JP2014097012A - Pretreatment method of biomass containing cellulose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply obtaining cellulose with low cost and energy saving, in pretreatment for regenerating cellulose from biomass such as lignocellulose in which the regeneration of cellulose is difficult by conventional methods.SOLUTION: In a set of processes that biomass such as lignocellulose containing cellulose is dispersed in an ion liquid, and cellulose regenerated by adding water is acted on an enzyme to saccharify cellulose, a pretreatment method of biomass containing cellulose comprises the steps of: heating an aqueous solution containing low concentration of peroxide; freeze-drying the heated biomass; and agitating and mixing the dried biomass in an ion liquid, and depositing cellulose from the ion liquid containing the biomass, thereby regenerating cellulose.

Description

  The present invention relates to a pretreatment method for biomass containing cellulose, and more particularly to a saccharification pretreatment method for cellulose for efficiently enzymatically degrading cellulose with cellulase.

  As a measure against global warming, the use of bioethanol has been devised from the perspective of carbon neutral and petroleum (fuel and basic chemicals) substitution. At present, bioethanol is produced using edible biomass such as corn and sugarcane as a raw material, and there is a limit to conventional production methods in which raw materials and food compete. Therefore, in order to eliminate the competition, a bioethanol production process using non-edible biomass such as bagasse, bay pine, and wood flour as a raw material has been studied. If non-edible part biomass can be used as a raw material, there are many advantages, such as the amount of bioethanol produced per unit cultivation area can be increased. However, most of the non-edible biomass such as bagasse, pine, and wood flour described above is lignin (a polymer compound in which a phenol compound forms a complex three-dimensional network structure) or hemicellulose (plant). Is a cellulose complex bound with a high molecular weight polysaccharide compound that constitutes the cell wall of the cell, and because of its strong binding property with lignin and hemicellulose, there is a concern that the cellulose in lignocellulose cannot be used efficiently. ing.

  Enzymatic saccharification is considered as one of the desirable saccharification methods for cellulosic biomass from the viewpoint of mild reaction conditions and high reaction selectivity. There are two major issues. This is because the cellulose in lignocellulose is bound to lignin and hemicellulose, so that it cannot be efficiently used as a substrate for enzyme reaction in the bound state, and the cellulose in lignocellulose is crystalline cellulose. For some reason, the enzymatic reaction by cellulase is very slow. Therefore, various studies have been made on the treatment of lignocellulose by some method before the enzymatic saccharification reaction, that is, pre-saccharification pretreatment, for practical application of the lignocellulose enzymatic saccharification process.

  For example, Patent Document 1 discloses a pretreatment method in which lignocellulose such as rice husk is frozen, mechanically pulverized, and then treated with alkali to remove lignin. In this method, a large amount of electric energy is required to perform pulverization using a machine, and complicated operation is essential because lignocellulose after pulverization is treated with an alkaline solution to remove lignin. Furthermore, since it is necessary to neutralize the alkali with an acid before the enzymatic saccharification reaction, it must be said that this is an expensive pretreatment method.

  Patent Document 2 discloses a pretreatment method in which lignocellulose such as rice husk is treated with hot water under pressure and then mechanically pulverized. Although this method does not require a lignin removal step, it requires a large amount of energy in the same manner as the method disclosed in Patent Document 1, and must be said to be a high-cost pretreatment method.

  On the other hand, Patent Document 3 discloses a pretreatment method in which lignocellulose such as cedar chips is digested with alkali and then bleached with oxygen or the like to remove lignin. In this method, since a high concentration of alkali is used for cooking, the subsequent neutralization washing is not easy, and a temperature of 150 ° C. or higher is required for cooking. It is hard to say that there is.

  The pretreatment methods exemplified above focus on removing lignin and hemicellulose bound to cellulose in lignocellulose. This is one of the problems mentioned above, because the cellulose in lignocellulose is bound to lignin and hemicellulose, so that it cannot be efficiently used as a substrate for enzyme reaction in the bound state. It is none other than to do. Many studies have been made so far in the field of pulp bleaching to decompose, remove and extract lignin and hemicellulose from lignocellulose. In fact, Patent Documents 4 and 5 disclose a method for removing lignin using a peroxide, and Patent Documents 6 and 7 disclose a method for extracting hemicellulose using an alkali. Patent Document 8 discloses a method for decomposing lignin using pressurized steam, and Patent Document 9 discloses a method for extracting hemicellulose using hot water. However, both methods require treatment at a high concentration and high temperature, and considering the complexity of the neutralization treatment and the amount of energy used, it is practical as a pretreatment method for the enzyme reaction. Is hard to say. That is, in order to use lignocellulose practically as a substrate for enzyme reaction, a method for removing and extracting lignin and hemicellulose from lignocellulose by a simple and energy-saving method is required.

  In order to decompose cellulose with an enzyme, specifically, to efficiently saccharify with cellulase, it is effective to relax the strong and dense crystal structure of cellulose so that cellulase can act easily. Patent Document 10 and Non-Patent Document 1 disclose and report a method for dissolving cellulose in an ionic liquid as the means. It is believed that by dissolving in an ionic liquid, the strong hydrogen bonding ability of the ionic liquid relaxes or breaks the hydrogen bonds inside and outside the cellulose, so that it is converted into a crystal structure in which cellulase is likely to act. Actually, cellulose is once dissolved in an ionic liquid, and after dissolution is confirmed, water, which is a poor solvent for cellulose, is added to precipitate cellulose. This technique is generally referred to as cellulose regeneration, and the regenerated cellulose is said to have a crystal structure in which the crystallinity of the original cellulose is relaxed and cellulase is likely to act.

