JP2009189291A - Saccharifying method for cellulose-containing biomass - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently depolymerizing a biomass composed of a plurality of carbohydrates containing cellulose to monosaccharides such as glucose and xylose as far as possible while suppressing excessive decomposition, thereby solving problems of conventional saccharification techniques. <P>SOLUTION: Provided is a method for saccharifying a biomass containing cellulose and other polysaccharides by using a series of processes comprising the addition of an aqueous solution of sulfuric acid having a sulfuric acid concentration of &ge;0.1 wt.% and &le;5 wt.% to the biomass, the hydrolysis of the biomass for 1 min to 3 hr at &ge;80&deg;C and &le;150&deg;C, the separation of the hydrolysis product into a solid and a liquid, and the enzymatic hydrolysis of the liquid fraction containing isolated oligosaccharides by contacting the fraction with an immobilized enzyme after adjusting the pH of the fraction. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

The present invention relates to a comprehensive saccharification technology for biomass containing cellulose and composed of a plurality of types of sugars. “Saccharification” in the present specification refers to a process of reducing the molecular weight of polysaccharides and oligosaccharides to monosaccharides such as glucose and xylose as much as possible. For the purpose of obtaining oligosaccharides as products, It does not indicate a “partial hydrolysis” step intended to reduce the molecular weight.
The present invention is to separate a biomass raw material into a sulfuric acid aqueous solution-soluble fraction containing sulfuric acid-soluble oligosaccharides and an insoluble fraction containing crystalline cellulose after dilute sulfuric acid hydrolysis under mild conditions with suppressed excessive decomposition. Features. The former is further saccharified by contacting the enzyme-immobilized product after pH adjustment, and the latter is saccharified by a process including hydrolysis of dilute sulfuric acid after concentrated sulfuric acid treatment.

In response to the growing global needs for biofuels, competition for the development of bioethanol production technology derived from carbohydrate-based biomass is taking place on a global scale. In particular, it is considered that the development of utilization technology of lignocellulosic biomass that does not compete with food resources can be the most important breakthrough not only in Europe and the United States but also in Japan.
In addition, when developing so-called biorefinery technology for producing chemical raw materials instead of petrochemical raw materials by converting biomass raw materials into monosaccharides, various carbohydrate resources contained in the raw materials are used as monosaccharides. The recovery process is important. When converting starch-based raw materials to ethanol, it has been evaluated that it is highly practical in terms of cost, energy efficiency, and LCA evaluation. However, there is much room for research and development when using lignocellulose raw materials. It is.

Also, when using potatoes, sweet potatoes, corn, rice, wheat, cassava, sago, etc. as a whole plant, or removing most of the sugar and starch in a simple process and saccharifying the remaining parts together Development of a simple saccharification method from a mixture containing both sugar and starch and fiber is required.
Furthermore, high seasonality is a problem at the stage of securing raw materials such as agricultural waste and primary processing waste, and there is a concern that the number of working days of the factory will be limited and the securing of employment will become unstable. Regardless of specific raw materials, there is a demand for the development of a saccharification system that operates stably throughout the year even when raw materials such as selected residues, weeds, and food waste are mixed to make the material heterogeneous.

  Saccharification technology of lignocellulose raw materials includes acid saccharification technology such as concentrated sulfuric acid method, dilute sulfuric acid two-step method, hydrochloric acid method, chemical pretreatment-enzyme such as dilute sulfuric acid explosion method, neutral water heat treatment method, alkali treatment method Saccharification technology has attracted attention.

  In particular, when saccharification is performed using a woody biomass raw material, a concentrated sulfuric acid method or a dilute sulfuric acid two-stage method has long attracted attention (see Non-Patent Document 1).

The concentrated sulfuric acid method is a method in which sulfuric acid at a concentration solubilizing cellulose is brought into contact with a raw material, and then the whole is diluted with water and hydrolyzed with dilute sulfuric acid to monosaccharides including other polysaccharides.
However, when recovering polysaccharides such as xylan that are susceptible to hydrolysis, the treatment conditions are too harsh and caution is required for overdegradation. Hyperdegradation products such as furfural are known as inhibitors in the ethanol fermentation process, and it is important to suppress their production. So far, a method has been devised in which pre- and pre-treatment (dilute sulfuric acid treatment) for recovering hemicellulose has been devised (Hiroyuki Suzuki, “Scientific Conversion of Biomass and Engineering Approach”, Takeda Shoten) (Issued in 2012)) When using one or more raw materials containing polysaccharides such as various hemicelluloses, pectin, starch, etc., the processing conditions for each raw material composition should be set to maximize the yield of monosaccharides while suppressing excessive decomposition. Need to optimize. In addition, after solubilizing cellulose by concentrated sulfuric acid treatment, it is necessary to lower the concentration of sulfuric acid to perform hydrolysis, so that the concentration of sulfuric acid to be recovered and reused is low, and energy is consumed in the concentration and reuse process. There is a drawback of doing. Furthermore, it is necessary to neutralize after separating and recovering the sugar solution.

