JP5403587B2 - Monosaccharide production method - Google Patents

Description

  The present invention relates to a method for obtaining monosaccharides from cellulosic biomass containing hemicellulose and lignin by enzyme treatment, and more specifically, a method for producing monosaccharides that undergoes hydrothermal treatment as a pre-stage for enzymatic saccharification of cellulosic biomass. It is about.

  From the viewpoint of preventing global warming, research and development for bioethanol production has been actively promoted. In particular, development of a saccharification / fermentation technology for cellulosic biomass that does not compete with food as a raw material is demanded.

  Cellulose biomass saccharification was mainly performed by hydrolyzing hemicellulose and cellulose in the biomass using sulfuric acid to monosaccharideize, but the corrosion resistance of the reactor material, sugar overdegradation, and sulfuric acid were used. In recent years, as a non-sulfuric acid method, research on a method for obtaining a monosaccharide by hydrolyzing with an enzyme after some kind of pretreatment has been carried out because of the large environmental load due to the above.

  As a pretreatment method, (1) a method of increasing the surface area of biomass by pulverization and reducing the crystallinity of cellulose, (2) decomposing hemicellulose and lignin by steaming / explosion treatment or hydrothermal treatment, cellulose (3) Method of solubilizing and removing hemicellulose by dilute sulfuric acid treatment, (4) Enzyme contact by mainly removing lignin by alkali treatment and swelling cellulose fiber (5) An organosolv method in which biomass is treated in an aqueous solution of an organic solvent (such as ethanol) or an organic acid (such as acetic acid) under high temperature and high pressure, and delignified, or (6) lignin by white rot fungi There is known a biological pretreatment method for decomposing DNA (see Non-Patent Document 1).

  Further, a pretreatment method combining these treatments, for example, a method of performing pulverization after hydrothermal treatment (see Patent Document 1), a method of performing hydrothermal treatment after delignification treatment with ozone (see Patent Document 2), presence of oxygen Below, the biomass is heat-treated in an alcohol solvent, a method in which most of the lignin is oxidatively decomposed to leave hemicellulose and cellulose as solids (see Patent Document 3), the biomass is hydrolyzed with dilute sulfuric acid, and then the residue is removed. A pretreatment method for delignifying in a mixed solution of hydrogen peroxide and an alkaline aqueous solution (see Patent Document 4) has been proposed. Furthermore, a method has been disclosed in which cellulose is reduced in molecular weight to a water-solubilizing component mainly composed of oligosaccharides by hydrothermal treatment, and monosaccharification is advanced by subsequent enzymatic saccharification (see Non-Patent Document 2).

JP 2006-136263 A (published June 1, 2006) JP 2006-141244 A (published June 8, 2006) Japanese Patent Laying-Open No. 2008-5832 (released on January 17, 2008) JP 2008-43328 A (published February 28, 2008)

"Production of ethanol from biomass such as rice straw", supervised by Yasuo Igarashi and Takashi Saiki, published by Regional Resource Recycling Technology Center, pp.61-64 (2008) Journal of the Japan Institute of Energy 86: pp.712-717 (2007)

  However, there are still many items to be improved such as the yield of enzymatic saccharification, energy required for pretreatment, cost and time, and post-treatment of chemicals used, and all of these matters can be satisfied. A pretreatment method has not yet been developed.

  This invention is made | formed in view of said problem, The objective is to provide the efficient processing method for manufacturing a monosaccharide from cellulosic biomass by a saccharification enzyme.

  The hydrothermal treatment method is a promising pretreatment method with a low environmental load that can decompose and remove part of hemicellulose and lignin in a short time of several minutes to several tens of minutes without using chemicals. It has been known that solubilization of hemicellulose proceeds with an increase in hydrothermal treatment temperature, but excessive decomposition of the solubilizate proceeds with an increase in hydrothermal treatment temperature, and when the treatment temperature reaches 240 ° C. or higher, It was also known to break down. Therefore, hydrothermal treatment as a pretreatment method for enzymatic saccharification is usually studied at 230 ° C. or lower. Such hydrothermal treatment method was effective in terms of enzymatic saccharification yield for soft biomass (rice straw, wheat straw, etc.), but was less effective for woody biomass, especially conifers (non-patented) Reference 2).

