7 with calcium carbonate was measured with a liquid chromatograph under the same conditions as described above, and the cellulose content was calculated from the measured sugar concentration.

<Saccharification process (saccharification of woody biomass)>
In a glass container with a capacity of 200 mL, dry biomass 5 g of woody biomass powder, 100 mL of 200 mmol / L citrate buffer (pH 4.8) adjusted to pH 4.8 and the amount of sulfosuccinic acid type activator (S3) described in Table 2 ) Or comparative additive (H3) was added and mixed uniformly for 60 minutes. Subsequently, cellulase preparation [trade name: Accelerase 1000, manufactured by Genencor Kyowa Co., Ltd.] 15 FPU was added and sealed, and left in a constant temperature bath at 40 ° C. for 144 hours.
Subsequently, the filtrate obtained by taking out from the thermostatic bath and solid-liquid separation with a glass filter was introduced into a liquid chromatograph, and the monosaccharide concentration in the filtrate was quantified. The saccharification rate (%) per cellulose content was calculated from the quantified monosaccharide concentration and the cellulose content contained in the woody biomass powder, and listed in Table 2.
The dry weight of the woody biomass powder is measured by drying under reduced pressure at a drying temperature of 50 ± 3 ° C. and a pressure of 1 to 10 kPa, and the sugar concentration is measured by a liquid chromatograph using the same apparatus as described above. It was.

<Manufacture of fermented ethanol>
Subsequently, production of fermented ethanol was carried out by the following method.
Into a 250 mL glass container containing a stirrer, 90 g of each sugar solution obtained by saccharification treatment of woody biomass powder, 5 g of nutrient salts, and 5 g of inoculation solution of yeast [trade name: Superstart, Alltech Co., Ltd.] In addition, ethanol fermentation was performed at 35 ° C. and 100 rpm for 50 hours. After completion of the fermentation, the fermented ethanol concentration (g / L) in the liquid was quantified by liquid chromatography under the following conditions and listed in Table 2.
As nutrients, a solution of corn steep liquor 5 g / L, urea 0.8 g / L, potassium dihydrogen phosphate 0.2 g / L was used, and the inoculum was cultured in YM medium for 8 hours. The body was adjusted with physiological saline so that the OD value (absorbance) was 1.

Quantification of the fermented ethanol concentration by liquid chromatography was performed under the following conditions.
Model: Waters Alliance 2695
Detector: RID
Analysis software: Waters Empower2
Eluent: 0.01 N Sulfuric acid eluent flow rate: 0.6 mL / min
Column temperature: 60 ° C
Injection volume: 20 μL
Column: Bio-Rad Aminex HPX-87P (300 x 7.8 mm)

  In Table 2, the numbers in <> represent the concentration of the anionic active agent or additive added to the woody biomass powder, and the numbers in () and H3 are the same as in Table 1.

  The enzymatic saccharification method (Example 16) of the present invention had a significantly higher saccharification rate than the enzymatic saccharification method (Comparative Example 4) using a comparative additive. That is, it has been confirmed that the enzymatic saccharification method of the present invention improves the reaction efficiency between cellulose and hydrolase contained in woody biomass, and can saccharify cellulose contained in woody biomass with an excellent saccharification rate. Moreover, the manufacturing method (Example 16) of the fermented ethanol of this invention has a large fermented ethanol concentration compared with the manufacturing method of the fermented ethanol of the comparative example (Comparative Example 4), and fermented ethanol using wood and building waste materials as raw materials ( It was confirmed that it was useful for the production of bioethanol.

  The method for saccharification of woody biomass of the present invention can efficiently saccharify cellulose contained in woody biomass, and is useful for producing fermented ethanol (bioethanol) using wood and building waste materials as raw materials. Fermented ethanol is attracting attention as a clean energy source.

Claims (2)

A pulverizing step (1) for pulverizing at least one woody biomass selected from wood chips and waste building materials to obtain a woody biomass powder; and a polymeric activator (1), a polymeric activator (2), In the presence of a hydrolase and at least one anionic active agent selected from the group consisting of a polymeric activator (3) and a sulfosuccinic acid activator (S) represented by the general formula (1) A method for enzymatic saccharification of woody biomass, comprising a saccharification treatment step (2) for obtaining a sugar by hydrolyzing woody biomass powder.

However, the polymeric activator (1) is a monomer represented by R ¹ OCOCH═CHCOOR ² {R ¹ and R ² are a hydrogen atom (H), ammonium (NH ₄ ), sodium atom (Na) or Formula: R ³ O— (A ¹ O) n ¹ —O— (R ³ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, A ¹ O is an oxyalkylene group having 2 to 4 carbon atoms, and n ¹ is 1. Or at least one of R ¹ and R ² is represented by the formula: R ³ O— (A ¹ O) n ¹ —O—. }, Maleic acid (salt) {in the case of salt, ammonium salt and / or sodium salt} and styrene as essential constituent monomers,
The polymer type activator (2) is composed of (meth) acrylic acid ester of ethylene oxide and / or 2-90 mol adduct of alkanol having 1 to 4 carbon atoms and (meth) acrylic acid (salt) {salt of The ammonium salt or sodium salt} as an essential constituent monomer,
The polymer type activator (3) comprises (meth) propenylene ether of 9 to 33 mol adduct of alkanol having 1 to 4 carbon atoms and / or propylene oxide and maleic anhydride as essential constituent monomers. Become.

R ⁴ and R ⁵ are each an alkyl group having 6 to 8 carbon atoms, M ¹ is an alkali metal atom or ammonium (NH ₄ ), C is a carbon atom, H is a hydrogen atom, O is an oxygen atom, and S is a sulfur atom. . Fermented ethanol obtained by fermenting sugar to obtain fermented ethanol after saccharification treatment step (2) of the enzymatic saccharification method for woody biomass according to claim 1 or simultaneously with this saccharification treatment step (2) Manufacturing method.