JPS60251897A - Method of pretreatment for enzymatic hydrolysis of cellulosic material - Google Patents

Abstract

PURPOSE:To improve a hydrolysis ratio of needle-leaf tree with an enzyme, by irradiating a cellulosic material with electron rays or gamma-rays, treating it with a halogen, delignifying it. CONSTITUTION:A cellulosic material consisting of needle-leaf tree such as pine, Japanese cedar, etc. is irradiated with 10<6>-10<8> rad electron rays or gamma-rays, immersed in an aqueous solution containing a halogen (e.g., Cl) or a halogen compound (e.g., hypochlorous acid), so that an aromatic nucleus of lignin is halogenated. The cellulosic material is then immersed in an alkali aqueous solution containing NaOH, KOH, NH3, etc. and delignified at >=100 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Technical Field The present invention relates to a pretreatment method for enzymatic hydrolysis of cellulosic materials.

(b) Technological embarrassment 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, dietary supplements, and industrial raw materials.

As a method for decomposing cellulose into glucose, enzymatic hydrolysis is being used because the reaction proceeds under mild pressure and temperature conditions, is specific, and does not cause secondary decomposition.

However, natural cellulose found in wood, rice straw, wheat straw, corn stalks, leaves, bagasse, paper, etc. is generally highly crystalline and is mixed with lignin, ash, etc., so it cannot be degraded by enzymatic hydrolysis. It has the drawbacks of strong resistance to oxidation, slow reaction rate, and low decomposition rate.

In order to eliminate this drawback, prior to enzymatic hydrolysis, blast crushing, mechanical crushing using a ball mill, etc., high-temperature heating crushing, γ
Increasing the amorphous nature of cellulose, such as by destroying it by radiation irradiation,
Pretreatments include physical pretreatments that increase the reaction surface area, and chemical methods such as breaking the crystalline structure of cellulose with reagents such as phosphoric acid, sulfuric acid, zinc chloride, cadoxene, and sodium hydroxide, and dissolving and removing lignin. It is being carried out as

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 stronger resistance to enzymatic hydrolysis than herbaceous and hardwood materials due to differences in physical structure, degree of polymerization of lignin species and cellulose, and degree of crystallinity. However, it is difficult to improve the decomposition rate even with simple physical or chemical pretreatment.

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, the present invention is directed to applying electron beams or gamma rays to softwood materials such as cedar and pine in advance at a rate of 10'ra 6 or more and 10''ra.
This is a pretreatment method for enzymatic hydrolysis of cellulosic materials, which is characterized by irradiating the cellulosic material with a halogen or a halogen compound, followed by delignification treatment with an alkaline aqueous solution. , it is possible to increase the enzymatic hydrolysis rate of softwood materials such as pine.

Halogen treatment of cellulosic materials containing lignin, such as wood, causes halogenation of the aromatic nucleus of the lignin.
It is known that alkali causes lignin to dissolve.

The effect is especially great when the halogen is chlorine, and chlorination of lignin with chlorine water, hypochlorous acid, its salts, etc. is known. Chlorine substitution also occurs at this position, as well as demethylation and phenyl ether hydrolysis. It is known that lignin that has been decomposed in this way can be dissolved and removed by an alkaline aqueous solution.

This chlorination reaction can proceed even at room temperature.

If the chlorination as described in E is carried out sufficiently 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 an I-lium 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 remove the adsorption of enzymes to the substrate remaining after saccharification, which is an obstacle to enzyme recovery. For this purpose, 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 temperature is necessary.
Even in cellulosic materials such as softwood materials such as cedar and pine whose lignin is sufficiently chlorinated, unless delignification treatment is performed with an alkaline aqueous solution at 100°C or higher, the enzymatic hydrolysis rate of the substrate will exceed 70%. You can't get it.

The present inventors have determined in advance that 10'ra 6 or more 10"ra
By irradiating coniferous wood materials such as cedar and pine with electron beams of less than It has been discovered that an enzymatic hydrolysis rate of 70% or more can be obtained.

The amount of Klason lignin in cellulosic material is 10'r
It has been reported that the amount of Klason lignin is slightly reduced by irradiation with electron beams or γ-rays of 6 or more, but the reduction in the amount of Klason lignin is due to the lignin contained in cellulosic materials being irradiated with electron beams or γ-rays. 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.

Cell [ ] - Cellulose in carbonaceous materials also undergoes a decrease in crystallinity and degree of polymerization when irradiated with electron beams or gamma rays.
There is little effect at irradiation doses below ♂rad, and lignin is more reactive through chlorine and alkali treatments;
By appropriately selecting conditions, it is possible to remove only lignin and to subject most of the cellulose contained in the original cellulosic material to enzymatic hydrolysis.