  Patent Document 11 discloses a pretreatment method including a step of bringing a hydrophobic ionic liquid into contact with lignocellulose. However, the cellulose lignin and hemicellulose are combined only by allowing the hydrophobic ionic liquid to permeate. It does not lead to undressing. In this method, since the crystal relaxation of cellulose does not proceed as a result, it cannot be said that the effect of promoting saccharification reaction is sufficient, and it cannot be said that the effective use of lignocellulosic biomass is sufficiently achieved. Patent Document 12 discloses a method in which woody biomass, so-called lignocellulose, is immersed in an ionic liquid and irradiated with ultrasonic waves to remove lignin and hemicellulose from lignocellulose and relax the crystallinity of cellulose. Although a treatment method is disclosed, since a device for generating ultrasonic waves must be provided separately and a large amount of electric energy is required for ultrasonic irradiation, this method is also a practical pretreatment method for enzyme reaction I can't say that.

  As mentioned above, the method of reducing the crystallinity of cellulose using an ionic liquid is effective. However, when lignocellulose, which is a biomass containing impurities that are difficult to remove, is used as a substrate, it is simple and low cost. The energy-saving and practical pretreatment method is a process of removing and extracting impurities such as lignin and hemicellulose from lignocellulose, etc., and the crystallinity of cellulose obtained thereby is relaxed with ionic liquid, and cellulase works It is considered that the method has a step of changing to a crystal structure that is easy to form.

  However, few examples have been disclosed or reported on such a simple, low-cost, energy-saving and practical pretreatment method. As described above, a high concentration chemical is used in the process of removing and extracting lignin and hemicellulose from lignocellulose. At present, it is a high-cost, high-energy method such as using a process at a high temperature. In other words, if there is a method that can remove and extract impurities such as lignin and hemicellulose from biomass such as lignocellulose in a simple, low-cost and energy-saving manner, construct a useful saccharification pretreatment method using lignocellulose as a substrate. And a very useful process for producing bioethanol from non-edible biomass including lignocellulose and the like can be constructed.

JP 55-045306 JP 2006-136263 A JP 2010-136702 A Japanese Patent Laid-Open No. 1-222187 JP-A-8-503749 JP-A 64-040502 JP-A-2-001701 JP-A-4-146281 JP-A 64-063033 Special table 2005-506401 JP 2010-220490 A JP 2012-086154 A JP 2009-201394 A JP 2011-149124 A JP 2012-005359 A

R. D. Rogers et al. Am. Chem. Soc., 124 (18), 4974-4975, 2002. R. D. Rogers et al., Green Chem. 5,443-447, 2003. C. A. Schall et al., Biotechnol. Bioeng. 95 (5), 904-910, 2006. Yasuno et al., Biosci. Biotech. Biochem. , 61 (11), 1944-1946, 1997. K. Shill et al., Biotechnol. Bioeng., 108 (3), 511-520, 2011.

  An object of the present invention is to provide a pretreatment method for biomass containing cellulose in order to saccharify cellulose contained in biomass such as lignocellulose with cellulase in a simple, low-cost, energy-saving and efficient manner.

  The present inventors disperse biomass such as lignocellulose in an ionic liquid, add a poor solvent such as water to regenerate cellulose, and saccharify cellulose by allowing the enzyme to act on the regenerated cellulose. In a series of processes, we conducted intensive studies to solve the above problems. As a result, a first step of heat-treating biomass containing cellulose such as lignocellulose in an aqueous solution containing a low-concentration peroxide, a second step of drying the heat-treated aqueous solution by freeze-drying or the like, Before including a third step of regenerating cellulose by dispersing the dried biomass, that is, the cellulose mixture obtained by drying in an ionic liquid, stirring and mixing, and then adding a poor solvent such as water to precipitate the cellulose. In the treatment method, by performing the second step after the first step, it becomes possible to remove lignin and hemicellulose from the biomass mainly composed of lignocellulose in an easy, low-cost and energy-saving manner, and then the third step. By performing the process after the second process, the cellulose taken out from the biomass is efficiently regenerated, and as a result, Found to improve the saccharification efficiency of cellulose by enzymes performed after, the present invention has been accomplished.

That is, the present invention is as follows.
<1> A method for treating biomass containing cellulose, the first step of heat-treating the biomass in an aqueous solution containing peroxide, the second step of drying the heat-treated aqueous solution, and drying And a third step of regenerating the cellulose by precipitating the cellulose from the ionic liquid containing the biomass and mixing the biomass containing the biomass with the first step, the second step This is a pretreatment method for biomass containing cellulose, which is processed in the order of the step and the third step.
<2> The pretreatment method according to <1>, wherein the aqueous solution containing the biomass and the peroxide heat-treated in the first step is directly dried in the second step.
<3> The pretreatment method according to <1> or <2>, wherein in the second step, the method of drying the aqueous solution is a vacuum freeze-drying method.
<4> The pretreatment method according to <1>, wherein in the third step, cellulose is precipitated by adding water to the ionic liquid obtained by stirring and mixing the biomass.
<5> The pretreatment method according to <1>, wherein the biomass is lignocellulose further containing lignin and / or hemicellulose.
<6> The pretreatment method according to <5>, wherein the lignocellulose is wood, rice straw, wheat straw, rice husk, bay pine, and / or powder thereof.
<7> The pretreatment method according to <1>, wherein the cation portion of the ionic liquid is an imidazolium cation, a pyridinium cation, and / or an ammonium cation.
<8> The pretreatment method according to <7>, wherein the imidazolium cation is 1-ethyl-3-methylimidazolium ion.
<9> The anion site of the ionic liquid is an ion containing a chloride ion, a phosphate ion, a formate ion, an acetate ion, and / or a perfluoroalkyl group. This is a pre-processing method.
<10> The pretreatment method according to <1>, wherein the ionic liquid is 1-ethyl-3-methylimidazolium acetate.
<11> The pretreatment method according to <1>, wherein in the first step, the peroxide is hydrogen peroxide and / or peracetic acid.
<12> The pretreatment method according to <1>, wherein in the first step, the concentration of the peroxide in the aqueous solution is 1% by weight to 8% by weight.
The present invention also provides:
<13> A method for producing monosaccharides, disaccharides, and / or oligosaccharides, which includes a cellulose decomposing step of enzymatically decomposing regenerated cellulose with cellulase through the pretreatment method described in <1> above.