The dilute sulfuric acid two-stage method is a method in which a polysaccharide such as hemicellulose that is susceptible to hydrolysis is first decomposed, and then the conditions are tightened to hydrolyze the cellulose.
However, in this method, it is necessary to pay attention to the possibility of hyperdegradation depending on the type of sugar because hydrolysis is performed to monosaccharide by hydrolysis in the first step. In the second stage, glucose is produced from cellulose by slightly strengthening the conditions for dilute sulfuric acid hydrolysis. However, since the reaction conditions are severe, it is necessary to stop the reaction at the stage where excessive decomposition is suppressed. And the yield of glucose derived from cellulose is lowered. Furthermore, the saccharified solution in the first stage dilute acid hydrolysis and the saccharified solution in the second stage dilute acid hydrolysis must be neutralized separately, resulting in the use of a large amount of chemical solution.

Regarding herbaceous biomass, since cellulose tends to be exposed, pretreatment-enzymatic saccharification techniques such as dilute sulfuric acid explosion method, steam explosion method, and alkali treatment method using an enzyme have attracted attention. In each case, enzymatic saccharification of cellulose produced as a solid after pretreatment is positioned as the main saccharification step.
However, cell wall polysaccharides that are susceptible to hydrolysis, amorphous parts of cellulose, sugar in pomace, some starches, etc. are solubilized in the treatment process, and some of them are excessively decomposed and their efficiency Recovery becomes difficult. The pretreatment by steam explosion method under acidic conditions improves the enzymatic saccharification efficiency of cellulose, but the equipment cost at the time of scale-up and the corrosion of the high-pressure treatment equipment by sulfuric acid become problems, and The problem remains. The idea of pretreating corn stover with hot water and maximizing the solubilization rate while decomposing hemicellulose, mainly xylan, into monosaccharides, and suppressing excessive decomposition has been published in the literature (see Non-Patent Document 2). However, it takes advantage of the advantage of using the hot water pretreatment that pH adjustment is unnecessary, and the dilute sulfuric acid treatment method has not been studied using the solubilization rate as an index.

  In addition, no attempt has been made for a saccharification method in which a solubilized saccharide is recovered by a dilute acid pretreatment, and saccharification is performed using an immobilized enzyme using the saccharide as a substrate to improve enzyme utilization efficiency.

In biomass, a plurality of types of carbohydrates having different hydrolysis characteristics are generally present. It is extremely difficult to optimize the process of hydrolysis to monosaccharides according to the characteristics of the polysaccharide, and when considering the use of multiple herbaceous biomass raw materials with seasonality, changes in raw materials It is necessary to construct a highly versatile system that can handle the above.
Furthermore, in addition to glucose, galactose, etc. that can be assimilated by yeast, xylose, arabinose, galacturonic acid, rhamnose, etc., which are constituents of cell wall polysaccharides, can be recovered to expand the scope of application of new conversion technologies. Become.

Hiroyuki Suzuki, “Scientific Conversion of Biomass and Engineering Approach”, Takeda Shoten, published in 2012 Mosier Nathan et al., Bioresour. Technol., 96, 1986-1993 (2005)

  The present invention solves the above-mentioned problems of the prior art, and suppresses the excessive decomposition of biomass composed of a plurality of types of carbohydrates including cellulose to monosaccharides such as glucose and xylose as much as possible. In addition, an object is to provide a method for efficiently reducing the molecular weight.

As a result of intensive studies to achieve the above object, the present inventors have (1) treated rice straw and other biomass with a 1 wt% sulfuric acid aqueous solution and hydrolyzed with an immobilized enzyme. (2) Glucose can be recovered by treating rice straw with an aqueous sulfuric acid solution and treating the residue with concentrated sulfuric acid, and (3) reusing the recovered liquid, The inventors found that sugar contained in the reaction solution before reuse can be obtained as a hydrolyzate, and the present invention has been completed based on these findings.
That is, the present invention is as follows.
(1) To a biomass containing cellulose and other polysaccharides, a sulfuric acid aqueous solution having a sulfuric acid concentration of 0.1 wt% or more and 5 wt% or less is added and hydrolyzed at 80 ° C or more and 150 ° C or less for 1 minute to 3 hours, and then solid-liquid. A biomass saccharification method comprising a series of steps of separating and separating a liquid fraction containing a free oligosaccharide, after adjusting the pH, and contacting with an enzyme-immobilized product to hydrolyze the enzyme.
(2) To the hydrolyzate described in (1) above, the sugar remaining in the insoluble fraction is solubilized as a partial decomposition product of cellulose in an aqueous sulfuric acid solution having a sulfuric acid concentration of 65 wt% or more, and then the aqueous sulfuric acid solution and the partial cellulose decomposition A biomass saccharification method comprising a series of steps of separating a product and hydrolyzing and saccharifying the separated cellulose partial degradation product containing dilute sulfuric acid with dilute sulfuric acid.
(3) including the step of using the solution of the partially decomposed cellulose remaining in the dilute sulfuric acid separated in (2) as at least a part of the dilute sulfuric acid supply source in the dilute sulfuric acid hydrolysis described in the above (1). This is a biomass saccharification method characterized by the following.

