JP2014195417A - Method for preparing raw material for enzymatic saccharification, sugar producing method and ethanol producing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing raw material for enzymatic saccharification, which can impart to vegetable biomass sufficient reactivity for enzymatic saccharification and can sufficiently decrease the usage of ammonia.SOLUTION: First, liquid ammonia or ammonia including liquid ammonia is fed to a reaction vessel storing lignocellulose-containing vegetable biomass at a temperature of from -40°C to 80°C, then the temperature of the reaction vessel is raised to from 60°C to 135°C and to a temperature higher than the reaction vessel temperature when starting the feed, for treatment of the biomass with ammonia including gaseous and liquid phase ammonia (ester bond cleavage). After that, the temperature in the reaction vessel is cooled down to from -40°C to 40°C, and additional liquid ammonia or ammonia including liquid ammonia is introduced into the vessel in such manner that mass of liquid phase ammonia/dry mass of the vegetable biomass becomes 0.1-1.1, to treat the biomass with ammonia including liquid phase ammonia at a temperature from -40°C to 40°C (crystalline transition).

Description

  The present invention relates to a method for producing a raw material for enzymatic saccharification used when producing sugar from plant biomass containing lignocellulose, a method for producing sugar from the raw material, and a method for producing ethanol from the sugar.

  In recent years, as part of global warming countermeasures, ethanol has been produced from plant biomass containing lignocellulose (a complex composed of cellulose, hemicellulose, and lignin) such as woody biomass and herbaceous biomass, and will be used as various fuels and chemical raw materials. Attempts to do so are widely made. The production of ethanol from a plant biomass containing lignocellulose (hereinafter sometimes simply referred to as “plant biomass”) is, for example, a method in which the collected plant biomass is decomposed into monosaccharides in the saccharification step and then the sugar obtained. Can be converted to ethanol by fermentation using a microorganism such as yeast.

  In addition, not only ethanol fermentation but also fermentation using various microorganisms can be used to produce various chemical raw materials such as organic acids. As an example, lactic acid used as a raw material for biodegradable polymers can be produced by subjecting the sugar to lactic acid fermentation.

  Conventionally, saccharification of plant biomass has often been performed using concentrated sulfuric acid, but it is desired to reduce the amount of sulfuric acid used from the viewpoint of reducing environmental burden. Therefore, in recent years, saccharification using enzymes has been widely studied as a means to replace saccharification with concentrated sulfuric acid. Enzymatic saccharification is a desirable means from the viewpoint of environmental impact. For this enzymatic saccharification, for the purpose of facilitating the action of the enzyme on cellulose, plant biomass is pretreated and lignocellulose is used. It is necessary to separate cellulose and lignin in it.

  Conventionally, as a pretreatment method for plant biomass, steaming treatment with dilute sulfuric acid, pressurized hot water, or the like is generally used (for example, see Patent Documents 1 to 4 below). However, as described above, the use of sulfuric acid is not preferable from the viewpoint of reducing the environmental load.

  On the other hand, it is known that treatment of plant biomass with ammonia improves its chemical reactivity or biochemical reactivity (see, for example, Patent Documents 5 and 6 and Non-Patent Documents 1 and 2 below). ).

  In lignocellulose in plant biomass, lignin and hemicellulose are bonded by a covalent bond (such as an ester bond), and hemicellulose and cellulose interact through a hydrogen bond, thereby strengthening cellulose and lignin. It is said that this lignin inhibits the action of the enzyme on cellulose. And, one of the mechanism of action that improves the efficiency of enzymatic saccharification by treating plant biomass with ammonia is that at least a part of lignin is caused by amidation cleavage of the ester bond between the lignin and hemicellulose by ammonia. It is based on being separated from cellulose. Hereinafter, the action of improving the efficiency of enzymatic saccharification based on the separation of lignin from cellulose by cleavage of the ester bond between lignin and hemicellulose may be referred to as “chemical action”. The amidation cleavage reaction of the ester bond is more likely to proceed as the treatment temperature for ammonia treatment is higher.

  In addition, when lignocellulose is treated with ammonia, it is also known that the enzymatic saccharification efficiency is improved by transfer of the crystal form of cellulose by ammonia apart from the separation of lignin and cellulose. That is, cellulose constituting plant biomass exists mainly as cellulose type I crystals, but when the cellulose type I crystals come into contact with ammonia, it has a lower crystal density than cellulose type I crystals, and acts as an enzyme. It is known to transfer to cellulose III type crystals that are more susceptible (see, for example, Patent Document 7 below). Hereinafter, the effect of improving the efficiency of enzymatic saccharification based on cellulose crystal type transfer may be referred to as “physical effect”.

  It is known that the physical action is manifested when cellulose type I crystals come into contact with ammonia in a liquid phase or a supercritical state. On the other hand, ammonia in the gas phase has no influence on cellulose from the structural viewpoint (see Non-Patent Document 1). This is thought to be because cellulose I-type crystals cannot be transferred to cellulose III-type crystals because gas-phase ammonia cannot form a cellulose-ammonia intermediate with cellulose (Non-Patent Document 2). reference).

  Therefore, conventionally, in the ammonia treatment of the plant biomass, if both chemical action and physical action are to be used effectively at the same time, the plant biomass and ammonia are introduced into the high-pressure vessel to express the chemical action. In order to promote the cleavage reaction of the bond between lignin and hemicellulose, the inside of the high-pressure vessel is heated, and in order to promote the transition of the cellulose crystals to develop the physical action, ammonia is used in the high-pressure vessel. The treatment was carried out in the liquid phase, or the treatment was carried out in a supercritical state under the condition of higher temperature and pressure.

JP 2006-075007 A JP 2004-121055 A JP-T-2002-541355 JP 2002-159954 A U.S. Pat. No. 4,600,590 JP 2008-161125 A

Sendich E.M. Laser M .; Kim S .; Alizadeh H .; Laureano-Perez L. , Dale B. and Lynd L. Recent process impulses for the ammonia fiber expansion (AFEX) process and researching reduction in minimum ethanol selling price bioresource 84 A. J. et al. Barry, F.M. C. Peterson & A. J. et al. King, x-Ray Studies of Reactions of Cellulose in Non-Aqueous Systems Interaction of Cellulose and Liquid Ammonia, J.A. Am. Chem. Soc. , 58, 333 (1936)

  By the way, in the treatment of plant biomass with ammonia, ammonia is usually separated and recovered from the treated plant biomass and reused. In the process of separation / recovery and reuse of ammonia, an operation of vaporizing liquid phase ammonia or liquefying gas phase ammonia is required, including purification of ammonia. Here, since ammonia is a substance having a very large latent heat of vaporization (1262 kJ / kg (0 ° C., 101.3 kPaA)), the energy per unit amount of ammonia required for these operations is large, and ammonia per unit amount of plant biomass. When the amount used increases, the energy consumption required for the separation, recovery and reuse of the ammonia increases, and the economic rationality of the process is lost. Moreover, although it is recycled, the use of a large amount of ammonia itself causes an increase in cost. Therefore, in the production of a raw material for enzymatic saccharification from plant biomass including an ammonia treatment step, the production of sugar using the raw material, and the production of ethanol from the sugar, the reduction of the amount of ammonia used in the ammonia treatment is a commercialization of the process. It is extremely important for us.