<Biomass>
In the pretreatment method and the like of the present invention, biomass containing cellulose is used. Specifically, the biomass is, for example, lignocellulose further containing cellulose alone and lignin and / or hemicellulose as long as cellulose can be extracted. Examples of the cellulose alone include type I cellulose which is natural cellulose, type II cellulose obtained by dissolving natural cellulose once, then washing the solvent with water and drying, and amorphous amorphous cellulose. The cellulose in the biomass may contain a plurality of these. Examples of lignocellulose include woody or herbaceous biomass containing lignin and / or hemicellulose, such as wood, rice straw, wheat straw, rice husk, bagasse, bay pine and / or powders thereof. In the present invention, these biomasses can be used alone or in combination of two or more.

<Pretreatment method>
The pretreatment method of the present invention includes a first step of heat-treating biomass containing cellulose in a low-concentration peroxide aqueous solution, and an aqueous solution of biomass containing cellulose obtained by the treatment of the first step. A second step of drying and a third step are included. In the third step, the cellulose containing the biomass obtained by drying is stirred in the ionic liquid, and the cellulose is precipitated from the mixed solution containing the biomass containing the cellulose and the ionic liquid obtained there, A cellulose regeneration step for regenerating cellulose. Hereinafter, components used in these steps and each step will be described.

<Peroxide>
The peroxide used in the present invention is not particularly limited, and examples thereof include hydrogen peroxide, peracetic acid, organic peroxide, monopersulfuric acid, and chlorine peroxide. Among them, preferred peroxides are hydrogen peroxide and peracetic acid, and particularly preferred peroxides are peracetic acid. Moreover, in this invention, you may use a peroxide individually or in combination of 2 or more types. Furthermore, chlorine dioxide etc. can be used besides a peroxide.

<Peroxide addition concentration>
In the first step, the peroxide addition concentration in the aqueous solution in which the biomass containing cellulose is heat-treated is preferably 1% by weight to 8% by weight. More preferably, it is 1 to 5% by weight, and most preferably 1 to 3% by weight. When the peroxide addition concentration is 1% by weight or less, the effect of removing impurities such as lignin from biomass containing cellulose by addition of peroxide is not observed. In addition, if it is 8% by weight or more, there is a concern about cost increase due to the use of a high concentration of peroxide, and a decrease in aqueous solution pH due to the addition of peroxide becomes remarkable, so a new neutralization step must be incorporated. As a result, the method becomes expensive and is not preferable. Furthermore, the present invention includes a second step of heat-treating biomass containing cellulose in an aqueous peroxide solution and drying the aqueous solution. In the case of drying a high-concentration peroxide aqueous solution, in particular, as described later. In the case of rapid freeze-drying, it is necessary to ensure safety without using a high concentration of peroxide because the peroxide may be rapidly decomposed.

<Drying process>
In the second step of the present invention, the aqueous solution heat-treated in the first step is dried. In this step, it is preferable to directly dry the treated aqueous solution as it is. “Drying directly as it is” means to dry without adding any volatile components such as water from the aqueous solution without adding anything to the aqueous solution after completion of the first step. Say. The drying method is not particularly limited, and general drying or lyophilization by heating or warm air can be applied, but a more preferable drying method is lyophilization. This is because, in lyophilization, the object to be dried is once frozen, so that a substance that is flammable, chemically unstable and easily decomposed, for example, a peroxide handled in the present application, can be safely dried.

  In the present invention, it is preferable to heat the aqueous solution below the flash point of the object to be dried using, for example, warm air or a hot plate. According to such a drying method, safety can be secured without freezing the aqueous solution. When drying by techniques other than freeze-drying, the aqueous solution is heated to 40 to 90 ° C, preferably about 40 to 60 ° C. Moreover, in this invention, in order to shorten drying time, after removing the supernatant of the aqueous solution heat-processed at the 1st process, it can also dry using various methods.

<Freeze drying>
Freeze-drying refers to what is performed by a so-called general vacuum freeze-drying technique. That is, it is a method of cooling an object to minus 30 ° C. or lower, more preferably about minus 40 ° C., freezing it, placing it in a vacuum, sublimating moisture, and removing and drying. Although the method of cooling to minus 30 ° C. or lower is not particularly limited, a refrigerant such as liquid nitrogen is generally used because it is preferable to rapidly cool and freeze. In this way, the peroxide can be safely removed from the aqueous solution by rapidly drying without applying heat.