  According to the present invention, biomass including cellulose and composed of a plurality of types of sugars can be efficiently reduced to a monosaccharide such as glucose and xylose as much as possible while suppressing excessive decomposition. A method is provided.

Hereinafter, the present invention will be described in detail.
The present invention relates to a biomass saccharification method, and to a biomass containing cellulose and other polysaccharides, a sulfuric acid aqueous solution having a sulfuric acid concentration of 0.1 wt% or more and 5 wt% or less is added, and the temperature is 80 ° C. or more and 150 ° C. or less for 1 minute to 3 hours. After liquid hydrolysis, the liquid fraction containing free oligosaccharide is contacted with enzyme-immobilized product after pH adjustment to hydrolyze the enzyme, and the sugar remaining in the insoluble fraction is appropriately adjusted to a sulfuric acid concentration of 65 wt. After solubilizing cellulose with an aqueous sulfuric acid solution of at least%, the aqueous sulfuric acid solution and cellulose are separated, and the separated cellulose is hydrolyzed by a saccharification method using dilute sulfuric acid.

  The biomass in the present invention is a renewable organic resource excluding fossil resources. Cellulose-containing biomass includes crops, trees, weeds and other higher plants, seaweeds, and separation, mixing, physical, chemical or biological treatments for industrial purposes. The resource which gave etc. is mentioned. For example, soybean as a plant, soybean (seed) as a part thereof, soybean foliage and roots, soybean milk boiled and squeezed soy beans, and squeezed okara are all biomass raw materials.

As the biomass material in the present invention, those containing polysaccharides other than cellulose, such as starch, hemicellulose, and pectin, are used in addition to cellulose.
Examples of biomass raw materials containing cellulose and other polysaccharides include rice, rice straw, rice husk, wheat, straw, corn, corn stover, bagasse, other monocotyledonous stems, bamboo, straw, beans, sago palm, dicotyledonous herbaceous plants For example, stems and leaves, hardwoods, conifers, cassava pomace, paper sludge, waste paper, filter paper powdered cellulose and the like.
Examples of biomass raw materials that accumulate both cellulose and starch include rice, straw, corn, beans, sago palm, and wheat.
Examples of biomass raw materials that accumulate both cellulose and sugar (sucrose) include sugarcane, beet, sorghum and the like. These plant-based biomass raw materials may use the entire plant body or only a part of seeds, stems, leaves, roots and the like.
As biomass, starch can be used, and the residue after one or more starch separations can be used.
Moreover, as a biomass, the residue after sucrose squeezing once or several times can be used including sucrose.
In addition, these biomass raw materials can be used individually by 1 type or in combination of 2 or more types.
In particular, since the present invention is characterized by solubilizing easily degradable carbohydrates other than cellulose by dilute acid hydrolysis, it can be used as a saccharification process for a wide range of raw materials.

  Some biomass raw materials have high perishability. In such a case, while reducing humidity by drying, performing lactic acid fermentation, spraying / immersing acid such as dilute sulfuric acid to lower pH, storing at low temperature, etc., while suppressing microbial contamination and carbohydrate degradation It can be stored. Further, in order to increase the surface area of the raw material, allow the dilute sulfuric acid solution to permeate rapidly, and to quickly release the soluble carbohydrates, it is desirable to pulverize the raw material in a dry or wet manner.

  In the present invention, an aqueous sulfuric acid solution having a sulfuric acid concentration of 0.1 wt% or more and 5 wt% or less is added to biomass containing cellulose and other polysaccharides, and hydrolyzed under conditions of 80 ° C. or more and 150 ° C. or less for 1 minute to 3 hours. A dilute sulfuric acid hydrolysis step is performed.

Unlike the hydrolysis performed in the first stage of the dilute sulfuric acid two-stage method described above, the dilute sulfuric acid hydrolysis step in the present invention is partially or when a sugar susceptible to hydrolysis is hydrolyzed with dilute sulfuric acid. The whole is recovered as a soluble oligosaccharide. Examples of sugars that are susceptible to hydrolysis include starch, sugar, soluble oligosaccharides, hemicellulose, and pectin.
When a readily degradable saccharide is hydrolyzed with dilute acid, the saccharide is converted by a route of solubilization → decomposition into a monosaccharide → overdegradation (conversion to furfurals or tar). Previous dilute sulfuric acid hydrolysis conditions were determined to maximize the recovery of monosaccharides, but this method optimizes the conditions for several readily degradable carbohydrates with different hydrolysis characteristics. Is extremely difficult to perform.
On the other hand, in the present invention, since the purpose is to once collect easily decomposable saccharides and partially decomposed products thereof in the liquid phase, for the main easily degradable carbohydrates, “solubilization → simple A wide range of conditions during “decomposition into sugars” can be set as reaction conditions, and the conditions under which most of the main readily degradable carbohydrates are solubilized can be searched.