  However, in the conventional production of enzyme saccharification raw material from plant biomass including an ammonia treatment step, production of sugar using the raw material, and production of ethanol from the sugar, Not enough consideration has been made.

  Furthermore, according to the study by the present inventors, it has been found that it is very difficult to reduce the amount of ammonia used until the economic rationality is satisfied in the conventional method.

  For example, in the treatment of plant biomass with liquid phase ammonia, ammonia is usually a gas-liquid mixed phase in a pressure vessel. At this time, if a predetermined amount of liquid phase ammonia is present in the pressure vessel, the higher the temperature in the vessel, the higher the pressure in the gas phase (saturated vapor pressure of ammonia at that temperature). As the vapor density increases, the amount of ammonia in the gas phase increases and the total amount of ammonia in the container increases. That is, the amount of ammonia required per unit amount of plant biomass to be treated increases.

  On the other hand, when the temperature in the pressure vessel is lowered, the amount of total ammonia required to bring the amount of liquid phase ammonia present in the vessel to the predetermined amount is reduced. It is difficult to develop an effective action. If a sufficient chemical action is to be expressed by this method, an operation unsuitable for commercial implementation is required in which treatment is performed for a very long time in a pressure vessel.

  The present invention has been made in view of the above circumstances, an enzyme capable of imparting sufficient reactivity for enzymatic saccharification to plant biomass and capable of sufficiently reducing the amount of ammonia used. It aims at providing the manufacturing method of the raw material for saccharification, the method of manufacturing saccharide | sugar efficiently using the said raw material, and the method of manufacturing ethanol from the said saccharide | sugar.

  As a result of intensive studies to achieve the above object, the present inventors first obtained the following knowledge.

  That is, the physical action in the treatment of plant biomass with ammonia can be efficiently expressed by bringing plant biomass into contact with liquid phase ammonia in a specific proportion of the amount of plant biomass. found. Further, it has been found that the physical action by the treatment using liquid phase ammonia is hardly affected by the treatment temperature and the treatment time and is sufficiently exhibited even at a low temperature and in a short time. On the other hand, it has been found that the chemical action in ammonia treatment appears even when ammonia is in the gas phase.

  And based on said knowledge, the present inventors tried to perform ammonia treatment in multiple times, aiming at the solution of the subject of reduction of ammonia usage-amount. As a result, surprisingly, by combining two specific ammonia treatments, plant biomass can be given sufficient reactivity for enzymatic saccharification, and the amount of ammonia used can be drastically reduced. I found that I can do it. That is, in the first stage ammonia treatment, treatment is performed with relatively high temperature ammonia to advance the ester bond cleavage reaction. In the second stage ammonia treatment, treatment with liquid ammonia or ammonia containing liquid ammonia is carried out at a relatively low temperature to advance the transition of the crystal form of cellulose.

  Furthermore, the present inventors have compared the case where the treatment is performed only with gaseous ammonia by performing the treatment in the first stage ammonia treatment under the condition that a small amount of liquid phase ammonia exists in addition to the gaseous phase ammonia. Thus, it has been found that the cleavage of the ester bond proceeds more efficiently.

  However, when supplying the liquid or the ammonia containing the liquid to the reaction tank (pressure vessel) containing the plant biomass is maintained at a high temperature exceeding 80 ° C. (especially the processing temperature, typically 120 ° C.). Since the vaporization of ammonia proceeds, it is necessary to supply a large amount of ammonia to make liquid phase ammonia exist in the container. Moreover, since the ammonia partial pressure rises, it is necessary to improve the pressure resistance performance of the reaction tank, leading to an increase in apparatus cost.

  Therefore, as a result of further studies, the present inventors kept the reaction vessel containing the plant biomass at a relatively low temperature (−40 to 80 ° C.), and supplied liquid ammonia or ammonia containing liquid ammonia thereto, After that, the temperature of the reaction vessel is raised to the treatment temperature, so that even when the same treatment temperature and the same amount of ammonia are supplied, the pressure is higher than that when liquid ammonia or ammonia containing liquid ammonia is supplied at the high temperature. The present inventors have found that the amount of liquid ammonia present in the container increases and the ammonia partial pressure decreases, and the present invention has been achieved.

That is, the present invention is a method for producing a raw material for enzyme saccharification, in which a plant biomass containing lignocellulose is treated with ammonia to obtain a raw material for enzyme saccharification,
Liquid ammonia or ammonia containing liquid ammonia is supplied to a reaction vessel containing plant biomass containing lignocellulose at a reaction vessel temperature of −40 ° C. to 80 ° C., and then within a range of 60 ° C. to 135 ° C. The temperature of the reaction vessel is increased to a treatment temperature that is higher than the reaction vessel temperature at the time of supply, and the plant biomass is treated with ammonia containing a gas phase and a liquid phase at the treatment temperature, and lignocellulose is obtained. A first step of cleaving at least part of the ester bond between lignin and hemicellulose constituting
After the first step, the inside of the reaction vessel is cooled to a reaction vessel temperature of −40 ° C. to 40 ° C., and the ratio of the mass of liquid ammonia to the dry mass of the plant biomass (mass of liquid ammonia / plant biomass Treatment of the plant biomass with ammonia containing liquid phase at −40 ° C. to 40 ° C. by adding liquid ammonia or ammonia containing liquid ammonia so that the dry mass) is 0.1 to 1.1 A second step of obtaining a raw material for enzyme saccharification by transferring at least a part of the crystal form of cellulose constituting lignocellulose from type I to type III, and
The manufacturing method of the raw material for enzyme saccharification provided with this is provided.

  In the first step, the reaction vessel is set so that the ratio of the mass of liquid ammonia present in the reaction vessel at the treatment temperature to the dry mass of the plant biomass is 0.05 to 0.25. It is preferable to supply liquid ammonia or ammonia containing liquid ammonia. By satisfying the above condition, it is possible to effectively achieve both efficient cleavage of the ester bond and reduction of the amount of ammonia used.

  Moreover, this invention provides the manufacturing method of saccharide | sugar provided with the process of saccharifying the raw material for enzyme saccharification obtained by the manufacturing method of the raw material for enzyme saccharification of the said invention with an enzyme.

  In the sugar production method of the present invention, by using the enzyme saccharification raw material with improved saccharification efficiency obtained by the method for producing enzyme saccharification raw material, sugar can be produced efficiently by enzymatic saccharification.

  Moreover, this invention provides the manufacturing method of ethanol provided with the process of fermenting the saccharide | sugar obtained by the manufacturing method of the said saccharide | sugar.

  In the method for producing ethanol of the present invention, ethanol can be efficiently produced from plant biomass as a starting material by using the sugar obtained by the method for producing sugar.

  According to the present invention, it is possible to solve conventional problems, and to provide a raw material for enzymatic saccharification in which the efficiency of enzymatic saccharification is sufficiently enhanced by effectively utilizing both chemical action and physical action. A method for producing a raw material for enzyme saccharification by ammonia treatment of plant biomass containing lignocellulose, which can reduce the amount of ammonia used than before and can further reduce the treatment time in a high-pressure vessel , A method for efficiently producing sugar using the raw material, and a method for producing ethanol from the sugar can be provided.