  In order to efficiently produce monosaccharides from cellulosic biomass, the present inventors have further intensively studied a hydrothermal treatment method as a pretreatment method for enzymatic saccharification, and as a result, the enzymatic saccharification of the cellulose fraction of the residue by hydrothermal treatment. However, as a condition for improving the enzymatic saccharification, it is found that the hydrothermal treatment temperature is extremely important than the hydrothermal treatment time. Even in the case of high-temperature hydrothermal treatment, it has been found that if it is a short time, there is little loss due to excessive decomposition of cellulose, and an efficient pretreatment method can be constructed, and the present invention has been completed.

  That is, the monosaccharide production method according to the present invention includes a first hydrothermal treatment step in which cellulosic biomass is heat-treated with first pressurized hot water, and a first hydrothermal treatment obtained in the first hydrothermal treatment step. A solid-liquid separation step of separating the product into a soluble fraction and a solid residue, a second hydrothermal treatment step of heat-treating the solid residue with a second pressurized hot water, and the soluble fraction, An enzymatic saccharification step in which the second hydrothermal treatment product obtained in the hydrothermal treatment step 2 is saccharified with an enzyme, and the temperature of the second pressurized hot water is the enzymatic saccharification of the cellulose component in the cellulosic biomass. The temperature is improved.

  In the method for producing monosaccharides according to the present invention, the temperature of the second pressurized hot water is preferably 240 to 270 ° C. Moreover, in the monosaccharide manufacturing method which concerns on this invention, it is preferable that the temperature of 1st pressurized hot water is 210-220 degreeC.

  In addition, it is preferable that the 1st hydrothermal treatment material used for a solid-liquid separation process is cooled rapidly after completion of hydrothermal treatment. Moreover, it is preferable that the 2nd hydrothermal treatment material used for an enzyme saccharification process is cooled rapidly after completion of hydrothermal treatment.

  In the enzyme saccharification step of the method for producing monosaccharides according to the present invention, the saccharification of the soluble fraction and the second hydrothermally treated product may be performed separately or mixed together.

  In the monosaccharide production method according to the present invention, the cellulosic biomass is preferably woody biomass.

  By using the present invention, the enzymatic saccharification of the cellulose component in the cellulosic biomass is remarkably improved, and the hemicellulose component is also solubilized and can be efficiently contacted with the saccharifying enzyme. Can be manufactured.

It is a figure which shows the flow of the process process in the monosaccharide manufacturing method which concerns on one Embodiment of this invention. It is a figure which shows the flow of the process process in the monosaccharide manufacturing method which concerns on one Embodiment of this invention.

  One embodiment of the present invention is described below with reference to FIGS.

  The present invention provides a method for producing monosaccharides from cellulosic biomass. In the monosaccharide production method according to the present invention, the cellulosic biomass is hydrothermally treated, the obtained hydrothermally treated product is separated into a soluble fraction and a residue, and the soluble fraction is subjected to an enzyme treatment, The residue is further hydrothermally treated at a high temperature, and the obtained hydrothermally treated product is treated with an enzyme. That is, the monosaccharide production method according to the present invention includes a first hydrothermal treatment step in which cellulosic biomass is heat-treated with first pressurized hot water, and a first hydrothermal treatment obtained in the first hydrothermal treatment step. A solid-liquid separation step of separating the product into a soluble fraction and a solid residue, a second hydrothermal treatment step of heat-treating the solid residue with a second pressurized hot water, and the soluble fraction, An enzymatic saccharification step in which the second hydrothermal treatment product obtained in the hydrothermal treatment step 2 is saccharified with an enzyme, and the temperature of the second pressurized hot water is the enzymatic saccharification of the cellulose component in the cellulosic biomass. The temperature is improved.