<Ionic liquid>
Although the ionic liquid used in the 3rd process (mixing process) of this invention is not specifically limited, A hydrophilic ionic liquid and a hydrophobic ionic liquid can be used.

<Cation site of ionic liquid>
Although the cation site | part which comprises the hydrophilic ionic liquid and hydrophobic ionic liquid used for this invention is not specifically limited, A heterocyclic onium cation and an ammonium cation are preferable. Among them, preferred cation sites are imidazolium cations and pyridinium cations, and the most preferred cation sites are imidazolium cations.
Examples of the imidazolium cation include 1-ethyl-3-methylimidazolium ion, 1-butyl-3-methylimidazolium ion, 1-propyl-3-methylimidazolium ion, 1-pentyl-3-methylimidazolium. Ion, 1-hexyl-3-methylimidazolium ion, 1-heptyl-3-methylimidazolium ion, 1-octyl-3-methylimidazolium ion, 1-nonethyl-3-methylimidazolium ion, 1-decyl- Dialkylimidazolium cations such as 3-methylimidazolium ion, 1- (1,2 or 3-hydroxypropyl) -3-methylimidazolium ion, 1,2,3-trimethylimidazolium ion, 1,2-dimethyl- 3-propylimidazolium ion, 1-buty 2,3-dimethyl-imidazolium trialkyl imidazolium cation ions, and the like.
Examples of the pyridinium cation include N-propylpyridinium ion, N-butylpyridinium ion, 1-butyl-4-methylpyridinium ion, 1-butyl-2,4-dimethylpyridinium ion, and the like.
Examples of the ammonium cation include aliphatic quaternary ammonium ions such as trimethylpropylammonium ion, trimethylhexylammonium ion, tetrapentylammonium ion, diethyltrimethyl (2-methoxyethyl) ammonium ion, N-butyl-N- And alicyclic quaternary ammonium ions such as methylpyrrolidinium ion.

<Anion site of ionic liquid>
The anion site constituting the hydrophilic ionic liquid and the hydrophobic ionic liquid used in the present invention is not particularly limited, but the anion site constituting the hydrophilic ionic liquid is a halogen anion such as chloride ion, bromide ion or iodide ion. Carboxylic acid anions such as formate ion, acetate ion and propionate ion, sulfate ion and phosphate ion are preferred. Among them, preferred anion sites are chloride ion, formate ion, acetate ion, and phosphate ion, and the most preferred anion site is acetate ion. In addition, the anion site constituting the hydrophobic ionic liquid is preferably a fluorinated alkyl group-containing anion, and more preferably an anion site containing a perfluoroalkyl group.

<Combination of anion and cation>
The ionic liquid is a combination of a cation and an anion, and a conventionally known cation and anion containing the above-described cation moiety or anion moiety can be used in appropriate combination. In addition to commercially available ionic liquids, those synthesized by known methods can also be used. The synthesis method is not particularly limited, and the cation may be once converted into a salt and then reacted with the anion of the ionic liquid to be obtained, or once converted into a hydroxide and then neutralized with an acid containing an anion.

<Use of ionic liquid>
The said ionic liquid used for this invention may be used individually or in combination of 2 or more types. Moreover, it is preferable that melting | fusing point of the ionic liquid used for this invention is 100 degrees C or less, and 80 degrees C or less is more preferable. More preferably, it is 40 degrees C or less, Most preferably, it is 20 degrees C or less. This is because cellulose can be regenerated at a lower temperature, energy used in the cellulose regeneration process can be reduced, and sugar excessive decomposition due to heating can be prevented.

<Biomass concentration>
The concentration of biomass containing cellulose used in the present invention is not particularly limited. However, when cellulose is added to the ionic liquid, the viscosity of the ionic liquid actually increases. It is preferable to adjust the concentration appropriately. For example, as biomass, a pine pine obtained by heat treatment with an aqueous peroxide solution and freeze-drying the aqueous solution is used, and 1-ethyl-3-methylimidazolium acetate (1-ethyl-3-methylimidazolium) is used as an ionic liquid. In the case of using acetate), it is preferable that the concentration of bay pine is 10% by weight or less with respect to the ionic liquid heated to 120 ° C., and when the operation such as stirring becomes difficult due to the increase in viscosity of the ionic liquid Further dilution is preferred.

<First step>
In the first step of the present invention, the heating temperature of the aqueous solution containing biomass and peroxide is preferably 70 to 90 ° C, more preferably 70 to 80 ° C. In consideration of the reaction between biomass and peroxide, it is preferable that the heating temperature is high. However, since most of the medium is water, heating above the boiling point of water is a safety and cost. This is because it is not preferable in terms of the aspect. The heat treatment time is preferably 5 to 8 hours, more preferably 6 to 7 hours. In consideration of the reaction between biomass and peroxide, it is preferable that the heat treatment time is long, but long-time treatment at a high temperature promotes decomposition of the peroxide, which is preferable in terms of safety. By not.

<Second step>
In the second step of the present invention, when the heat-treated aqueous solution is cooled at −30 ° C. or lower as described above, the cooling time is preferably adjusted appropriately according to the amount and physical properties of the object to be frozen. Moreover, it is preferable to adjust suitably also according to states, such as the quantity of the target object to dry and water content, also about drying time.