<First Embodiment: Method for Producing Enzymatic Saccharification Raw Material>
The method for producing an enzyme saccharification raw material according to the first embodiment of the present invention is a method for producing an enzyme saccharification raw material for obtaining a raw material for enzyme saccharification by treating plant biomass containing lignocellulose with ammonia,
Liquid ammonia or ammonia containing liquid ammonia is supplied to a reaction vessel containing plant biomass containing lignocellulose at a reaction vessel temperature of −40 ° C. to 80 ° C., and then within a range of 60 ° C. to 135 ° C. The temperature of the reaction vessel is increased to a treatment temperature that is higher than the reaction vessel temperature at the time of supply, and the plant biomass is treated with ammonia containing a gas phase and a liquid phase at the treatment temperature, and lignocellulose is obtained. A first step of cleaving at least part of the ester bond between lignin and hemicellulose constituting
After the first step, the inside of the reaction vessel is cooled to a reaction vessel temperature of −40 ° C. to 40 ° C., and the ratio of the mass of liquid ammonia to the dry mass of the plant biomass (mass of liquid ammonia / plant biomass Treatment of the plant biomass with ammonia containing liquid phase at −40 ° C. to 40 ° C. by adding liquid ammonia or ammonia containing liquid ammonia so that the dry mass) is 0.1 to 1.1 A second step of obtaining a raw material for enzyme saccharification by transferring at least a part of the crystal form of cellulose constituting lignocellulose from type I to type III, and
Is provided.

  Hereinafter, the manufacturing method of the raw material for enzyme saccharification concerning this embodiment is explained in full detail.

(Plant biomass)
The plant biomass used as a raw material in the method for producing a raw material for enzyme saccharification of the present embodiment is not particularly limited as long as it contains lignocellulose, and can be appropriately selected according to the purpose. For example, “waste biomass” obtained as a residue resulting from production activities such as agriculture and forestry, “resource crop biomass” obtained by intentionally cultivating for the purpose of obtaining energy and the like can be used. Examples of the “waste biomass” include waste building materials, thinned wood, rice straw, straw, rice husk, bagasse, etc., and the “resource crop biomass” is intended to use, for example, celluloses. Birch, eucalyptus, poplar, acacia, willow, cedar, switchgrass, napiergrass, Eliansus, Miscanthus, Susuki, Reed canarygrass and the like that are cultivated as Plant biomass is also classified into “woody biomass” derived from trees, “herbaceous biomass” derived from grass, and the like. In the present invention, both woody biomass and herbaceous biomass can be used. Plant biomass may be used individually by 1 type and may use 2 or more types together. In addition, the cellulose which comprises the lignocellulose contained in untreated plant biomass has taken the form of the cellulose I type crystal | crystallization fundamentally.

(Plant biomass process)
As plant biomass, collected ones may be used as they are, but from the viewpoint of ease of handling and ammonia treatment efficiency, it is necessary to use them after they are made into particles of a certain size by cutting or grinding. desirable. In this case, the size of the plant biomass particles after granulation is not particularly limited, and can be appropriately selected according to the ease of handling as particles. The following is preferable, and 3 mm or less is more preferable. If the mesh size of the mesh exceeds 5 mm, the efficiency of ammonia treatment described later may be reduced. On the other hand, pulverization as a unit operation is low in energy efficiency. If plant biomass is pulverized into fine particles, the energy required for pulverization increases and the economic rationality of the process is impaired. Accordingly, the amount of energy consumed for pulverization is preferably, for example, 1 MJ or less per kg of plant biomass dry mass, and the size of the particles may be selected according to the energy consumed for pulverization.

  There is no restriction | limiting in particular as an apparatus used for particle formation, According to the objective, it can select suitably, For example, a wheelie mill, a cutter mill, a hammer mill, a pin mill etc. can be used.

(Plant biomass drying process)
Collected plant biomass usually retains moisture. The plant biomass that retains moisture may be subjected to ammonia treatment as it is. However, in order to sufficiently improve the efficiency of enzyme saccharification of the obtained material for enzyme saccharification by effectively using physical action, plant biomass is used. It is preferable to use after drying.

  Presumed that when a certain amount or more of water is present in the system in which plant biomass is treated with ammonia, the production of cellulose-ammonia intermediates necessary for crystal type transition tends to be suppressed. Is done. Therefore, in the crystal-type transition step in the method for producing a raw material for enzyme saccharification according to this embodiment, when the amount of water in the system exceeds a predetermined level, the transition of cellulose I-type crystals to cellulose III-type crystals does not proceed sufficiently. . Therefore, in order to advance the transition of the cellulose crystal and to fully develop the physical action, it is preferable to reduce the water content in the system to a predetermined level or less during the crystal type transition in the second step. For this, it is preferable to dry the plant biomass and reduce its water content and use it for ammonia treatment.

In the second step, the amount of water contained in the plant biomass and ammonia preferably satisfies the following formula (1). For this purpose, when the plant biomass is subjected to the second step, it is preferable that the water content in the plant biomass satisfies at least the following formula (1).
Water mass / (dry weight of plant biomass + water mass) ≦ 0.30 (1)

Furthermore, in the second step, it is preferable that the amount of water contained in the plant biomass and ammonia satisfies the following formula (2). For that purpose, when subjecting the dried plant biomass to the second step, the water content in the plant biomass preferably satisfies at least the following formula (2).
Water mass / (dry mass of plant biomass + mass of water) ≦ 0.15 (2)

  The method for drying plant biomass is not particularly limited and may be appropriately selected depending on the intended purpose.However, plant biomass is destroyed or oxidized when dried at high temperatures in the atmosphere. Drying, natural drying, drying by ventilation at 100 ° C. or lower, drying under reduced pressure, drying using dimethyl ether, and the like are preferable.

  When plant biomass is granulated, drying may be before or after, but is preferably after granulation for handling.

  The plant biomass may be dried between the first step and the second step, but is preferably performed before the first step.

(First step)
In the first step, first, liquid ammonia or ammonia containing liquid ammonia is supplied to a reaction vessel in which plant biomass containing lignocellulose is accommodated at a reaction vessel temperature of −40 ° C. to 80 ° C. Next, the reaction vessel temperature is raised to a treatment temperature that is in the range of 60 ° C. to 135 ° C. and higher than the reaction vessel temperature at the time of supply. Then, the plant biomass is treated with ammonia containing a gas phase and a liquid phase at the treatment temperature to cleave at least a part of the ester bond between lignin and hemicellulose constituting the lignocellulose. As the lignin / hemicellulose bond, there is an ether bond in addition to the ester bond, but the cleavage of the ester bond in the first step may involve the cleavage of the ether bond.

  Here, in the first step of the present embodiment, liquid ammonia in a reaction vessel in which plant biomass containing lignocellulose is accommodated at a reaction vessel temperature of −40 ° C. to 80 ° C. (preferably outside air temperature to 60 ° C.). Alternatively, ammonia containing liquid ammonia is supplied, and then the reaction vessel temperature is within a range of 60 ° C. to 135 ° C. (preferably 80 ° C. to 130 ° C.) and is higher than the reaction vessel temperature at the time of supply. The configuration in which the temperature is raised is based on the inventors' original knowledge described later.

