JPH0217157B2 - - Google Patents

Description

[Detailed description of the invention]

(a) Technical field The present invention relates to a pretreatment method for enzymatic hydrolysis of cellulosic materials. (b) Technical background In recent years, there have been widespread attempts to decompose cellulose, which is abundant in nature, to produce glucose, which is a resource for energy, food, and industrial raw materials. As a method for decomposing cellulose into glucose, enzymatic hydrolysis is being used because the reaction proceeds under hidden pressure and temperature conditions, is specific, and does not cause secondary decomposition. However, natural cellulose contained in wood, rice straw, wheat straw, corn stalks, leaves, bagasse, paper, etc. is generally highly crystalline.
Furthermore, because it is mixed with lignin, ash, etc., it exhibits strong resistance to enzymatic hydrolysis, and has the disadvantage of slow reaction rate and low decomposition rate. In order to eliminate this drawback, prior to enzymatic hydrolysis, it is necessary to increase the amorphous nature of cellulose, such as mechanical crushing using a ball mill, high-temperature heat crushing, destruction by γ-ray irradiation, or use physical pretreatment methods to increase the reaction surface area. Chemical methods are used as pretreatments, such as treatment, destruction of the crystalline structure of cellulose with reagents such as phosphoric acid, sulfuric acid, zinc chloride, cadoxene, and sodium hydroxide, and dissolution and removal of lignin. The above pretreatment method is effective for herbs such as rice straw, wheat straw, bagasse, and hardwoods such as beech and oak. By doing so, it is possible to obtain almost 100% enzymatic hydrolysis of the cellulose contained. However, softwood materials such as cedar and pine have physical structure,
Due to differences in the degree of polymerization and crystallinity of lignin species and cellulose, it exhibits stronger resistance to enzymatic hydrolysis compared to herbs and hardwoods, and cannot be treated with simple physical or chemical pretreatment. However, it is difficult to improve the decomposition rate. This invention was obtained from the above-mentioned viewpoint as a result of studies on a pretreatment method for improving the enzymatic hydrolysis rate of softwood materials such as cedar and pine. (c) Disclosure of the invention That is, this invention involves irradiating softwood materials such as cedar and pine in advance with electron beams or gamma rays at a dose of 10 ⁶ rad or more and 10 ⁸ rad or less, and then treating the material with chlorine or chlorinated water. is a pretreatment method for enzymatic hydrolysis of cellulosic materials, which is characterized by subsequent delignification treatment with an alkaline aqueous solution, and this pretreatment increases the enzymatic hydrolysis rate of softwood materials such as cedar and pine. It is possible. It is known that when a cellulosic material containing lignin, such as wood, is treated with halogen, the aromatic nucleus of the lignin is halogenated, and the lignin becomes dissolved by the alkali. The effect is especially great when the halogen is chlorine, and chlorination of lignin with chlorine water, hypochlorous acid, its salts, etc. is known, and the main reaction is chlorine substitution at the C ₅ and C ₆ positions of the aromatic nucleus. At the same time, chlorine substitution also occurs at the C ₁ position, and demethylation reaction and hydrolysis of phenyl ether also occur. It is known that lignin that has been decomposed in this way can be dissolved and removed with an alkaline aqueous solution. This chlorination reaction can proceed even at room temperature.
If the above-mentioned chlorination is sufficiently carried out and a part of the chlorinated lignin is dissolved and removed with an alkaline aqueous solution, the enzymatic hydrolysis rate can be dramatically increased. For the delignification treatment at this time, an alkaline aqueous solution such as a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, or an ammonia aqueous solution can be used. However, in order to increase the substrate concentration during enzymatic hydrolysis, which is necessary to obtain a highly concentrated sugar solution, and to eliminate adsorption of the enzyme to the substrate residue after saccharification, which is an obstacle to enzyme recovery, It is desirable to remove non-hydrolyzable substances contained in the substrate, such as lignin and ash, as much as possible. For this purpose, alkaline treatment at high temperatures is necessary, and even in cellulosic materials such as cedar and pine, which have sufficiently chlorinated lignin,
Unless delignification treatment is performed with an alkaline aqueous solution at 100°C or higher, it is not possible to obtain an enzymatic hydrolysis rate of 70% or higher for the substrate. The present inventors have previously irradiated softwood materials such as cedar and pine with an electron beam of 10 ⁶ rad or more and 10 ⁸ rad or less. It was discovered that an enzymatic hydrolysis rate of 70% or more of the substrate can be obtained by pretreatment called delignification treatment with . The amount of Klason lignin in cellulose material is