  The sulfuric acid concentration during dilute sulfuric acid hydrolysis varies depending on the type of biomass raw material, but falls within the range of 0.1 wt% to 5 wt%, preferably 0.5 wt% to 5 wt%, more preferably 1 wt% to 5 wt%. Should. When the concentration is low, the polysaccharide solubilization efficiency is lowered, and it is necessary to set conditions such as making the reaction temperature and pressure harsh and extending the reaction time. In addition, if the concentration exceeds 5 wt%, the neutralization cost of the reagent is increased, and the influence on the entire conversion cost is increased.

Conditions such as reaction temperature, pressure, and reaction time vary depending on the type of biomass raw material and the sulfuric acid concentration, so it is difficult to determine unambiguously, but the reaction temperature is 80 ° C to 150 ° C and the reaction time is warm. A range of 1 minute to 3 hours including the time is desirable.
For example, in the case of a raw material with a low degree of lignin deposition, such as beet pulp and potato pulp, the reaction proceeds sufficiently even at about 100 ° C. When the hydrolysis reaction is performed at 100 ° C for about 1 hour, it is not necessary to use a pressure vessel, so an open-type vessel can be used, thereby reducing the capital investment cost and improving work safety. It becomes possible. On the other hand, when wood powder such as cedar powder and oak powder is used as a raw material, the degree of lignin deposition is large. It is desirable to perform the treatment under relatively severe conditions such as the following.

  In the first stage hydrolysis reaction, since it is not necessary to recover the sugar as a monosaccharide, the reaction conditions are not only mild, but also the equipment cost of the reaction tank and the use of acid are suppressed. This produces the advantage of suppressing the excessive decomposition reaction.

In the present invention, after performing the hydrolysis reaction of the first stage, solid-liquid separation is performed, and the liquid fraction containing free oligosaccharide is hydrolyzed by contacting with the enzyme-immobilized product after pH adjustment. In addition, the sugar remaining in the insoluble fraction is appropriately solubilized with a sulfuric acid aqueous solution having a sulfuric acid concentration of 65 wt% or more, and then the aqueous sulfuric acid solution and cellulose are separated, and the separated cellulose is hydrolyzed by a saccharification method using dilute sulfuric acid. Here, solid-liquid separation may be performed by centrifugation or the like, for example, centrifugation may be performed at 20,300 × g for about 3 minutes.

The liquid fraction containing free oligosaccharide obtained by solid-liquid separation is hydrolyzed by contacting with an enzyme-immobilized product after pH adjustment.
That is, the soluble oligosaccharide obtained by solid-liquid separation is immobilized after neutralizing it in the range of pH 4 to 9, preferably pH 4 to 6, in consideration of the reactivity and stability of the immobilized enzyme. Hydrolysis to monosaccharides by contact with enzymes.

  As the neutralization of pH, a method using an ion exchange resin, a method using an alkali such as lime and ammonia, a method of performing electrodialysis, and the like can be considered.

Since the reaction between the soluble oligosaccharide and the enzyme is a liquid-liquid reaction, the cost of the enzyme can be reduced by using a bioreactor having the enzyme immobilized thereon. In particular, in order to efficiently enzymatically decompose hemicellulose and pectin having a complex structure, it is necessary to use a cocktail in which a plurality of enzymes are mixed according to the characteristics of the raw material. It is extremely difficult to stably supply the enzyme, and the production cost of the optimized enzyme cocktail corresponding to the raw material is extremely high. Cellulo-oligosaccharides are known to remain when cellulose is hydrolyzed, and methods for immobilizing β-glucosidase to suppress enzyme reaction inhibition and improve monosaccharide yield are studied. (Tu et al., Biotechnol. Lett., 28, 151-156 (2006)), however, it is not envisaged to produce oligosaccharides from readily degradable polysaccharides by dilute acid hydrolysis. Thus, saccharification has not been performed with a focus on easily hydrolyzable polysaccharides of lignocellulosic biomass.
The present invention is characterized by optimizing the yield of the soluble fraction regardless of the amount of monosaccharides produced in order to moderate the reaction conditions, and this process is most efficient by using an immobilized enzyme. To do.

  As for the enzyme to be immobilized, for example, α-amylase, β-amylase, amyloglucosidase, α-D-glucosidase, xylanase, glucomannanase, β-D-xylosidase, β-D-glucosidase, α-L-arabinofurano Sidase, α-D-glucuronidase, β-L-arabinofuranosidase, β-D-mannosidase, acetyl xylan esterase, feruloyl esterase, β-glucanase, β-galactanase, arabinanase, pectin methyl esterase, pectin acetyl esterase, Examples include polygalacturonase, pectinase, α-D-galacturonase, α-D-galactosidase, endoglucanase, cellobiohydrolase I, and cellobiohydrolase II. In addition, for example, when Jerusalem artichoke containing fructan is used as a biomass raw material, the enzyme cocktail to be used depends on the characteristics of the sugar contained in the raw material so that saccharification efficiency is improved by immobilizing and using a fructan degrading enzyme. The composition is different.