  That is, first, the cleavage of the ester bond does not necessarily require liquid-phase ammonia, and can proceed even with gas-phase ammonia that does not contain a liquid phase. By carrying out the treatment, the cleavage of the ester bond can proceed more efficiently as compared with the case where the treatment is carried out only with gaseous ammonia.

  However, in a state where the supply of liquid ammonia or ammonia containing liquid ammonia to the reaction tank (pressure vessel) containing the plant biomass is kept at a high temperature exceeding 80 ° C. (especially treatment temperature, typically 120 ° C.). When this is done, the vaporization of ammonia proceeds, so that a large amount of ammonia needs to be supplied in order for liquid phase ammonia to be present in the reaction vessel. Moreover, since the ammonia partial pressure rises, it is necessary to improve the pressure resistance performance of the reaction tank, leading to an increase in apparatus cost.

  Therefore, in the first step of the present embodiment, the reaction tank in which the plant biomass is accommodated is kept at a relatively low temperature (−40 to 80 ° C.), and liquid ammonia or ammonia containing liquid ammonia is supplied thereto, and then the treatment temperature is reached. A configuration in which the temperature of the reaction vessel is raised is adopted. By adopting such a configuration, the amount of liquid ammonia existing in the pressure vessel even at the same processing temperature and the same ammonia supply amount as compared with the case where liquid ammonia or ammonia containing liquid ammonia is supplied at the high temperature. Since the ammonia partial pressure decreases, it is possible to achieve both efficient cleavage of the ester bond and reduction of the amount of ammonia consumed.

  The supply amount of liquid ammonia or ammonia containing liquid ammonia in the first step is not particularly limited as long as the effects of the present invention are not impaired, but the mass of liquid ammonia present in the reaction vessel at the treatment temperature is not limited. The reaction vessel so that the ratio of the plant biomass to the dry mass (the mass of ammonia in the liquid phase / the dry mass of the plant biomass) is preferably 0.05 to 0.25, more preferably 0.06 to 0.15. It is preferable to supply liquid ammonia or ammonia containing liquid ammonia. By satisfying the above condition, it is possible to effectively achieve both efficient cleavage of the ester bond and reduction of the amount of ammonia used.

  Moreover, it is preferable that the quantity with respect to the plant biomass of the total ammonia in a 1st process is below the quantity with respect to the plant biomass of the total ammonia in a 2nd process. When the total amount of ammonia in the first step and the second step satisfies the above condition, the total amount of ammonia used in the first step and the second step can be reduced. Moreover, even if such conditions are selected, the cleavage of the ester bond can proceed and the chemical action can be sufficiently expressed.

  One preferable embodiment for carrying out the first step is to use a pressure vessel as a reaction tank and perform ammonia treatment in a relatively high ammonia partial pressure and a relatively high temperature for a relatively short time. According to this aspect, it is possible to reduce the capacity of the pressure vessel that increases the equipment cost and to increase the productivity.

  In the aspect of the first step using the pressure vessel, the ammonia partial pressure is preferably 0.5 MPaA to 4 MPaA, more preferably 1.0 MPaA to 2.0 MPaA. When the ammonia partial pressure is less than 0.5 MPaA, the ester bond cleavage tends not to proceed at a sufficient rate. On the other hand, when the ammonia partial pressure exceeds 4 MPaA, the amount of ammonia used tends to increase.

  In the embodiment of the first step using the pressure vessel, the treatment temperature is 60 to 135 ° C, preferably 80 to 135 ° C, and most preferably 100 to 135 ° C. When the treatment temperature is lower than 60 ° C., it takes a long time for the ester bond cleavage to proceed, and in an apparatus using a pressure vessel with a reduced capacity, the productivity tends to decrease. On the other hand, when the processing temperature exceeds 135 ° C., the supply amount of ammonia necessary to make liquid phase ammonia exist increases.

The amidation cleavage of the ester bond between lignin and hemicellulose in the ester bond cleavage step can be confirmed by FT-IR spectroscopy. More specifically, the absorption in the vicinity of 1662 cm ⁻¹ in the FT-IR spectrum is attributed to the amide group generated in the plant biomass after the ester bond cleavage step, and can be used as an indicator of the amidation cleavage of the ester bond. . Furthermore, the ratio of the area of absorption attributed to the amide group to the area of absorption attributed to the aromatic ring of lignin (near 1515 cm ⁻¹ ) is calculated, and this may be used as an index of the degree of amidation cleavage of the ester bond. it can. In addition, in this invention, the FT-IR spectrum by the reflection method was measured about the range of wave numbers 400-4000 cm < ^-1 > using NICOLET380 made from Thermo.

  The cleavage of the ester bond between lignin and hemicellulose in the ester bond cleavage step can also be confirmed by extraction of hemicellulose with hot water. Hemicellulose, whose ester bond with lignin is cleaved by ammonia treatment, is difficult to dissolve in cold water, but easily dissolved in hot water. On the other hand, hemicellulose in plant biomass that has not been subjected to the ester bond cleavage step is not extracted into hot water. Therefore, the plant biomass after being subjected to the ester bond cleavage step is extracted with hot water, and the ratio of the amount of extracted hemicellulose to the total amount of hemicellulose contained in the plant biomass (hereinafter referred to as “extraction rate”). It was studied to use it as an index for the cleavage of the ester bond between lignin / hemicellulose. In addition, it is thought that the hemicellulose which the ester bond between lignin cleaved does not melt | dissolve in cold water is due to the hydrogen bond between hemicellulose and cellulose.

The specific procedure for extraction of hemicellulose with the hot water is as follows.
That is, first, 0.5 g of a plant biomass sample after being subjected to the ester bond cleavage step is suspended in 20 ml of 50 mM acetate buffer (pH 4.5) and heated in a 100 ° C. hot water bath for 10 minutes. And extract. Then, about 1 mg of a commercially available enzyme containing hemicellulase as a protein amount is added to 10 ml of the filtrate and shaken at 37 ° C. and 200 rpm for 24 hours to perform enzymatic degradation of the extracted hemicellulose. The concentration of xylose in the obtained enzyme decomposition solution is quantified by high performance liquid chromatography using a Shodex SUGAR SP0810 (trade name) column. On the other hand, the raw material plant biomass is separately saccharified by sulfate saccharification, and the amount of xylose produced is similarly quantified. This xylose can be regarded as having been produced by hydrolysis of substantially all hemicellulose contained in the raw plant biomass. The extraction rate of hemicellulose is calculated by dividing the amount of xylose obtained by hot water extraction and enzymatic saccharification by the amount of xylose derived from total hemicellulose in the raw plant biomass obtained by the sulfate saccharification method.

(Second step)
After the first step, first, the inside of the reaction vessel is cooled to a reaction vessel temperature of −40 ° C. to 40 ° C. Next, liquid ammonia or liquid ammonia in the reaction tank is set so that the ratio of the mass of liquid phase ammonia to the dry mass of plant biomass (the mass of liquid phase ammonia / the dry mass of plant biomass) is 0.1 to 1.1. Add ammonia, including liquid ammonia. Then, the plant biomass is treated with ammonia containing a liquid phase, and at least a part of the crystal form of cellulose constituting lignocellulose is transferred from type I to type III to obtain a raw material for enzyme saccharification.