However, under conditions where 60% or more of the cellulosic material is dissolved and removed by treatment with a halogen or halogen compound followed by delignification treatment with an alkaline aqueous solution, the presence of non-hydrolyzable substances such as lignin and ash contained in the cellulosic material In other words, part of the cellulose content in the cellulose material is also dissolved and removed, and in enzymatic hydrolysis, the cellulose content contained in the original cellulose material is rate will drop. In order to ensure that most of the cellulose contained in the original cellulose material is enzymatically hydrolyzed and that as little residue as possible remains during enzymatic hydrolysis,
In the delignification treatment with an alkaline aqueous solution following treatment with a halogen or halogen compound, it is desirable that the weight dissolved and removed is 40-60% of the original cellulosic material.

Examples of the present invention will be described below along with comparative examples.

Comparative Example Cedar that had been made into 2-3 cm planer scraps was ground in a hammer mill using a 1 mm diameter round-hole screen, and then 42-80 mesh particles were sized using a sieve. Put 3.47 into 200yf water,
After stirring for 10 minutes while generating chlorine by adding 0.8 g of sulfuric acid, the mixture was filtered through a 200-mesh stainless steel screen and thoroughly washed with water.

50ν of the sample remaining on the stainless steel screen! 0.
Treatment was carried out by immersion in 2, 0.5 and 1.0 N aqueous sodium hydroxide solutions at various temperatures, and after 1 hour, an equivalent amount of 10% acetic acid aqueous solution was added to neutralize and stop the reaction, and the reaction was stopped again at 200 N. Filtration and water washing were performed using a mesh stainless steel screen.

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.

The sample pretreated above was placed in a 100 wl Mayer flask, and added with +1 H4, so that the total dry weight was 2%.
Add 0.1 molar acetate buffer of 9 and add cellulose hydrolase (trade name, Cellulase Onozuka R-10) to 1%, and perform enzymatic hydrolysis in a reciprocating culture shaker at 45°C. After 48 hours, the amount of glucose produced in the reaction solution was measured, and the enzyme hydrolysis rate for the substrate and the enzyme hydrolysis rate for the sample were calculated.

Weight of produced glucose vs. substrate enzyme hydrolysis rate = X100 (%) Weight of substrate bone dry weight of produced glucose vs. sample enzyme hydrolysis rate - xloo (%) Sample bone dry weight before treatment Note that the amount of glucose in the reaction solution is fixed. The glucose measurement was performed using a glucose measuring device (Yellow Spring Instrument YSI model 23A) using an enzyme membrane.

The results obtained are shown in Table 1.

Example 2 to 33 of cedar which had been made into planer waste were prepared using a Kotscroft electron beam accelerator (3,0, M, V, 33111A).
24 and 48M rad electron beam irradiation was carried out using a 24- and 48-Mrad electron beam irradiation, which was then crushed in a hammer mill using a 1 mmφ round-hole screen, and then sized into 42 to 80 mesh particles using a sieve, which were used as samples for treatment. Pretreatment and enzymatic hydrolysis were performed in the same manner as in the comparative example.

The results are shown in Table 1.

Table 1 From the above table, when using the unirradiated sample of the comparative example, 70
While an alkaline treatment temperature of 100°C or higher is required to obtain a substrate enzymatic hydrolysis rate of 24M r
When irradiated with an ad electron beam, the temperature was 70 to 90°C, and when irradiated with an electron beam of 48M rad, the alkaline treatment temperature was around room temperature, which was 30 to 50°C. Enzyme hydrolysis efficiency was obtained, and the effect of electron beam irradiation was clearly observed.

Patent applicant: New Fuel Oil Development Technology Research Association Agent: Kazu 1) Akira

Claims (1)

[Claims] (1) Electron beam or gamma ray is applied in advance to 10'r a 6 or more 1
1. A pretreatment method for enzymatic hydrolysis of a cellulosic material, which comprises treating a cellulosic material with a density of 1 rad or less with a halogen or a halogen compound, and then delignifying it with an alkaline aqueous solution. (2) A pretreatment method for enzymatic hydrolysis of cellulosic materials according to claim 1, wherein the halogen or halogen compound is chlorine water. (3) The pretreatment method for enzymatic hydrolysis of cellulosic material according to claim 1, wherein the halogen or halogen compound is chlorine generated by the reaction between bleached flour and sulfuric acid. (4) The pretreatment method for enzymatic hydrolysis of cellulosic material according to any one of claims 1 to 3, wherein the cellulosic material is a softwood material such as cedar or pine. .