  As described above, the present inventors have found that the enzymatic saccharification of the cellulose fraction of the residue by hydrothermal treatment is drastically improved after the time point when the solubilized product becomes excessively decomposed. The present invention employing a heat treatment method has been completed. The temperature that improves the enzymatic saccharification of the cellulose component in the cellulosic biomass is a temperature at which excessive decomposition of the solubilized product obtained by conventional hydrothermal treatment becomes significant. That is, the second pressurized hot water is at a higher temperature than the first pressurized hot water. Therefore, as shown in FIGS. 1 and 2, the first hydrothermal treatment step and the second hydrothermal treatment step are also referred to as a low-temperature hydrothermal treatment step and a high-temperature hydrothermal treatment step, respectively. The heat-treated product and the second hydrothermal-treated product are also referred to as a low-temperature hydrothermal product and a high-temperature hydrothermal product, respectively. In addition, the temperature suitable for the 1st and 2nd pressurized hot water of this invention is mentioned later in this specification.

  For those skilled in the art who are fully aware that the solubilization of the solubilized product progresses as the hydrothermal treatment temperature rises and that the hydrothermal treatment temperature is 240 ° C. or higher, cellulose is degraded. It is not easy to adopt a two-stage hydrothermal treatment method as the pretreatment method, or to adopt a hydrothermal treatment at a higher temperature than the first hydrothermal treatment as the second hydrothermal treatment. By adopting such a two-stage hydrothermal treatment, the present invention significantly improves the enzymatic saccharification of the cellulose component in the cellulosic biomass and improves the yield of monosaccharides.

  Typical examples of the cellulosic biomass used as a raw material for the present invention include woody biomass (wood, thinned wood, etc. (eg, eucalyptus, bay pine, cedar, etc.)) and herbaceous biomass (eg, rice straw, wheat straw). , Bagasse, bamboo, etc.). These may be in a dry state or a wet state, but are preferably pulverized in advance to about 5 mm or less in order to simplify the operation in the hydrothermal process.

  “Pressurized hot water” is hot water that has been pressurized to a saturated vapor pressure or higher and kept in a liquid state. When used in this specification, high-temperature and high-pressure water at 100 ° C. or higher and below the critical point is used. Intended. One skilled in the art will readily appreciate that water can exist in a liquid state by maintaining the pressure of the reaction system above the vapor pressure at that temperature. Examples of the processing method using pressurized hot water include, but are not particularly limited to, batch processing and slurry flow (continuous) processing. Water has the property that the ionic product increases (maximum at around 250 to 300 ° C.) as the temperature rises, and is considered to act effectively on the hydrolysis reaction.

  In the low-temperature hydrothermal treatment step for solubilizing hemicellulose from cellulosic biomass, pressurized hot water (first pressurized hot water) 3 to 10 times on a dry weight basis with respect to the raw material is used for 2 to 20 minutes, Preferably, it is brought into contact with the raw material for 4 to 10 minutes. This solubilizes part of hemicellulose and lignin. If the treatment time is shorter than the above, the desired effect is remarkably lowered, which is not preferable. A treatment time longer than the above is not preferable because excessive decomposition of hemicellulose increases. The temperature of the first pressurized hot water is preferably 180 to 230 ° C, and more preferably 210 to 220 ° C. If the treatment temperature is lower than the above, the desired effect is remarkably lowered, which is not preferable. A treatment temperature higher than the above is not preferable because excessive decomposition of hemicellulose increases. The pressure of the first pressurized hot water is preferably not more than twice the saturated vapor pressure. When the pressure is higher than twice the saturated vapor pressure, there is no effect of increasing the pressure even though the thickness of the reaction vessel is unnecessarily thick, and it is not preferable from the viewpoint of apparatus cost. In addition, the amount of heat for heating the reactor increases, which is not preferable from the viewpoint of energy. When the pressure is equal to or lower than the saturated vapor pressure, water becomes vapor and the hydrolysis ability is significantly reduced.