In the present embodiment, after the ester bond cleavage is performed in the first step, the crystal type transition is performed in the second step for the following reason.
That is, when the crystal type transition is performed on the plant biomass in which the ester bond is not cleaved, the cellulose III type crystal once generated by the crystal type transition is easily transferred again to the cellulose I type crystal by coming into contact with moisture. On the other hand, when at least part of the ester bond between lignin and hemicellulose is cleaved by ester bond cleavage, the cellulose type III crystal formed by the crystal type transition becomes relatively stable, and cellulose I type by contact with moisture Transition to the crystal again is suppressed.

  In the second step, the plant biomass containing lignocellulose is treated with ammonia containing a liquid phase, and at least a part of the crystal form of cellulose constituting the lignocellulose is transferred from type I to type III. In the present invention, “lignocellulose” means not only a complex of lignin, hemicellulose, and cellulose contained in untreated plant biomass, but also at least an ester bond between lignin and hemicellulose by an ester bond cleavage step. The complex partly cleaved and the untreated plant biomass are subjected to a crystal transition process, and include the complex in which at least a part of cellulose type I crystals is transferred to type III crystals.

  In the second step, it is necessary for ammonia to contain a liquid phase. In order to transfer at least a part of cellulose type I crystals to type III crystals, it is necessary to produce a cellulose-ammonia intermediate. For the production of the intermediate, the cellulose comes into contact with liquid phase ammonia. This is because it is necessary. Accordingly, if liquid phase ammonia is present, at least a part of the cellulose I-type crystal can be transferred to the III-type crystal.

  The ammonia containing the liquid phase in the second step is usually gas-liquid mixed phase ammonia. This is because it is difficult to make the inside of the pressure vessel filled with liquid ammonia without having a gas phase portion, and in such a case, the amount of ammonia to be used depends on the physical action. Is unfavorable because it tends to increase beyond the minimum amount required to make the best use of.

  In the second step, the crystal transition temperature is −40 ° C. to 40 ° C., and the ratio of the liquid ammonia mass to the dry mass of the plant biomass at the treatment temperature (liquid ammonia mass / plant biomass Liquid ammonia or ammonia containing liquid ammonia is added to the reaction vessel so that the dry mass) is 0.1 to 1.1.

  In the second step, the amount of ammonia in the liquid phase necessary for crystal type transition is hardly affected by temperature. On the other hand, when ammonia is in a gas-liquid mixed phase, the amount of ammonia in the gas phase varies depending on the temperature and decreases as the temperature decreases. Furthermore, the progress of the crystal type transition is hardly affected by the temperature, and proceeds rapidly even at a low temperature. Therefore, from the viewpoint of reducing the amount of ammonia used, the temperature in the crystal transition process is preferably low. On the other hand, the energy consumption of the refrigerator increases as the ammonia is cooled to a lower temperature. Therefore, from the balance between reducing the amount of ammonia used by lowering the temperature and increasing the energy consumption of the refrigerator, the ratio of the treatment temperature and the mass of liquid ammonia at that treatment temperature to the dry mass of plant biomass is determined. It is preferable to do. Specifically, the crystal transition temperature is -40 ° C to 40 ° C, preferably -10 ° C to 20 ° C. Moreover, the ratio of the mass of ammonia in the liquid phase / the dry mass of the plant biomass is 0.1 to 1.1 as described above, and preferably 0.4 to 0.8. In addition, as long as the processing temperature of crystal type transition is in the range of −40 ° C. to 40 ° C., it may be the same as or different from the temperature when the reaction vessel temperature is cooled after the first step.

  The ammonia partial pressure in the second step is not particularly limited as long as it is a partial pressure necessary for the presence of liquid phase ammonia at the treatment temperature. The partial pressure is a saturated vapor pressure of ammonia at the temperature, and naturally varies depending on the processing temperature. As a preferable ammonia partial pressure when the processing temperature is −40 to 40 ° C., 0.07 to 1.6 MPaA is exemplified. can do.

  In the second step, the processing time for crystal type transition is not particularly limited. Since the crystal transition by the ammonia treatment including the liquid phase proceeds rapidly, the treatment time is, for example, 0.01 to 1 hour.

In the second step, the amount of water contained in the plant biomass and ammonia preferably satisfies the following formula (1).
Water mass / (dry weight of plant biomass + water mass) ≦ 0.30 (1)

Furthermore, in the second step, it is preferable that the amount of water contained in the plant biomass and ammonia satisfies the following formula (2).
Water mass / (dry mass of plant biomass + mass of water) ≦ 0.15 (2)

  Furthermore, in the example using Elianthus as raw material biomass, all cellulose I-type crystals are transferred to III-type crystals by the presence of about 40% by mass or more of liquid phase ammonia with respect to the dry mass of plant biomass. Can be made. Therefore, in order to minimize the amount of ammonia to be used while maximizing the physical action by transferring almost all the I-type crystals to the III-type crystals, the liquid phase ammonia present in the crystal-type transition step is used. The amount of is preferably about 35 to about 50 mass% with respect to the dry mass of the plant biomass. The ratio of the amount of liquid phase ammonia to the amount of the preferred plant biomass varies depending on the type of plant biomass, the content of cellulose, the apparatus used for ammonia treatment and the operating conditions thereof, etc. It is preferred to determine the amount.

  As described in the explanation relating to the “plant biomass drying process”, when the amount of water present in the system in the crystal transition process increases, the transition of cellulose I crystals to III crystals tends to be suppressed. And in order to accelerate | stimulate transition to the III type crystal of a cellulose I type crystal, it is preferable that the moisture content which exists in plant biomass and ammonia satisfy | fills said Formula (2). When the water content does not satisfy the formula (2), even when plant biomass is treated with ammonia containing a liquid phase, the transition of cellulose type I crystals to type III crystals tends to hardly proceed.

Furthermore, the amount of water present in plant biomass and ammonia is expressed by the following formula (3):
Water mass / (dry weight of plant biomass + water mass) ≦ 0.15 (3)
It is preferable to satisfy.

  When the amount of water present in plant biomass and ammonia satisfies the above formula (3), in the cellulose constituting lignocellulose, all of the cellulose type I crystals tend to be transferred to type III crystals, and the physical action Can be fully expressed.

  The transition from cellulose type I crystal to type III crystal in the second step can be confirmed by performing X-ray diffraction (XRD) analysis on the plant biomass before and after the treatment. More specifically, in the XRD pattern, peaks detected around 2θ = 16 ° (101) and 2θ = 22 ° (020) are peaks attributed to the I-type crystal, and around 2θ = 12 ° ( 101) and peaks detected at around 2θ = 21 ° (020) are peaks attributed to type III crystals, and the presence or absence of each crystal type can be determined by the presence or absence of these peaks. In addition, when the present inventors performed the process by gaseous-phase ammonia to the plant biomass containing lignocellulose, the peak attributed to a III type crystal | crystallization is not detected in the XRD pattern about the plant biomass after a process. That is, it has also been confirmed that crystal type transition does not occur in the treatment with gaseous ammonia.