<Mixing process>
In the mixing step, which is the previous stage of the third step of the present invention, the dried biomass is stirred in the ionic liquid, preferably at 100 to 130 ° C, more preferably at 110 to 120 ° C. This is because the temperature at the time of stirring is made higher than the boiling point of water to improve the solubility of biomass in the ionic liquid, but the stirring treatment at high temperature promotes the decomposition of the ionic liquid, Further, this is not preferable in terms of cost. Moreover, although stirring time is adjusted according to the temperature of an ionic liquid, Preferably it is 1-3 hours, More preferably, it is 1-2 hours. In consideration of the dissolution of biomass, it is preferable that the agitation treatment time is long, but long-time treatment at a high temperature is not preferable in terms of safety and cost because it promotes decomposition of the ionic liquid. by. In addition, although it adjusts suitably as mentioned above about the biomass concentration in a mixing process, it is 1 to 10 weight% in general, More preferably, it is 3 to 7 weight%.

<Cellulose regeneration process>
In the cellulose regeneration process, which is the latter stage of the third process of the present invention, cellulose is precipitated and regenerated from the mixed liquid of ionic liquid and biomass. In this cellulose regeneration step, the poor solution of cellulose, for example, water, methanol, ethanol, propanol, ethylene glycol, aprotic solvent dimethyl sulfoxide, dimethylacetamide, N-methylpyrrolidone, aprotic polar solvent, It is preferable to add acetone, ethyl acetate, diethyl ether, tetrahydrofuran, nonpolar solvents such as dichloromethane, chloroform, and benzene. More preferably, water is added to the mixture as a poor solvent. In addition, it is preferable that the temperature of water etc. used when regenerating cellulose is heated to the same level as the solution temperature. For example, when water is added at the time of cellulose regeneration, if the temperature difference between the water to be added and the solution is 20 ° C. or less, more preferably 10 ° C. or less, a gel is hardly formed when the solution and water are mixed. On the other hand, if the temperature difference is 30 ° C. or more, a cellulose gel is formed at the time of regeneration, and it is difficult for the enzyme to act in the subsequent enzyme reaction, which may slow the reaction rate.

<Ionic liquid removal process>
Although attempts have been made to saccharify cellulose solubilized in ionic liquid with cellulase, Non-Patent Document 2 reports that cellulase is inactivated in ionic liquid. In Non-Patent Document 3, cellulose is dissolved in an ionic liquid, regenerated by adding a poor solvent such as water, and then the regenerated cellulose is further washed with water to remove the ionic liquid. It has been reported that it is easily degraded by cellulase in water. That is, in the cellulose material before the enzyme reaction, it is preferable to remove the ionic liquid to at least 10 wt% or less, preferably 5 wt% or less, more preferably less than 1 wt%.

  In addition, it is desired that the regenerated cellulose material before the enzyme reaction is not completely dehydrated but wet. The concentration of the solvent mainly composed of water in the regenerated cellulose material before the enzyme reaction is preferably 10% by weight or less, more preferably 3% by weight or less. As a method for separating a solid component (such as a product insoluble in cellulose and water) and a liquid component (such as an ionic liquid or water) from a slurry obtained by adding water to a mixed liquid of ionic liquid and biomass, for example, , Centrifugal precipitation and filtration (gravity filtration, vacuum filtration, pressure filtration, centrifugal filtration, squeezing filtration), dialysis, and the like, which are general solid-liquid separation methods. In addition, it can also be separated using an ion exchange resin as disclosed in Patent Document 13. The above-mentioned solid-liquid separation methods can also be used in combination, for example, the dehydration method normally used in the production of paper as disclosed in Patent Document 14 can be mentioned. The liquid component can be removed with a roll press after removing the liquid component.

<Sugar production method and cellulose decomposition step>
The present invention includes methods for producing monosaccharides, disaccharides, and / or oligosaccharides. The sugar production method of the present invention includes a cellulose decomposition step in which cellulose regenerated through the above-mentioned pretreatment method is enzymatically decomposed with cellulase. According to this method, hydrolysis (saccharification) of the regenerated cellulose is promoted by each step of the pretreatment. Therefore, simpler, lower cost, energy saving and more efficient than the conventional method, the monosaccharide that is a cellulose degradation product, It is possible to produce sugars and / or oligosaccharides.

<Cellulase>
Cellulase is a general term for various enzymes that act to hydrolyze cellulose to glucose. The cellulase used in the present invention is not particularly limited, and examples thereof include β-1,4-endoglucanase, glucan 1,4-β-glucosidase, cellulose 1,4-β-cellobiosidase, and β-glucosidase. The cellulase may be naturally derived or artificially modified. Although it does not specifically limit as a thing of natural origin, It is preferable to use the cellulase etc. which are derived from the genus Trichoderma or Aspergillus. In the present invention, the above-mentioned cellulases can be used alone or in combination of two or more. In addition, cellulases having different origins can be used in combination.

<Buffer solution>
The buffer solution used for the enzyme reaction is not particularly limited, but it is preferable to use a buffer solution having a buffer capacity in a range that matches the pH range suitable for the enzyme activity, including the optimum pH of the cellulase to be used. Since the preferred pH of general cellulase is about 4 to 6, for example, citrate buffer (citrate and sodium citrate), acetate buffer (acetic acid and sodium acetate), citrate-phosphate buffer (citrate) The pH and pH of the cellulase working environment are preferably set to 4 or more and 8 or less, and preferably set to pH 5 or more and 6 or less by adjusting the concentration and pH of the buffer solution appropriately using acid and sodium dihydrogen phosphate). More preferred.