Here, when the biomass contains starch, the enzyme to be immobilized on the enzyme immobilization product may contain at least one of α-amylase, β-amylase, amyloglucosidase, and α-D-glucosidase. preferable.
Moreover, when biomass contains hemicellulose, as an enzyme to be immobilized on the enzyme immobilization product, xylanase, glucomannanase, β-D-xylosidase, β-D-glucosidase, α-L-arabinofuranosidase, It preferably contains at least one of α-D-glucuronidase, β-L-arabinofuranosidase, β-D-mannosidase, acetyl xylan esterase, feruloyl esterase, β-glucanase, and β-galactanase. .
Next, when the biomass contains pectin, the enzyme to be immobilized on the enzyme immobilization product is arabinanase, α-L-arabinofuranosidase, pectin methylesterase, pectin acetylesterase, polygalacturonase, pectinase, α It is preferable that at least one of -D-galacturonase and α-D-galactosidase is included.
Further, when the biomass contains β-glucan such as cellulose, the enzyme immobilized on the enzyme immobilization product is at least one of endoglucanase, cellobiohydrolase I, cellobiohydrolase II, and β-glucosidase. It is preferable that one is included.

  By using enzymes that ensure safety such as food grade and feed grade as these enzymes, it is considered that the acceptability at the time of use / treatment of sugar solution or waste solution after saccharification can be ensured.

  As a reaction method using a bioreactor, a method of flowing a sugar solution through a column on which an enzyme is immobilized, a method of contacting a sugar solution in a reaction tank to which beads having immobilized enzyme are added, and the like can be considered.

  The conditions for hydrolysis in contact with the enzyme-immobilized product vary depending on the type of enzyme used and it is difficult to determine uniquely. Usually, however, the reaction temperature is about 37 ° C. to 50 ° C., and the reaction time is 0.5 hours. To 4 hours.

As described above, in the present invention, after dilute sulfuric acid hydrolysis, the liquid phase containing soluble sugar and the solid phase containing insoluble sugar are separated.
Then, as described above, the liquid phase containing soluble sugar, that is, the liquid fraction containing free oligosaccharide, is contacted with the enzyme-immobilized product after pH adjustment and hydrolyzed to monosaccharide.

On the other hand, highly crystalline cellulose is not hydrolyzed to oligosaccharides under dilute sulfuric acid hydrolysis conditions, and remains in the solid phase.
Therefore, in the present invention, it is necessary to change the reaction conditions after solid-liquid separation to solubilize and recover the sugar from the solid phase fraction.

In the present invention, an improved method of the concentrated sulfuric acid method is used to obtain sugar from a solid phase mainly composed of cellulose.
The concentrated sulfuric acid method is a process in which an insoluble polysaccharide such as a cellulose chain is swollen and hydrolyzed by bringing a raw material dried to some extent into contact with sulfuric acid having a concentration of 65 wt% or more at a temperature around room temperature. A method of spraying concentrated sulfuric acid on raw materials such as wood chips is also known (New Energy and Industrial Technology Development Organization “Development item“ Development of high-efficiency conversion technology for biomass energy / Development of technology for producing ethanol for fuel using ethanol fermentation technology, etc., “FY2001-2005 Results Report”, March 2006). Hydrolysis occurs when water is added thereto for dilution, the polysaccharide is further reduced in molecular weight, and is appropriately hydrolyzed to monosaccharide by heating. The separation of monosaccharide and sulfuric acid can be carried out continuously by using not only ordinary ion exchange column chromatography but also ion exchange column chromatography using a pseudo moving bed.
However, as described above, a large amount of dilute sulfuric acid is by-produced, and there is a problem that the recovery and concentration process is costly.

Therefore, in the present invention, a technique for separating and recovering only cellulose solubilized in the presence of an aqueous sulfuric acid solution having a concentration of 65 wt% or more from lignin and sulfuric acid is applied, and the cellulose recovered in the form of a partially decomposed product such as cellooligosaccharide Was saccharified to a monosaccharide with acid.
As a method for recovering such partially decomposed products, a method is used in which cellulose is adsorbed on a material having affinity for cellulose solubilized in a sulfuric acid aqueous solution with a concentration of 65 wt% or higher, and then recovered by reducing the sulfuric acid concentration. can do.