It has been reported that irradiation with electron beams or gamma rays of 10 ⁶ rad or more causes a slight decrease in the amount of Klason lignin. This shows that the reactivity of lignin is increased, and this invention utilizes this improvement in the reactivity of lignin by irradiation with electron beams or γ-rays. Cellulose in cellulosic materials also undergoes a decrease in crystallinity and degree of polymerization when irradiated with electron beams or gamma rays, but there is little effect at irradiation doses of 10 ⁸ rad or less, and lignin is improved through chlorine treatment and alkali treatment. is more reactive, and by selecting the conditions, only lignin can be removed, and most of the cellulose contained in the original cellulose material cannot be subjected to enzymatic hydrolysis. It is possible. However, under conditions where more than 60% of the cellulosic material is dissolved and removed by treatment with chlorine or chlorinated water followed by delignification treatment with an alkaline aqueous solution, non-hydrolyzable substances such as lignin and ash contained in the cellulosic material may be removed. more than the content will be dissolved and removed, that is, a portion of the cellulose in the cellulose material will also be dissolved and removed,
In enzymatic hydrolysis, the rate of enzymatic hydrolysis of cellulose contained in the original cellulosic material decreases. In order to ensure that most of the cellulose contained in the original cellulosic material is enzymatically hydrolyzed and that as little residue remains as possible during the enzymatic hydrolysis, in the delignification treatment with an alkaline aqueous solution following the treatment with chlorine or chlorinated water. , the weight removed by dissolution is 40 to 60 of the original cellulosic material.
% is desirable. Examples of the present invention will be described below along with comparative examples. Comparative example: After grinding cedar into 2 to 3 cm planer scraps using a hammer mill using a 1 mm diameter round hole screen,
The sample to be processed is sized into 42 to 80 mesh particles using a sieve, and 2 g of the sample is mixed with 3.4 g of bleached powder in 200 ml of water, and by adding 0.8 sulfuric acid to this,
After stirring for 10 minutes while generating chlorine, the mixture was filtered through a 200-mesh stainless steel screen and thoroughly washed with water. Pour 50ml of the sample remaining on the stainless steel screen.
The treatment was carried out by immersion in 0.2, 0.2 and 1.0 normal sodium hydroxide aqueous solutions at various temperatures. After 1 hour, an equivalent amount of 10% acetic acid aqueous solution was added to neutralize and stop the reaction, and the reaction was again immersed in a 200 mesh stainless steel screen. Washed with excess water. The weight and moisture content of the obtained sample were measured, and the percentage of weight removed from the original sample by pretreatment and the removal rate were calculated. Removal rate = (1 - absolute dry weight of the sample after treatment / absolute dry weight of the sample before treatment) x 100 (%) Place the pretreated sample in a 100 ml Meyer flask, and adjust the total dry weight to 2%. PH
Add 4.9 of 0.1 molar acetate buffer and add cellulose hydrolase (trade name, Cellulase Onozuka R-10).
was added to a concentration of 1%, and enzymatic hydrolysis was carried out in a reciprocating culture shaker at 45°C. After 48 hours, the amount of glucose produced in the reaction solution was measured, and the rate of enzymatic hydrolysis relative to the substrate and relative to the sample was measured. The enzymatic hydrolysis rate was calculated. Enzyme hydrolysis rate for substrate = Weight of glucose produced / Absolute dry weight of substrate x 100 (%) Enzyme hydrolysis rate for sample = Weight of glucose produced / Absolute dry weight of sample before treatment x 100 (%) The amount of glucose in the reaction solution was measured using a glucose measuring device (Yellow Spring Instrument YSImodel 23A) using an immobilized enzyme membrane. The results obtained are shown in Table 1. Example A Kotskucroft type electron beam accelerator (3.0.MeV, 33mA) was applied to cedar that had been made into 2 to 3 cm planer scraps in advance.
Electron beam irradiation at 24 and 48 Mrad was carried out using a 1 mm φ round hole screen, and the samples were sized into 42 to 80 meshes using a sieve. Pretreatment and enzymatic hydrolysis were performed in the same manner as in the comparative example. The results are shown in Table 1.

【table】

[Table] From the table above, when using the non-irradiated sample of the comparative example, it is necessary to obtain a substrate enzymatic hydrolysis rate of 70% or more.
While an alkaline treatment temperature of 100°C or higher is required, when electron beam irradiation at 24 Mrad is performed,
When 48 Mrad electron beam irradiation is performed at a temperature of 70 to 90°C, an enzymatic hydrolysis rate of more than 70% for the substrate can be obtained by using an alkali treatment temperature of 30 to 50°C, which is around room temperature. The effect of radiation irradiation was clearly observed.

Claims (1)

[Claims] 1. A cellulosic material that has been previously irradiated with electron beams or gamma rays at a dose of 10 ⁶ rad or more and 10 ⁸ rad or less is treated with chlorine or chlorine water, and then delignified with an alkaline aqueous solution. A pretreatment method for enzymatic hydrolysis of cellulosic materials, characterized by: 2. The pretreatment method for enzymatic hydrolysis of a cellulosic material according to claim 1, wherein the cellulosic material is a softwood material such as cedar or pine.