  In the solid-liquid separation step of separating the low temperature hydrothermal treatment product from the low temperature hydrothermal treatment step into a soluble fraction containing hemicellulose sugars and a solid residue enriched with cellulose, the low temperature water obtained in the low temperature hydrothermal treatment step is used. The heat-treated product is separated into a soluble fraction and a solid residue by a method such as filtration or centrifugation. The soluble fraction contains hemicellulose-based saccharides solubilized in the form of monosaccharides to oligosaccharides, and cellulose is concentrated in the solid residue.

  In addition, it is preferable that the low-temperature hydrothermally-treated product subjected to the solid-liquid separation step is rapidly cooled after the hydrothermal treatment is completed.

  In the high-temperature hydrothermal treatment step in which the solid residue obtained in the solid-liquid separation step is further treated with high-temperature pressurized hot water, the solid residue obtained in the solid-liquid separation step is 3 to 10 times on a dry weight basis. Of pressurized hot water (second pressurized hot water) is contacted with the solid residue for 1 to 10 minutes, preferably 2 to 6 minutes. Thereby, the residual lignin is decomposed, and the affinity between the enzyme and cellulose in the subsequent enzymatic saccharification step is increased. If the treatment time is shorter than the above, the desired effect is remarkably lowered, which is not preferable. A treatment time longer than the above is not preferable because excessive decomposition of cellulose increases. The temperature of the second pressurized hot water may be any temperature that improves the enzymatic saccharification of the cellulose component in the cellulosic biomass, and when used in the present specification, it is higher than 230 ° C. and lower than 280 ° C. Although it is intended, 240-270 ° C is more preferred. If the treatment temperature is lower than the above, the desired effect is remarkably lowered, which is not preferable. A treatment temperature higher than the above is not preferable because excessive decomposition of cellulose increases. Moreover, it is preferable that the pressure of 2nd pressurized hot water is 2 times or less of a saturated vapor pressure. When the pressure is higher than twice the saturated vapor pressure, there is no effect of increasing the pressure even though the thickness of the reaction vessel is unnecessarily thick, and it is not preferable from the viewpoint of apparatus cost. In addition, the amount of heat for heating the reactor increases, which is not preferable from the viewpoint of energy. When the pressure is equal to or lower than the saturated vapor pressure, water becomes vapor and the hydrolysis ability is significantly reduced.

  The subsequent enzymatic saccharification step is a step in which the soluble fraction obtained in the solid-liquid separation step and the high-temperature hydrothermal treatment product obtained in the high-temperature hydrothermal treatment step are saccharified by an enzyme and hydrolyzed to a monosaccharide. The enzyme treatment of the soluble fraction and the high-temperature hydrothermally treated product may be performed together or separately (FIGS. 1 and 2 respectively).

  In addition, it is preferable that the high-temperature hydrothermally-treated product subjected to the enzymatic saccharification step is rapidly cooled after the hydrothermal treatment is completed.