Specifically, for example, it can be performed using a material that adsorbs the cellulose partial decomposition product in the presence of high-concentration sulfuric acid, such as activated clay.
Here, as the material for adsorbing the cellulose partial decomposition product, an inorganic material prepared through a mineral acid treatment step is used as an active ingredient, and specific examples thereof include activated clay.
A material that adsorbs a partially decomposed cellulose, which contains an inorganic substance prepared through mineral acid treatment, such as activated clay, has a low binding property to monosaccharides, so the monosaccharides can easily dissociate. , It will be released toward the dilute sulfuric acid solution.
Cellulose is adsorbed to the material using a material having an affinity for solubilized cellulose in a sulfuric acid aqueous solution having a concentration of 65 wt% or more, preferably 72 wt% or more, and the sulfuric acid aqueous solution and cellulose partial decomposition product (solubilized sugar) Then, the cellulose is eluted from the material by reducing the concentration of the aqueous sulfuric acid solution as much as possible.
This cellulose partial decomposition product is further hydrolyzed with dilute sulfuric acid. Conditions such as sulfuric acid concentration, reaction temperature, pressure, reaction time, etc. when performing dilute sulfuric acid hydrolysis are the same as in dilute sulfuric acid hydrolysis in the first stage. Cellulose or oligosaccharide whose crystallinity has been greatly reduced by partial degradation is rapidly degraded by dilute acid.

The concentration of sulfuric acid recovered by this process becomes high, and the concentration cost for reuse can be suppressed.
As described above, cellulose recovered as a solubilizate by the latter method can be hydrolyzed with dilute sulfuric acid to be hydrolyzed to a monosaccharide.
A solution containing both cellulose and dilute sulfuric acid (solution of partially decomposed cellulose remaining in dilute sulfuric acid) eluted from an adsorbent material such as activated clay with reduced concentration of sulfuric acid is hydrolyzed with dilute acid in the first stage. It can be used as an acid for time.
In this case, in the dilute acid hydrolysis step in the first stage, the partially decomposed product of cellulose is also reduced in molecular weight. By immobilizing the cellulolytic enzyme system on the column, the yield of glucose is further improved.
This method makes it possible to improve the efficiency of the neutralization step of sulfuric acid obtained from the two-stage process, and separates the sugar solution from the hemicellulose fraction and the sugar solution from the cellulose fraction, as in the dilute sulfuric acid two-step method. It is possible to improve the neutralization step.

Such a comprehensive saccharification system characterized by recovering cellulose as a polysaccharide or a partially decomposed product while minimizing dilution of concentrated sulfuric acid has not been proposed so far.
Similar to the known concentrated sulfuric acid method, in the present invention, the conditions are controlled so that concentrated sulfuric acid works effectively by dehydrating the solid phase portion before the concentrated sulfuric acid treatment and controlling the water content. Is desirable. For lignin obtained as a by-product, it is possible to use a processing technique such as turning it into fuel as a dehydrated cake.

The present invention is a process suitable for recovering polysaccharides in a plurality of types of biomass with high efficiency. Part of the starch remaining in the starch squeezed residue remains in the cell wall and is not easily eluted by heating under neutral conditions. On the other hand, the residual starch is efficiently liberated by heating in dilute acid.
Thus, the present invention is also suitable for conversion of raw materials containing starch and the entire plant body. As for the sugar remaining in the squeezed residue, the stirring proceeds to the inside with the progress of the dilute sulfuric acid decomposition of the cell wall component, and elution is accompanied with it, so that the soluble carbohydrate can be efficiently recovered. Thus, since it is the reaction method corresponding to various raw materials, it can respond flexibly to generation | occurrence | production of a highly seasonal agricultural residue, a primary processing residue, etc.

When efficiently saccharifying biomass and resource crops as whole crops, most of starch and sugar can be recovered from biomass as high-purity fermentable glucose and sucrose. .
For example, when using whole rice with grain, the whole is crushed and the whole amount is saccharified by the method of the present invention, or the grain part is ground by dry pulverization or wet pulverization. After separation and collection as a powder, pulverization residue such as rice husks and saccharified leaves are saccharified by the method of the present invention, whereby the sugar derived from starch remaining together with sugar derived from lignocellulose can be collected.

  The starch initially separated by the latter method can be efficiently saccharified by saccharification using a known enzyme saccharification technique or the like. In an industrial starch or sugar recovery process, it is common to recover sugars several times to obtain a high yield, but the amount of liquid used, processing cost, etc. are improved according to the number of times.

  In the saccharification process of the present invention for the purpose of comprehensive utilization of the whole biomass raw material, the starch and sugar recovery process is omitted or simplified, and the cost is reduced, and the starch and sugar remaining in the cell wall portion are subjected to dilute sulfuric acid treatment. Thus, it can be saccharified and recovered together with the cell wall constituting sugar.

As described above, the biomass can be saccharified.
Ethanol can be produced by adding ethanol-producing bacteria to the saccharified product thus obtained for fermentation. As the ethanol-producing bacteria, those usually used for ethanol production can be used. Specific examples include Saccharomyces cerevisiae.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.