<Temperature during enzyme reaction>
The temperature during the enzyme reaction is not particularly limited, but is preferably selected according to the optimum temperature of the cellulase used. A suitable temperature for a general cellulase is about 35 ° C to 60 ° C.

<Method for quantifying cellulose degradation product>
The amount of cellulose degradation products can be evaluated by measuring the amount of reducing sugar produced by cellulose degradation and the amount of cellulose degradation products themselves. As the quantification method of reducing sugar, the Somogyi-Nelson method and the Park-Johnson method are known, and the degradation rate (saccharification rate) of cellulose is determined by quantifying the reducing sugar decomposed from cellulose using these methods. Can be evaluated. As a method for measuring the amount of cellulose degradation product itself, Patent Document 15 discloses a method for quantifying water-soluble sugar, which is a cellulose degradation product, by HPLC (high performance liquid chromatography). Although methods for quantifying by HPLC after derivatizing sugar have been reported in No. 4, the degradation rate (saccharification rate) of cellulose is evaluated by quantifying cellulose degradation products using these methods. You can also.

<Quantification method of lignin>
Lignin is a polymer compound in which a phenol compound forms a complex three-dimensional network structure. Therefore, the amount of lignin can be quantified by measuring the absorbance of the aromatic compound in the UV-Vis (ultraviolet-visible) region. Non-Patent Document 5 reports a method for quantifying the lignin concentration in an aqueous solution from ultraviolet absorbance at 280 nm using Klarson lignin as a standard substance. By using this method, the lignin concentration in an aqueous solution is evaluated. Can do.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to a following example.

  1.0 g of water was added to 50 mg of lignocellulose bay pine (cellulose content 50% by weight), and 50 μL of 40% by weight peracetic acid (hereinafter abbreviated as PAA, manufactured by Mitsubishi Gas Chemical Co., Ltd.) was added. The mixture was heated and stirred at 80 ° C. for 6 hours (peracetic acid concentration was about 1.9% by weight). After completion of heating and stirring, the aqueous solution containing bay pine and PAA was rapidly frozen in liquid nitrogen and vacuum lyophilized. Subsequently, 1.0 g of 1-ethyl-3-methylimidazolium acetate (hereinafter abbreviated as [EMIm] [OAc]; manufactured by Sigma-Aldrich), which is an ionic liquid, was added to the lyophilized product, and 120 ° C. for 1 hour. Stir with heating. To the obtained reaction liquid, 5.0 mL of water heated to 90 ° C. was added to precipitate and regenerate cellulose, and then centrifuged at 4000 rpm for 20 minutes, and the supernatant (water, ionic liquid, water-soluble sugar and PAA mixture) and precipitates (lignocellulose, regenerated cellulose and other poorly soluble substances). The supernatant was extracted, 5.0 mL of water was added to the remaining precipitate, and centrifugation was repeated three times under the same conditions as described above, and finally the supernatant was removed. The amount of lignin in the supernatant extracted four times in the previous operation was quantified using a UV-Vis spectrophotometer, and the concentration of lignin removed from bay pine, that is, about 20 mL, which is the total amount of supernatant extracted four times. The concentration of lignin was calculated. As a result, the removed lignin concentration was 0.98 mg / mL (see Table 1 described later).

Subsequently, 10 mg of 10 mM citrate-sodium citrate buffer (pH: 5.0) and 20 mg of cellulase C8546 (manufactured by Sigma-Aldrich) derived from Trichoderma reesei ATCC 26921 were added to the precipitate obtained in the previous operation. The cellulase concentration was adjusted to 2 mg / mL (4 units / mL). The mixture was incubated at 40 ° C. and 200 rpm, and the slurry was collected 1 hour and 25 hours after addition of cellulase, and heated with a block heater at 100 ° C. for 20 minutes to inactivate the enzyme. The glucose and cellobiose concentrations in each of the obtained collected liquids were quantified by HPLC, the glucose conversion rate and the cellobiose conversion rate were calculated according to formulas (1) and (2), and the sum was defined as the saccharification rate ( See Table 2 below). As a result, the glucose conversion rate in this operation was 37.7% 1 hour after the addition of cellulase and 85.7% after 25 hours. The cellobiose conversion rate in this operation was 41.1% 1 hour after addition of cellulase and 0% after 25 hours (all decomposed and converted to glucose). Therefore, the saccharification rate was 78.8% 1 hour after addition of cellulase and 85.7% after 25 hours (see Table 2 described later).

  The same operation as in Example 1 was performed except that freeze-drying in Example 1 was changed to hot-air drying. Specifically, 1.0 g of water was added to 50 mg of bay pine, 50 μL of 40 wt% peracetic acid was added thereto, and the mixture was heated and stirred at 80 ° C. for 6 hours. After the heating and stirring, only the supernatant was carefully removed from the aqueous solution containing bay pine and PAA, and the residue was dried for 24 hours in a blast constant temperature dryer (internal temperature: 50 ° C.). Subsequently, 1.0 g of [EMIm] [OAc] was added to the dried product, and the mixture was heated and stirred at 120 ° C. for 1 hour. To the obtained reaction liquid, 5.0 mL of water heated to 90 ° C. was added to precipitate and regenerate cellulose, and then centrifuged at 4000 rpm for 20 minutes, and the supernatant (water, ionic liquid, water-soluble sugar and PAA mixture) and precipitates (lignocellulose, regenerated cellulose and other poorly soluble substances). The supernatant was extracted, 5.0 mL of water was added to the remaining precipitate, and centrifugation was repeated three times under the same conditions as described above, and finally the supernatant was removed. In Example 2, since the supernatant was removed from the aqueous solution before drying in the second step, it was assumed that lignin removed from bay pine could not be quantified accurately, and a UV-Vis spectrophotometer was used. The lignin was not quantified (see Table 1 described later).