  The soluble fraction obtained in the solid-liquid separation step is an oligosaccharide and / or monosaccharide derived from hemicellulose, and the constituent sugar varies depending on the raw material used. For example, from rice straw, wheat straw and most hardwoods, aqueous solutions of oligosaccharides and / or monosaccharides with pentose xylose and arabinose as unit sugars are obtained. Coniferous wood also includes oligosaccharides and / or monosaccharides containing hexose sugars such as mannose, glucose and galactose, and oligosaccharides and / or simple sugars containing pentose sugars such as xylose and arabinose. An aqueous solution in which sugar is mixed is obtained. On the other hand, the high-temperature hydrothermal treatment product obtained in the high-temperature hydrothermal treatment step is a suspension of cellulose containing glucose as a constituent sugar and a decomposition product of lignin. Therefore, as shown in FIG. 1, in the case where the soluble fraction and the high-temperature hydrothermally treated product are hydrolyzed together by an enzyme for monosaccharification treatment, both hemicellulase and cellulase are used to simultaneously saccharify hemicellulose and cellulose. Need to be added in the saccharification treatment. In addition, as shown in FIG. 2, when the soluble fraction and the high-temperature hydrothermally treated product are separately saccharified with an enzyme, hemicellulase is applied to the soluble fraction, and cellulase is applied to the high-temperature hydrothermally treated product. Need to add. In the enzymatic saccharification treatment shown in FIG. 1 and FIG. 2, it is important to appropriately select and use an enzyme preparation containing a large amount of hemicellulase that effectively decomposes the components of the raw material hemicellulose. . Specifically, it is desirable to use a hemicellulase preparation containing xylanase, xylosidase, arabinofuranosidase and the like for saccharification of soluble fractions derived from rice straw, wheat straw and most hardwoods. In addition, it is desirable to use a hemicellulase preparation containing mannanase, mannosidase and the like for saccharification of the soluble fraction derived from conifers.

[Example 1]
A two-stage hydrothermal treatment and enzymatic saccharification test according to the present invention were carried out using Eucalyptus, a kind of hardwood.

  1.6 g of eucalyptus powder ground to 2 mm or less and distilled water in a stainless steel reactor (outer diameter 19.05 mm x inner diameter 15.75 mm x length 100 mm, horizontal type, thermocouple, pressure sensor, valve) After charging 14.4 g and replacing the air in the reactor with nitrogen gas, an initial pressure of 0.5 MPa was applied using nitrogen gas, the valve was closed and the reactor was sealed, and subjected to low-temperature hydrothermal treatment. The reactor was put into an oil bath heated to 215 ° C. and heat-treated for 5 minutes while being shaken in the horizontal direction. Thereafter, the reactor was immediately transferred into a water bath and rapidly cooled. During this time, the temperature in the reactor reached 210 ° C. in about 2 to 3 minutes, and thereafter maintained at 210 ° C., and then decreased to 100 ° C. or less in about 30 seconds by water cooling. After further cooling to 30 ° C. or lower, the gas in the system was drained, and the contents of the reactor, that is, the low-temperature hydrothermally treated product were all washed out with distilled water, filtered under reduced pressure through a glass filter, and separated into a solution and a residue. The reaction solution and the residue were lyophilized, the yield was determined, the soluble fraction was subjected to enzymatic saccharification, and the residue was subjected to the next high-temperature hydrothermal treatment.

  In the high-temperature hydrothermal treatment step, 1 g of the above dry residue and 9 g of distilled water were added to a 14 mL stainless steel reactor (outer diameter 19.05 mm × inner diameter 14.83 mm × length 80 mm, vertical type, thermocouple, pressure sensor, valve After replacing the air in the system with nitrogen gas, an initial pressure of 0.5 MPa was applied using nitrogen gas, the valve was closed, and the reactor was sealed. In the high-temperature hydrothermal treatment, the reactor is put into a salt bath that has been heated in advance so that the temperature in the reactor becomes 240, 250, and 260 ° C., respectively, and the heat treatment is performed while shaking the reactor in the vertical direction for 5 minutes. After that, the reactor was immediately transferred to a water bath and rapidly cooled. During this time, the temperature in the reactor reached the target temperature in about 1 to 2 minutes, and then maintained that temperature, and then decreased to 100 ° C. or less in about 30 seconds by water cooling. After the temperature in the reactor has decreased to 30 ° C. or lower, venting is performed, and all contents of the reactor are washed out with distilled water, and then freeze-dried, and 240 ° C./5 minutes, 250 ° C./5 minutes, 260 ° C. / 5 minutes of high-temperature hydrothermal treatment was recovered.