Example 1 (Immobilized enzyme treatment effect on neutralized product of each biomass raw material after dilute sulfuric acid treatment)
In a 1.5 ml plastic open container, take 10.0 mg each of ground rice straw, potato pulp powder, beet pulp powder or cassava starch squeeze, add 1 wt% sulfuric acid aqueous solution, close the cap, and stir with a vortex mixer Hydrolysis was performed in a heat block at 100 ° C. for 1 hour. Thereafter, centrifugation was performed at 20,300 × g for 3 minutes, and 0.5 ml of the supernatant was transferred to another container. This was neutralized to near neutrality with a small amount of 1N aqueous sodium hydroxide solution to obtain a neutralized sample.
Separately, two 50 ml Falcon tubes containing 1 ml of 1 M potassium phosphate buffer (pH 7.0) were prepared. 3 mg Viscozyme L (Novozyme Japan) and 3 mg Pectinex Ultra SP-L (Novozyme Japan) are added to one, and 20.8 mg amyloglucosidase (A9228, SIGMA) to the other 20.3 mg of α-amylase (A-6380, SIGMA) was added (referred to as PV and AA, respectively).
Subsequently, 500 mg of immobilized enzyme production resin (Oipergit C, Higuchi Shokai Co., Ltd.) was added to each, and shaken gently at room temperature for 36 hours.
Thereafter, each solution portion was discarded, and the resin portion was washed 5 times with 40 ml of cold water, and then suspended in 100 mM sodium acetate buffer (pH 5.0) to make 8 ml.
Place 200 μl of PV suspension in an Eppendorf tube and treat it with 1 wt% sulfuric acid at 100 ° C for 1 hour in the supernatant of the reaction mixture of rice straw (Straw), potato pulp (PP) or beet pulp (BP). 200 μl of Japanese product was added, and the reaction was performed at 37 ° C. for 1 hour at 12 revolutions per minute.
As a control, a sample obtained by adding 200 μl of 100 mM sodium acetate buffer (pH 5.0) instead of 200 μl of suspension to each neutralized product was used.
Similarly, for the suspension of AA, 200 μl of a neutralized sample of Cassava starch pomace (Cassava) was used.
After the reaction, the amount of reducing sugar in the supernatant was quantified by the Somogi-Nelson method using glucose as a standard substance. The results are shown in Table 1.
After the reaction, the Eppendorf tube containing the resin and the reaction solution is centrifuged to remove the supernatant, and then 1 ml of 50 mM sodium acetate buffer (pH 5.0) is added to the resin remaining in the precipitate. In addition, light agitation with a vortex mixer, centrifugation, and discarding the supernatant were repeated 6 times to wash the resin. Thereafter, the same buffer solution was added to make 200 μl, and this was used to conduct the above-described experiment for reacting with the neutralized product derived from each biomass raw material. The results are shown in Table 1.

  According to the results in Table 1, the saccharides solubilized after the dilute sulfuric acid treatment from each raw material increased the amount of reducing sugars by the action of the immobilized enzyme, suggesting that the immobilized enzyme is acting effectively. . In addition, the enzyme activity was maintained even after the enzyme-immobilized resin after the enzyme reaction was washed with a buffer solution and reused.

Example 2
3.00 g of rice straw powder was weighed out, put into a 100 ml glass bottle, 30 ml of 5 wt% sulfuric acid was added, and then treated at 121 ° C. for 60 minutes.
After returning this to room temperature, it was made up to 100 ml with ion-exchanged water. This was suspended and 3.33 ml was taken and placed in a 15 ml falcon tube. The supernatant was removed by centrifugation, and the precipitate was dried at 60 ° C. for 12 hours.
To this, 1 ml of 72 wt% sulfuric acid was added at room temperature, and the mixture was treated for 1 hour while stirring with a vortex mixer and suspending with a glass rod.
Using the supernatant obtained by centrifugation, this was placed in a spin column (UFC30LG00, Millipore) and centrifuged at 4700 × g, and 800 μl of the filtrate was recovered.
Dispense 200 μl of this filtrate into four spin columns with 150 mg of air-dried activated clay placed on the filter, let stand at room temperature for 15 minutes, and then centrifuge at 4700 × g for 30 minutes Went. After removing the filtrate (filtrate A, total amount 571 μl), add 200 μl of ion-exchanged water to each spin column and let stand at room temperature for 15 minutes in the same way, and then centrifuge at 4700 × g for 30 minutes. Separation was performed. After collecting the filtrate (filtrate B, total volume 730 μl), 100 μl of filtrate B was mixed with 5.00 mg rice straw powder, and 400 μl of ion-exchanged water was added (sample 1). In contrast, 400 μl of ion-exchanged water (Sample 2) and 5.00 mg of rice straw powder with 500 μl of ion-exchanged water (Sample 3) were prepared and stirred with a vortex mixer. The reaction was carried out at 60 ° C for 60 minutes. Thereafter, a part of the centrifugal supernatant was neutralized, and reducing sugar was quantified using glucose as a standard substance by the Somogi-Nelson method.

  As a result, 9.44 μmol of reducing sugar was generated in sample 1, whereas 1.62 μmol of sample 2 and 0.91 μmol of reducing sugar were generated in sample 3. As described above, the sugar solution containing the dilute acid collected after the concentrated sulfuric acid treatment was reused to carry out dilute acid hydrolysis of the rice straw powder.