  Subsequently, 10 mL of 10 mM citrate-sodium citrate buffer (pH: 5.0) and 20 mg of cellulase C8546 derived from Trichoderma reesei ATCC 26921 were added to the precipitate obtained in the previous operation, so that the cellulase concentration was 2 mg / It prepared so that it might become mL (4unit / mL). The mixture was incubated at 40 ° C. and 200 rpm, and the slurry was collected 1 hour and 25 hours after addition of cellulase, and heated with a block heater at 100 ° C. for 20 minutes to inactivate the enzyme. The glucose and cellobiose concentrations in each of the collected liquids were quantified by HPLC, and the glucose conversion rate, the cellobiose conversion rate, and the saccharification rate that was the sum thereof were calculated. As a result, the glucose conversion rate in this operation was 55.8% 1 hour after the addition of cellulase and 89.2% after 25 hours. The cellobiose conversion rate in this operation was 16.6% 1 hour after addition of cellulase and 0% after 25 hours (all were decomposed and converted to glucose). Therefore, the saccharification rate was 72.4% 1 hour after addition of cellulase and 89.2% after 25 hours (see Table 2 described later).

Comparative Example 1

  Substantially the same operation as in Example 1 was performed, except that the order of the heating and stirring treatment with the PAA aqueous solution in Example 1 and the heating and stirring treatment with [EMIm] [OAc] were reversed and lyophilization was not performed. Specifically, 1.0 g of [EMIm] [OAc] was added to 50 mg of bay pine, and the mixture was heated and stirred at 120 ° C. for 1 hour. Thereto, 50 μL of 40 wt% PAA was added, and the mixture was further heated and stirred at 80 ° C. for 6 hours. After completion of heating and stirring, 5.0 mL of water heated to 90 ° C. is added to an aqueous solution containing bay pine, ionic liquid and PAA to precipitate and regenerate cellulose, and then centrifuged at 4000 rpm for 20 minutes to obtain a supernatant ( Water, ionic liquid, water-soluble sugar and PAA mixture) and precipitates (lignocellulose, regenerated cellulose and other poorly soluble substances). The supernatant was extracted, 5.0 mL of water was added to the remaining precipitate, and centrifugation was repeated three times under the same conditions as described above, and finally the supernatant was removed. The amount of lignin in about 20 mL of the supernatant extracted four times in the previous operation was quantified using a UV-Vis spectrophotometer, and the concentration of lignin removed from bay pine was calculated. As a result, the removed lignin concentration was 0.47 mg / mL (see Table 1 described later).

  Subsequently, 10 mL of 10 mM citrate-sodium citrate buffer (pH: 5.0) and 20 mg of cellulase C8546 derived from Trichoderma reesei ATCC 26921 were added to the precipitate obtained in the previous operation, so that the cellulase concentration was 2 mg / It prepared so that it might become mL (4unit / mL). The mixture was incubated at 40 ° C. and 200 rpm, and the slurry was collected 1 hour and 25 hours after addition of cellulase, and heated with a block heater at 100 ° C. for 20 minutes to inactivate the enzyme. The glucose and cellobiose concentrations in each of the collected liquids were quantified by HPLC, and the glucose conversion rate, the cellobiose conversion rate, and the saccharification rate that was the sum thereof were calculated. As a result, the glucose conversion rate in this operation was 18.2% 1 hour after the addition of cellulase and 68.2% after 25 hours. The cellobiose conversion rate in this operation was 28.6% 1 hour after addition of cellulase and 0% after 25 hours (all were decomposed and converted to glucose). Therefore, the saccharification rate was 46.8% 1 hour after the addition of cellulase and 68.2% after 25 hours (see Table 2 described later).

Comparative Example 2

  The same operation as in Example 1 was performed except that the PAA aqueous solution in Example 1 was changed to water (no PAA was added). Specifically, 1.0 g of water was added to 50 mg of bay pine, and the mixture was heated and stirred at 80 ° C. for 6 hours. After completion of heating and stirring, an aqueous solution of bay pine was rapidly frozen with liquid nitrogen and vacuum lyophilized. Subsequently, 1.0 g of [EMIm] [OAc] was added to the lyophilized product, and the mixture was heated and stirred at 120 ° C. for 1 hour. To the obtained reaction solution, 5.0 mL of water heated to 90 ° C. was added to precipitate and regenerate cellulose, and then centrifuged at 4000 rpm for 20 minutes to obtain a supernatant (water, ionic liquid, water-soluble sugar). Mixture) and precipitates (lignocellulose, regenerated cellulose and other poorly soluble substances). The supernatant was extracted, 5.0 mL of water was added to the remaining precipitate, and centrifugation was repeated three times under the same conditions as described above, and finally the supernatant was removed. The amount of lignin in about 20 mL of the supernatant extracted four times in the previous operation was quantified using a UV-Vis spectrophotometer, and the concentration of lignin removed from bay pine was calculated. As a result, the removed lignin concentration was 0.30 mg / mL (see Table 1 described later).