  According to the above-described method for producing a raw material for enzymatic saccharification according to the first embodiment of the present invention, sufficient reactivity can be imparted to plant biomass for enzymatic saccharification, and the amount of ammonia used is sufficient. It becomes possible to reduce it.

  The first step may be replaced with a treatment other than the ammonia treatment for cleaving the ester bond between hemicellulose and lignin, for example, so-called alkali cooking treatment or steaming treatment with pressurized hot water. The efficiency of saccharification can be improved. However, since a large amount of water is used in these steps, the plant biomass after treatment includes a lot of water. As described above, in the crystal form transition step in the present embodiment, when the plant biomass contains moisture exceeding a predetermined ratio, the transition of the cellulose type I crystal to the type III crystal tends to be inhibited, It is necessary to dry the plant biomass before this step. Since drying of plant biomass containing a large amount of water requires a large amount of energy, the method of performing ester bond cleavage by a process other than the ammonia treatment tends to impair economic rationality.

<Second Embodiment: Method for Producing Sugar>
The sugar production method according to the second embodiment of the present invention includes at least an enzyme saccharification step for enzymatic saccharification of the enzyme saccharification raw material obtained by the enzyme saccharification raw material production method according to the first embodiment. Depending on the situation, other steps are included.

  Hereinafter, along with preferable embodiment, the manufacturing method of the saccharide | sugar of this invention is demonstrated in detail.

(Enzyme saccharification process)
The enzyme saccharification step according to the sugar production method of the present embodiment is a step in which the cellulose constituting he biomass and hemicellulose are hydrolyzed to obtain a monosaccharide by bringing the enzyme saccharification raw material into contact with the enzyme.

  The enzyme saccharification method used in the enzyme saccharification step is not particularly limited as long as an enzyme is used, and a known method can be appropriately selected. When a chemical saccharification method using sulfuric acid or the like is used, the yield of monosaccharides tends to decrease due to excessive decomposition, and a substance having an inhibitory action is likely to be produced in the fermentation process following saccharification. In contrast to the problem of the tendency and the discharge of environmentally hazardous substances such as sulfuric acid, mild conditions can be selected in the saccharification method using an enzyme. It tends not to occur.

  There is no restriction | limiting in particular as an enzyme used in the said enzyme saccharification process, According to the objective, it can select suitably, For example, a cellulase, cellobiase ((beta) -glucosidase), etc. are mentioned. An immobilized enzyme in which these enzymes are immobilized on an appropriate carrier or matrix can also be used.

  There is no restriction | limiting in particular as the usage-amount of the enzyme in the said enzyme saccharification process, Although it can select suitably according to the objective, For example, with respect to 1g of solid content dry mass in the said raw material for enzyme saccharification, 0.001 mg- 100 mg is preferable, 0.01 mg to 10 mg is more preferable, and 0.1 mg to 1 mg is still more preferable. If the amount of the enzyme used is less than 0.001 mg relative to 1 g of the solid content dry mass in the enzyme saccharification raw material, enzyme saccharification may be insufficient, and if it exceeds 100 mg, saccharification inhibition may occur. May happen. On the other hand, when the amount of the enzyme used is within the further preferable range, it is advantageous in that the amount of sugar obtained is larger than the amount of enzyme used.

  There is no restriction | limiting in particular as temperature in the said enzyme saccharification process, Although it can select suitably according to the objective, 10 to 70 degreeC is preferable, 20 to 60 degreeC is more preferable, and 30 to 50 degreeC is still more preferable. . If the temperature is lower than 10 ° C, enzyme saccharification may not proceed sufficiently, and if it exceeds 70 ° C, the enzyme may be deactivated. On the other hand, when the temperature is within the further preferable range, it is advantageous in that the amount of sugar obtained is larger than the amount of enzyme used.

  There is no restriction | limiting in particular as pH in the said enzyme saccharification process, Although it can select suitably according to the objective, For example, 3.0-8.0 are preferable, 3.5-7.0 are more preferable, 4. 0 to 6.0 is more preferable. If the pH is less than 3.0 or more than 8.0, the enzyme may be deactivated. On the other hand, when the pH is within the more preferable range, it is advantageous in that the amount of sugar obtained is larger than the amount of enzyme used.

  By the enzyme saccharification step, glucose is obtained as monosaccharide from cellulose contained in the enzyme saccharification raw material. Moreover, hexoses such as glucose, galactose and mannose and pentoses such as xylose and arabinose are produced from hemicellulose contained in the enzyme saccharification raw material.

(Other processes)
The sugar solution containing the monosaccharide obtained by the above enzymatic saccharification step may be directly subjected to the fermentation step described later. For example, a step of adjusting the pH of the sugar solution, a step of adjusting the sugar concentration, etc. Therefore, the sugar solution may be more suitable for fermentation.

  The sugar obtained by the sugar production method of the present embodiment can be used not only in the ethanol production method described later, but also as a raw material for producing other substances, for example, organic acids such as lactic acid.

<Third Embodiment: Method for Producing Ethanol>
The method for producing ethanol according to the third embodiment of the present invention includes at least a fermentation process (ethanol fermentation process) for fermenting sugar obtained by the sugar production method according to the second embodiment, and if necessary. Furthermore, other processes are included.

  Hereinafter, the ethanol production method according to this embodiment will be described in detail.

(Fermentation process)
The fermentation process according to the ethanol production method of the present embodiment is a process in which an ethanol fermentation microorganism is added to a sugar solution containing the sugar obtained by the sugar production method of the present invention to perform ethanol fermentation.

  There is no restriction | limiting in particular as said ethanol fermentation microorganisms, Although it can select suitably according to the objective, Bacteria of genus Zymomonas, such as yeast and Zymomonas mobilis, etc. are preferable, and yeast is more preferable.

  There is no restriction | limiting in particular as said yeast, Although it can select suitably according to the objective, Saccharomyces genus yeasts, such as Saccharomyces cerevisiae, are preferable. However, as described above, the hemicellulose that constitutes the plant biomass produces pentoses such as xylose and arabinose by enzymatic saccharification, but the natural yeast of the genus Saccharomyces has the ability to assimilate the pentose and produce ethanol. There is no waste. Therefore, in order to effectively utilize not only hexose but also pentose derived from hemicellulose and converting it to ethanol, yeast that has the ability to assimilate pentose and produce ethanol (pentose-utilizing yeast) It is also preferable to use There is no restriction | limiting in particular as said pentose utilization yeast, Although it can select suitably according to the objective, Pichia stipitis, Candida shihatae, etc. are preferable. In order to efficiently convert hexose sugar and pentose sugar to ethanol, a method of using a yeast of the genus Saccharomyces and the aforementioned pentose-utilizing yeast in combination is also preferably employed. In this case, the yeast of the genus Saccharomyces and the above-mentioned pentose-assimilating yeast may be fermented, or the glucose in the sugar solution is first assimilated by the yeast of the genus Saccharomyces, and then the above-mentioned pentose-assimilating yeast. You may assimilate pentose sugars.

  The yeast used in the fermentation process may be a natural yeast or a genetically modified yeast. In particular, it is possible to efficiently convert both hexose and pentose derived from cellulose and hemicellulose into ethanol by using a genetically modified yeast having the ability to assimilate both hexose and pentose. it can.