Example 3
3.00 g of rice straw powder was weighed out, put into a 100 ml glass bottle, 30 ml of 5 wt% sulfuric acid was added, and then treated at 121 ° C. for 60 minutes.
After returning this to room temperature, it was made up to 100 ml with ion-exchanged water. This was suspended and 3.33 ml was taken and placed in a 15 ml falcon tube. The supernatant was removed by centrifugation, and the precipitate was dried at 60 ° C. for 36 hours.
To this, 1 ml of 72 wt% sulfuric acid was added at room temperature, and the mixture was treated for 1 hour while stirring with a vortex mixer and suspending with a glass rod.
The supernatant obtained by centrifuging this was placed in a spin column (UFC30LG00, Millipore) and centrifuged at 4700 × g to collect the filtrate (filtrate 1).
200 μl of this filtrate was added to the same spin column in which 150 mg of activated clay was placed on the filter, allowed to stand at room temperature for 15 minutes, and then centrifuged at 4700 × g for 30 minutes. After collecting the filtrate (filtrate 2, total volume 134 μl), add 200 μl of ion-exchanged water to the spin column and let stand at room temperature for 15 minutes in the same manner, and then centrifuge at 4700 × g for 30 minutes. It was. The filtrate was recovered as filtrate 3 (178 μl). Similarly, 200 μl of ion-exchanged water was added and the mixture was allowed to stand at room temperature for 15 minutes and then centrifuged at 4700 × g for 30 minutes, and the filtrate was collected (filtrate 4, 154 μl). Filtrate 1 and 2 are diluted 8-fold with ion-exchanged water, filtrate 3 is diluted 2-fold with ion-exchanged water, and filtrate 4 is added by adding an equal amount of 72 wt% sulfuric acid and 6-fold amount of ion-exchanged water. Hydrolyzed samples were prepared. These were hydrolyzed at 100 ° C. for 60 minutes, and the amount of glucose produced after neutralization was quantified using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).

  As a result, 11.5 μmol of glucose was produced from 200 μl of filtrate 1, and 0.97 μmol, 5.29 μmol and 2.09 μmol of glucose were produced from the corresponding amounts of filtrate 2, filtrate 3 and filtrate 4, respectively. Assuming that each filtrate is hydrolyzed quantitatively, 64% of the glucose residues obtained from filtrate 1 are present in the ion-exchanged water eluate and converted to glucose by dilute sulfuric acid treatment. It has been suggested.

The present invention is expected to be a breakthrough in the saccharification process of herbaceous and woody lignocellulosic biomass.
In addition, the present invention includes saccharides and starches in addition to lignocellulose, which is efficient for raw materials with high sugar content and starch content after coarsely collecting saccharides and starch from biomass as a whole crop or resource crops. This is thought to lead to the development of a typical saccharification technology.
In biomass, a plurality of types of carbohydrates having different hydrolysis characteristics are generally present.
The present invention exhibits high effectiveness as a technique for optimizing the process of hydrolyzing to a monosaccharide according to the characteristics of the polysaccharide.
In addition, when considering using multiple herbaceous biomass raw materials and food wastes with seasonality, it is possible to propose a highly versatile comprehensive saccharification system that can cope with changes in raw materials.
Furthermore, in the present invention, a mild reaction using dilute sulfuric acid and a saccharide hydrolase is assumed. When considering the use of by-products and waste treatment, the environmental impact is low and the acceptability is high. -It is expected that a processing method can be provided.
Since saccharification products are finally produced under mildly acidic to neutral pH conditions, they can be converted into useful substances such as ethanol through fermentation processes using known hexose, pentose or uronic acid as raw materials. It becomes.
The present invention is expected to develop a regional energy production process and biorefinery process with mild reaction conditions.

Claims (3)

To a biomass containing cellulose and other polysaccharides, a sulfuric acid aqueous solution having a sulfuric acid concentration of 0.1 wt% or more and 5 wt% or less is added, hydrolyzed at 80 ° C or more and 150 ° C or less for 1 minute to 3 hours, and then solid-liquid separated. A biomass saccharification method comprising a series of steps in which a liquid fraction containing a free oligosaccharide is subjected to enzyme hydrolysis after contacting with an enzyme-immobilized product after pH adjustment. The saccharide remaining in the insoluble fraction is solubilized with the hydrolyzate according to claim 1 by dissolving cellulose in a sulfuric acid aqueous solution having a sulfuric acid concentration of 65 wt% or more as a partial decomposition product, and then separating the sulfuric acid aqueous solution and the cellulose partial decomposition product. The biomass saccharification method comprising a series of steps of hydrolyzing and saccharifying the separated cellulose partial decomposition product containing dilute sulfuric acid with dilute sulfuric acid. The step of using the solution of the partial decomposition product of cellulose remaining in the dilute sulfuric acid separated in claim 2 as at least a part of the dilute sulfuric acid supply source in the dilute sulfuric acid hydrolysis according to claim 1, Biomass saccharification method.