  The soluble fraction obtained by low-temperature hydrothermal treatment at 210 ° C./5 minutes contains hemicellulose and some lignin in eucalyptus, but this was subjected to enzymatic saccharification at 45 ° C. for 48 hours. The amounts of xylose, the main component, and other monosaccharides such as arabinose and galactose were quantified. As enzymatic saccharification conditions, commercially available cellulase preparation Acremonium cellulase (Meiji Seika) and commercially available hemicellulase preparation Optimash BG (Genencor) for degrading xylan were 0.03 g (10 FPU / g substrate equivalent) and 40 μL, respectively, per 1 g of substrate. In addition, the reaction was carried out at pH 5.0.

  A hydrothermal treatment residue obtained by subjecting the hydrothermal treatment residue obtained by low temperature hydrothermal treatment at 210 ° C./5 minutes to 240 ° C., 250 ° C. and 260 ° C. for 5 minutes, respectively, It was subjected to enzymatic saccharification over time, and the amounts of glucose and other monosaccharides such as xylose as the main components in the sugar solution were quantified. Enzymatic saccharification conditions were the same as those for the soluble fraction, that is, 0.03 g and 40 μL of commercially available cellulase preparation Acremonium cellulase and commercially available hemicellulase preparation Optimash BG were added per 1 g of substrate, respectively, and the reaction was performed at pH 5.0. It was.

  The results are shown in Table 1. In a low temperature hydrothermal treatment of eucalyptus at 210 ° C. for 5 minutes, 12.4 g of monosaccharide containing 10.5 g of xylose was obtained from the soluble fraction on the basis of 100 g of dry charged raw materials. By performing high-temperature hydrothermal treatment at 240 ° C. for 5 minutes, 36.9 g of monosaccharide containing 36.0 g of glucose is obtained in the enzymatic saccharification of the hydrothermally treated product, and 49.3 g of monosaccharide is obtained by combining the two. It was confirmed that

[Example 2]
Example 2 is an example in which the amount of cellulase added is increased to 40 FPU / g substrate for monosaccharideization. The eucalyptus raw material pulverized to 2 mm or less was subjected to the same low-temperature hydrothermal treatment, solid-liquid separation, and high-temperature hydrothermal treatment as in Example 1, and then commercial cellulase was used as the enzyme saccharification conditions for the soluble fraction and the high-temperature hydrothermal treatment. Preparation Acremonium cellulase and commercially available hemicellulase preparation Optimash BG were added at 0.12 g and 40 μL per gram of substrate, respectively, and reacted at pH 5.0. Other enzyme saccharification conditions are the same as in Example 1.

  The results are shown in Table 2. In this case, the maximum total monosaccharide yield is 52.9 g / 100 g when a combination of low-temperature hydrothermal treatment at 210 ° C./5 minutes and high-temperature hydrothermal treatment at 250 ° C./5 minutes. It was confirmed that the raw material was obtained.

[Comparative Example 1]
1.0 g of eucalyptus powder pulverized to 0.2 mm or less and 9.0 g of distilled water were charged into a stainless steel reactor having an internal volume of 14 mL used in the above Examples, and the same procedure as in the high temperature hydrothermal treatment of Examples 1 and 2 above. Then, hydrothermal treatment was performed for 5 minutes at 220 ° C., 240 ° C., 250 ° C. and 260 ° C., respectively. Each content was transferred onto a glass filter and filtered under reduced pressure to obtain a solution and a residue. The residue was further washed with water and then lyophilized to obtain the yield and subjected to enzymatic saccharification at 45 ° C. for 72 hours. The solution was a stock solution from which hemicellulose in eucalyptus and some lignin and cellulose molecular weight-reduced substances were eluted, and this was subjected to enzymatic saccharification at 45 ° C. for 48 hours. As the enzymatic saccharification conditions of the solution, 78 mg and 25 μL of the commercially available cellulase preparation Acremonium cellulase and the commercially available hemicellulase preparation Optimash BG were added per 10 mL of the substrate, respectively, and the reaction was performed at pH 5.0.