  Subsequently, 10 mL of 10 mM citrate-sodium citrate buffer (pH: 5.0) and 20 mg of cellulase C8546 derived from Trichoderma reesei ATCC 26921 were added to the precipitate obtained in the previous operation, so that the cellulase concentration was 2 mg / It prepared so that it might become mL (4unit / mL). The mixture was incubated at 40 ° C. and 200 rpm, and the slurry was collected 1 hour and 25 hours after addition of cellulase, and heated with a block heater at 100 ° C. for 20 minutes to inactivate the enzyme. The glucose and cellobiose concentrations in each of the collected liquids were quantified by HPLC, and the glucose conversion rate, the cellobiose conversion rate, and the saccharification rate that was the sum thereof were calculated. As a result, the glucose conversion rate in this operation was 5.8% 1 hour after the addition of cellulase and 69.0% after 25 hours. The cellobiose conversion rate in this operation was 20.7% 1 hour after the addition of cellulase and 0% after 25 hours (all were decomposed and converted to glucose). Therefore, the saccharification rate was 26.5% 1 hour after addition of cellulase and 69.0% after 25 hours (see Table 2 described later).

Table 1 summarizes the concentrations of lignin removed from bay pine in the above examples and comparative examples.

  As apparent from Table 1, in Example 1, it can be said that lignin was efficiently removed from bay pine. The lignin removal concentration in Example 1 was 0.98 mg / mL, whereas in Comparative Examples 1 and 2, the lignin removal concentration in the supernatant of the same amount (about 20 mL) was less than half that in Example 1. It is. That is, from Table 1, it was confirmed that lignin was removed from bay pine more efficiently in Example 1 than in Comparative Example.

Furthermore, the results of saccharification rates in the above-mentioned Examples and Comparative Examples are summarized in Table 2.

  As is clear from Table 2, in Examples 1 and 2, it can be said that the saccharification rate is higher than that of the comparative example at 1 hour after addition of cellulase and at 25 hours. The saccharification rates one hour after addition of the cellulase in Examples 1 and 2 were 78.8% and 72.4%, respectively, whereas in Comparative Examples 1 and 2, 46.8% and 26.5%, respectively. Because there was. Similarly, the saccharification rates 25 hours after addition of the cellulase of Examples 1 and 2 were 85.7% and 89.2%, respectively, while those of Comparative Examples 1 and 2 were 68.2% and 69, respectively. This is because it was 0.0%.

Moreover, from Table 1 and Table 2, in Examples 1 and 2, it was confirmed that the enzymatic degradation reaction of cellulose was performed more efficiently than the comparative example, and the saccharification rate was improved. In the Examples of the present application, it was suggested that the saccharification rate of cellulose is improved by applying any drying method such as hot air drying or freeze drying, but it has flammability, is chemically unstable and decomposes. From the viewpoint that safe substances can be dried more safely, freeze-drying can be said to be a more effective drying method. However, even in Example 2 adopting hot air drying, no safety problem occurred, and safety was ensured.

  From the above results, as a technique for improving the saccharification rate, in addition to the usefulness of the heating and stirring treatment with peroxide such as PAA aqueous solution, heating and stirring in the order of peroxide and ionic liquid such as [EMIm] [OAc] The usefulness of the process to process, the process of drying the heat-processed aqueous solution containing the biomass containing a cellulose and a peroxide, for example using a vacuum freeze-drying method was confirmed. And according to the pretreatment method of the present invention including these useful steps, impurities can be efficiently removed from biomass containing cellulose, and cellulose can be regenerated and saccharified easily and at low cost with energy saving. Is possible.

Claims (13)

  A method for treating biomass containing cellulose, the first step of heat-treating the biomass in an aqueous solution containing peroxide, the second step of drying the heat-treated aqueous solution, and the dried biomass And the third step of regenerating cellulose by precipitating cellulose from the ionic liquid containing biomass, and mixing the biomass containing cellulose with the first step and the second step. And the pre-processing method of the biomass containing a cellulose processed in order of the said 3rd process.   The pretreatment method according to claim 1, wherein the aqueous solution containing the biomass and the peroxide heat-treated in the first step is directly dried as it is in the second step.   The pretreatment method according to claim 1 or 2, wherein in the second step, the method for drying the aqueous solution is a vacuum freeze-drying method.   The pretreatment method according to claim 1, wherein in the third step, water is added to the ionic liquid obtained by stirring and mixing the biomass to precipitate cellulose.   The pretreatment method according to claim 1, wherein the biomass is lignocellulose further containing lignin and / or hemicellulose.   The pretreatment method according to claim 5, wherein the lignocellulose is wood, rice straw, wheat straw, rice husk, bay pine, and / or powder thereof.   The pretreatment method according to claim 1, wherein the cation portion of the ionic liquid is an imidazolium cation, a pyridinium cation, and / or an ammonium cation.   The pretreatment method according to claim 7, wherein the imidazolium cation is 1-ethyl-3-methylimidazolium ion.   The pretreatment method according to claim 1, wherein the anion portion of the ionic liquid is a chloride ion, a phosphate ion, a formate ion, an acetate ion, and / or an ion containing a perfluoroalkyl group.   The pretreatment method according to claim 1, wherein the ionic liquid is 1-ethyl-3-methylimidazolium acetate.   The pretreatment method according to claim 1, wherein in the first step, the peroxide is hydrogen peroxide and / or peracetic acid.   2. The pretreatment method according to claim 1, wherein, in the first step, the concentration of the peroxide in the aqueous solution is 1 wt% to 8 wt%.   A method for producing monosaccharides, disaccharides and / or oligosaccharides, comprising a cellulose decomposing step of enzymatically decomposing regenerated cellulose with cellulase through the pretreatment method according to claim 1.