  There are no particular restrictions on the amount of yeast used in the fermentation process, additives other than sugar, fermentation temperature, pH, fermentation time, etc., and known conditions can be appropriately selected and used. -7, As for fermentation temperature, about 20 to 37 degreeC is preferable.

  In addition, by performing fermentation at a temperature higher than usual using heat-resistant yeast, it is possible to perform fermentation efficiently without the need for cooling equipment and suppressing the growth of various bacteria. Examples of the thermostable yeast include thermostable yeast belonging to the genus Kloyveromyces such as Kleiberymyces marxianas. When using these heat-resistant yeasts, the temperature of fermentation can be about 37 ° C to 50 ° C.

  As the fermentation process, a so-called parallel double fermentation method in which the enzyme saccharification process and the fermentation process are simultaneously performed may be employed. By employing this parallel double fermentation method, the enzyme saccharification step and the fermentation step can be carried out as a single step, and ethanol can be produced by a simplified step. In the parallel double fermentation, the enzyme saccharification raw material obtained by the enzyme saccharification raw material production method of the present embodiment is used in the reaction system as it is in the reaction system. A microorganism for ethanol fermentation is added to perform enzymatic saccharification and ethanol fermentation.

(Other processes)
The ethanol production method according to the present invention preferably further comprises a purification step of separating and purifying ethanol from the medium containing ethanol obtained in the fermentation step. Through the purification step, ethanol is separated / purified from various substances contained in the fermentation medium and concentrated. The separation / purification method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the fermentation medium is first subjected to solid-liquid separation by centrifuging and / or filtering solids such as bacterial cells. A method of recovering an aqueous solution containing ethanol and then concentrating and purifying the ethanol by a method such as distillation or membrane separation is preferred.

  According to the ethanol production method of this embodiment, ethanol can be produced efficiently by using the sugar obtained using the enzyme saccharification raw material. Ethanol obtained by the ethanol production method can be suitably used as, for example, fuel ethanol, industrial ethanol, and the like.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

[Example 1]
<Manufacture of raw materials for enzymatic saccharification>
(Plant biomass)
Elianthus was used as a plant biomass containing lignocellulose. The harvested Eliansus was pulverized using a cutter mill while controlling the particle size with a screen having a mesh opening of 3 mm to obtain Eliansus particles. The average particle diameter of the particles was 678 μm as a median diameter (d50) measured by a laser diffraction method (apparatus: laser diffraction particle diameter measuring apparatus LMS-2000e (manufactured by Seishin Enterprise Co., Ltd.)). The Elianthus particles were dried in a vacuum dryer at 40 ° C. under a reduced pressure of 5 kPaA for a whole day and night. The moisture content of the obtained biomass was 0.9% by mass based on the total mass of the biomass.
(First step)
50 kg of the above biomass as a dry mass was charged into a reaction tank (pressure vessel with an internal volume of 2000 L) equipped with a stirrer, and the reaction tank was depressurized to -0.095 MPaG with a vacuum pump, and then nitrogen was supplied to 0 MPaG. Next, after the temperature in the reaction vessel was raised to 40 ° C., 18.3 kg of ammonia containing liquid ammonia was supplied using a pump while stirring the biomass. After supply of ammonia, the temperature of the reaction vessel was raised to 120 ° C., and ammonia treatment was performed for 2.0 hours. The ammonia partial pressure in the reaction tank at the time of the treatment is 1.1 MPaA. From the comparison between the temperature / pressure conditions and the supply amount of ammonia, 5.34 kg of ammonia exists in the liquefied state under the above conditions. The ratio of state ammonia to biomass was 0.103.
(Second step)
After the first step, the reaction vessel is cooled to about 20 ° C., liquid ammonia is additionally supplied so that the ratio of liquefied ammonia to biomass at 10 ° C. becomes 0.6, and then the biomass is stirred. And treated at 11.0 ° C. for 20 minutes. After the treatment, ammonia was exhausted. After evacuation, the reaction tank was depressurized to -0.095 MPaG with a vacuum pump to remove ammonia remaining in the biomass. After the removal of ammonia, the reaction vessel was pressurized to atmospheric pressure with nitrogen, and the enzyme saccharification raw material A was extracted from the reaction vessel.
<Manufacture of sugar (enzymatic saccharification)>
Using the enzyme saccharification raw material A, an enzyme saccharification reaction was performed by the following operation.
The enzyme saccharification raw material A precisely weighed so as to have a dry solid content of 500 mg is taken into a 50 mL centrifuge tube, and the sample concentration is 5% (dry mass / volume), and Accelerase DUET (registered trademark) (trade name) The enzyme saccharification reaction liquid was prepared so that it might become 125% (volume / dry mass) and pH4.5 (acetate buffer) with respect to the raw material for enzyme saccharification. This was subjected to an enzymatic saccharification reaction by shaking for 24 hours using a rotary shaker (200 rpm) in a thermostatic chamber at 50 ° C. After the reaction, the glucose concentration in the supernatant obtained by centrifugation was measured using Glucose CII Test Wako (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), and the glucose yield was calculated as a measure of the enzyme saccharification rate.
The glucose yield is defined by the following formula (4).
Glucose yield (%) = [glucose amount in enzyme saccharification reaction solution / (dry mass of enzyme saccharification raw material × total glucosylation rate / 100)] × 100 (4)
Here, the total glucosylation rate (%) is determined by (amount of glucose obtained when the biomass raw material is completely and completely hydrolyzed / dry mass of the biomass raw material) × 100, and is based on the biomass raw material standard. This corresponds to the theoretical yield of glucose.
The results obtained above are summarized in Table 1.
<Production of ethanol>
Biomass A is used as an enzyme saccharification raw material, and enzymatic saccharification is performed under the conditions equivalent to enzyme saccharification using a centrifuge tube in the above <sugar production (enzymatic saccharification)> (however, the enzyme saccharification raw material solid content concentration is 15 g / 100 ml). A sugar solution was obtained. The glucose concentration in the obtained sugar solution was 4.6 g / 100 ml, and the xylose concentration was 2.6 g / 100 ml.
Next, in a 50 ml Erlenmeyer flask, 15 ml of the sugar solution was inoculated with Saccharomyces yeast to which xylose assimilation ability was imparted so that the optical density OD600 would be 20, and cultured with shaking at 30 ° C. at 140 rpm. And fermented. All glucose was consumed 4 hours after the start of fermentation, and xylose was all consumed 48 hours later. The ethanol yield was 66%.

[Example 2]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction vessel temperature at the time of supplying ammonia in the first step was set to 60 ° C. to obtain a raw material B for enzyme saccharification. The ammonia partial pressure in the reaction vessel at 120 ° C. was 1.3 MPaA, and the ratio of liquefied ammonia to biomass was 0.096.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material B was used in place of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 1.

[Example 3]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction tank temperature at the time of supplying ammonia in the first step was set to 80 ° C. to obtain a raw material C for enzymatic saccharification. The partial pressure of ammonia in the reaction vessel at 120 ° C. was 1.3 MPaA, and the ratio of liquefied ammonia to biomass was 0.092.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material C was used in place of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 1.