  The amount of enzyme added to the hydrothermal residue and the saccharification reaction conditions were the same as in Example 1, that is, 0.03 g and 40 μL of Acremonium cellulase and Optimash BG were added per 1 g of substrate, and the result was adjusted to pH 5.0 as Comparative Example 1. It is shown in Table 3.

  As the total amount of monosaccharides, hydrothermal pretreatment at 240 ° C./5 minutes was the largest, resulting in a raw material of 35.7 g / 100 g. However, in this hydrothermal treatment condition, the amount of monosaccharides from the soluble fraction is small, and compared with Example 1, the total amount of monosaccharides is only about 70% of Example 1, and the monosaccharide pretreatment in one stage has a monosaccharide content. It was found that the yield was insufficient.

[Comparative Example 2]
The eucalyptus powder was subjected to a one-step hydrothermal pretreatment according to exactly the same method as in Comparative Example 1 to carry out enzymatic saccharification of the solution and the residue. However, the results when the amount of cellulase added to the residue is 40 FPU / g substrate are shown in Table 4 as Comparative Example 2.

  By increasing the amount of enzyme added, the amount of monosaccharides produced from the residue increased, and a hydrothermal pretreatment at 250 ° C./5 minutes resulted in a maximum total monosaccharide amount of 37.8 g / 100 g raw material. Compared to Example 2 with the same enzyme addition amount, the total monosaccharide amount was about 70% of Example 2.

[Example 3]
Next, two-stage hydrothermal pretreatment and enzymatic saccharification tests were performed according to the present invention using 2 mm or less powder of bay pine, which is a kind of conifer.

  The test method was the same as in Example 1, but the low-temperature hydrothermal treatment at the first stage was 220 ° C./5 minutes. The enzymatic saccharification conditions were also the same as in Example 1 except that 10 mg per 1 g of the substrate hemicellulase preparation cellulosin GM5 (HBI) for degrading glucomannan was added instead of Optimash BG.

  The results when the amount of cellulase added to the high-temperature hydrothermally treated product is 10 FPU / g substrate are shown in Table 5 as Example 3.

  In the case of bay pine, the monosaccharide from the soluble fraction obtained by low-temperature hydrothermal treatment contains mannose as the main component, and also contains glucose, xylose, galactose, and arabinose, and the amount thereof is generally larger than that of hardwood. Then, it was confirmed that a low-temperature hydrothermal treatment at 220 ° C./5 minutes yielded a 17.2 g / 100 g raw material monosaccharide containing 9.5 g / 100 g raw material mannose from the soluble fraction. In addition, the low-temperature hydrothermal treatment residue obtained at this time was subjected to high-temperature hydrothermal treatment for 5 minutes at 240 ° C., 250 ° C., 260 ° C., 270 ° C. and 280 ° C., respectively, and further saccharified with an enzyme of 10 FPU / g substrate. As a result of high-temperature hydrothermal treatment at 260 ° C. for 5 minutes, a maximum of 17.0 g of monosaccharides containing 16.9 g of glucose was obtained on the basis of 100 g of dry raw material, and the total amount of monosaccharides combined with monosaccharides from the soluble fraction It could be confirmed that 34.2 g / 100 g raw material monosaccharide was produced.

[Example 4]
A two-stage hydrothermal pretreatment was performed using the same bay pine sample according to exactly the same method as in Example 3. However, in the enzymatic saccharification of the soluble fraction and the high-temperature hydrothermally treated product, the results when the amount of cellulase added was 40 FPU / g substrate are shown in Table 6 as Example 4.

  In the case of bay pine, by increasing the amount of enzyme added, the amount of monosaccharides produced from the high-temperature hydrothermally treated product is greatly increased. The combination of low-temperature hydrothermal treatment at 220 ° C./5 minutes and high-temperature hydrothermal treatment at 260 ° C./5 minutes is 44 It was confirmed that a monosaccharide of .6 g / 100 g raw material was produced.