[Example 4]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction tank temperature at the time of ammonia supply in the first step was 80 ° C. and that the ammonia supply amount was 16.4 kg. A raw material D for saccharification was obtained. The ammonia partial pressure in the reaction vessel at 120 ° C. was 1.1 MPaA, and the ratio of liquefied ammonia to biomass was 0.092.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material D was used in place of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 1.

[Example 5]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction tank temperature at the time of ammonia supply in the first step was set to 80 ° C. and the ammonia supply amount was set to 15.4 kg. A raw material E for saccharification was obtained. In addition, the ammonia partial pressure of the reaction tank at 120 ° C. was 1.1 MPaA, and the ratio of liquefied ammonia to biomass was 0.075.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material E was used in place of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 1.

[Example 6]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction tank temperature at the time of ammonia supply in the first step was set to 60 ° C. and the ammonia supply amount was set to 14.6 kg. A raw material F for saccharification was obtained. The ammonia partial pressure in the reaction vessel at 120 ° C. was 1.1 MPaA, and the ratio of liquefied ammonia to biomass was 0.063.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was carried out in the same manner as in Example 1 except that the enzyme saccharification raw material F was used in place of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 1.

[Example 7]
<Manufacture of raw materials for enzymatic saccharification>
In the first step, the same as in Example 1 except that the reaction tank was filled with 75 kg of biomass as a dry mass, the reaction tank temperature at the time of ammonia supply was 80 ° C., and the ammonia supply amount was 16.4 kg. Thus, the first and second steps were carried out to obtain a raw material G for enzymatic saccharification. The ammonia partial pressure in the reaction vessel at 120 ° C. was 1.1 MPaA, and the ratio of liquefied ammonia to biomass was 0.061.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material G was used instead of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 1.

[Comparative Example 1]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction tank temperature at the time of ammonia supply in the first step was 120 ° C. and the ammonia supply amount was 13.0 kg. A saccharification raw material H was obtained. The partial pressure of ammonia in the reaction vessel at 120 ° C. was 1.2 MPaA, and the ratio of liquefied ammonia to biomass was 0.014.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material H was used in place of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 2.

[Comparative Example 2]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction tank temperature at the time of liquid ammonia supply in the first step was 110 ° C. and the ammonia supply amount was 13.0 kg. A raw material I for enzymatic saccharification was obtained. The partial pressure of ammonia in the reaction vessel at 120 ° C. was 1.2 MPaA, and the ratio of liquefied ammonia to biomass was 0.015.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material I was used in place of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 2.

[Comparative Example 3]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction tank temperature at the time of ammonia supply in the first step was 110 ° C. and the ammonia supply amount was 13.9 kg. A raw material J for saccharification was obtained. In addition, the ammonia partial pressure of the reaction tank at 120 ° C. was 1.2 MPaA, and the ratio of liquefied ammonia to biomass was 0.034.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material J was used instead of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 2.

[Comparative Example 4]
<Manufacture of raw materials for enzymatic saccharification>
The first and second steps were carried out in the same manner as in Example 1 except that the reaction tank temperature at the time of ammonia supply in the first step was set to 100 ° C. and the ammonia supply amount was set to 14.3 kg. A raw material K for saccharification was obtained. The ammonia partial pressure in the reaction vessel at 120 ° C. was 1.2 MPaA, and the ratio of liquefied ammonia to biomass was 0.037.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material K was used instead of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 2.

[Comparative Example 5]
<Manufacture of raw materials for enzymatic saccharification>
The same procedure as in Example 1 except that the reaction tank temperature at the time of ammonia supply in the first step was 120 ° C., the ammonia supply amount was 13.0 kg, and ammonia was supplied in a gas state at 120 ° C. The first and second steps were carried out to obtain a raw material L for enzymatic saccharification. The partial pressure of ammonia in the reaction vessel at 120 ° C. was 1.2 MPaA, and the ratio of liquefied ammonia to biomass was 0.014.
<Manufacture of sugar (enzymatic saccharification)>
An enzyme saccharification reaction was performed in the same manner as in Example 1 except that the enzyme saccharification raw material L was used instead of the enzyme saccharification raw material A, and the glucose yield was calculated.
The results obtained above are summarized in Table 2.

As shown in Table 1, according to Examples 1-7, it is possible to manufacture the raw material for enzyme saccharification which is excellent in enzyme saccharification efficiency, reducing the usage-amount of ammonia, and the obtained enzyme saccharification use It is clear that by performing enzymatic saccharification using raw materials, it is possible to efficiently produce sugar, and furthermore, by performing fermentation using the obtained sugar, ethanol can be produced efficiently. It became.

However, in a state where the supply of liquid ammonia or ammonia containing liquid ammonia to the reaction tank (pressure vessel) containing the plant biomass is kept at a high temperature exceeding 80 ° C. (especially treatment temperature, typically 120 ° C.). When this is done, the vaporization of ammonia proceeds, so that a large amount of ammonia needs to be supplied in order for liquid phase ammonia to be present in the container. Moreover, since the ammonia partial pressure rises, it is necessary to improve the pressure resistance performance of the reaction tank, leading to an increase in apparatus cost.

Claims (4)

A method for producing a raw material for enzyme saccharification, wherein a plant biomass containing lignocellulose is treated with ammonia to obtain a raw material for enzyme saccharification,
Liquid ammonia or ammonia containing liquid ammonia is supplied to a reaction vessel containing plant biomass containing lignocellulose at a reaction vessel temperature of −40 ° C. to 80 ° C., and then within a range of 60 ° C. to 135 ° C. The temperature of the reaction vessel is increased to a treatment temperature that is higher than the reaction vessel temperature at the time of supply, and the plant biomass is treated with ammonia containing a gas phase and a liquid phase at the treatment temperature, and lignocellulose is obtained. A first step of cleaving at least part of the ester bond between the constituent lignin and hemicellulose;
After the first step, the inside of the reaction vessel is cooled to a reaction vessel temperature of −40 ° C. to 40 ° C., and the ratio of the mass of liquid ammonia to the dry mass of the plant biomass (mass of liquid ammonia / plant biomass Liquid ammonia is added to the reaction vessel so that the dry mass) is 0.1 to 1.1, and the plant biomass is treated with ammonia containing a liquid phase at −40 ° C. to 40 ° C. to obtain lignocellulose. A second step of obtaining a raw material for enzymatic saccharification by transferring at least a part of the crystal form of cellulose constituting from type I to type III;
The manufacturing method of the raw material for enzyme saccharification provided with.   In the first step, in the reaction tank, the ratio of the mass of liquid phase ammonia present in the reaction tank at the treatment temperature to the dry mass of the plant biomass is 0.05 to 0.25. The method for producing a raw material for enzyme saccharification according to claim 1, wherein liquid ammonia or ammonia containing liquid ammonia is supplied.   A method for producing sugar, comprising a step of saccharifying an enzyme saccharification raw material obtained by the method for producing an enzyme saccharification raw material according to claim 1 or 2 with an enzyme. The manufacturing method of ethanol provided with the process of fermenting the saccharide | sugar obtained by the manufacturing method of the saccharide | sugar of Claim 3.