[Comparative Example 3]
1.0 g of bay pine powder pulverized to 0.2 mm or less and 9.0 g of distilled water were charged into a stainless steel reactor having an internal volume of 14 mL used in the above Examples, and the temperature was 220 ° C. in the same manner as in Comparative Examples 1 and 2. , 240 ° C., 250 ° C. and 260 ° C. for 5 minutes each. Each content was transferred onto a glass filter and filtered under reduced pressure to obtain a solution and a residue. The residue was further washed with water and then lyophilized to obtain the yield and subjected to enzymatic saccharification at 45 ° C. for 72 hours. The solution was a stock solution from which hemicellulose in bay pine, some lignin and a cellulose molecular weight-reduced product were eluted, and this was subjected to enzymatic saccharification at 45 ° C. for 48 hours. Enzymatic saccharification conditions were the same as those in Comparative Example 1, except that 6.25 mg of commercially available hemicellulase preparation cellulosin GM5 for degrading glucomannan instead of Optimash BG was added per 10 mL of substrate.

  The results when the amount of cellulase added to the hydrothermal residue is 10 FPU / g substrate are shown in Table 7 as Comparative Example 3.

  As the total monosaccharide amount, the hydrothermal pretreatment at 220 ° C./5 minutes, which produced a large amount of monosaccharide from the soluble fraction, was the most, but the value was 20 g / 100 g or less, and compared with Example 3. Then, it turned out that the monosaccharide yield is inadequate in one step of hydrothermal pretreatment.

[Comparative Example 4]
The bay pine powder was subjected to one-stage hydrothermal pretreatment according to exactly the same method as in Comparative Example 3 to perform enzymatic saccharification of the solution and the residue. However, the results when the amount of cellulase added to the residue was 40 FPU / g substrate are shown in Table 8 as Comparative Example 4.

  By increasing the amount of enzyme added, the amount of monosaccharides produced from the residue increased, and by hydrothermal pretreatment at 260 ° C./5 minutes, a maximum total monosaccharide amount of 28.8 g / 100 g raw material was obtained. Compared to Example 4 with the same enzyme addition amount, the total monosaccharide amount was about 65% of Example 4.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  By using the present invention, it is possible to produce monosaccharides from cellulosic biomass in high yield, which contributes greatly to the provision of biomass energy to replace fossil fuels, and is very useful for agriculture and agribusiness, It can also contribute to the production of petrochemical products starting from monosaccharides. Furthermore, since the present invention can be used for saccharification of woody biomass, it is also useful for activation of forestry.

Claims (5)

A method for producing monosaccharides from cellulosic biomass,
A first hydrothermal treatment step of heat-treating cellulosic biomass with first pressurized hot water;
A solid-liquid separation step of separating the first hydrothermal treatment product obtained in the first hydrothermal treatment step into a soluble fraction and a solid residue;
A second hydrothermal treatment step of heat-treating the solid residue with a second pressurized hot water, and the soluble fraction and the second hydrothermal treatment product obtained in the second hydrothermal treatment step with an enzyme Including an enzymatic saccharification step to saccharify,
Temperature of the second pressurized hot water, Ri temperature der to improve the enzymatic saccharification of cellulose component of cellulosic biomass,
The temperature of the first pressurized hot water is 180-230 ° C.,
Monosaccharide production method in which the temperature of the second pressurized hot water and said 240 to 270 ° C. der Rukoto. The method for producing monosaccharides according to claim 1, wherein the temperature of the first pressurized hot water is 210 to 220 ° C. The method for producing monosaccharides according to claim 1 or 2 , wherein in the enzymatic saccharification step, the soluble fraction and the second hydrothermally treated product are independently saccharified. The method for producing monosaccharides according to claim 1 or 2 , wherein in the enzymatic saccharification step, the soluble fraction and the second hydrothermally treated product are saccharified together. Monosaccharides method according to any one of claim 1 to 4, wherein the cellulosic biomass is wood-based biomass.

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