A method for removing lignin from lignocellulose raw materials using composite enzymes

Abstract

The invention discloses a method for removing lignin from a lignocellulose raw material, the method comprising the following steps: placing the lignocellulose raw material in a mixed solution of hydrogen peroxide and glacial acetic acid, adding a composite enzyme to react, and obtaining the lignocellulose raw material from which lignin is removed, wherein the composite enzyme is composed of perhydrolase and laccase. The method of the invention achieves the purpose of removing or separating lignin by adding hydrogen peroxide and acetic acid to produce a synergistic effect with the composite enzyme to oxidize toxic and difficult-to-degrade organic compounds, and the whiteness of the lignocellulose raw material treated by the method can reach 76ISO, and a finished product with high whiteness and high lignin removal rate can be obtained by a one-step method.

Description

Technical Field

[0001] The invention belongs to the technical field of biomass, and in particular relates to a method for removing lignin from lignocellulose raw materials. Background Art

[0002] In lignocellulosic raw materials, its composition usually includes three parts: lignin, cellulose and hemicellulose. Among them, lignin, cellulose and hemicellulose are cross-linked with each other, and lignin is rigid and not easy to corrode, and usually plays a supporting role in cells.

[0003] The traditional method for removing lignin is the strong alkali removal method. However, this treatment method has problems such as the inability to regenerate and circulate waste liquid, residual metal ions, and environmental pollution during the reaction process. The processed lignin raw materials require pre-treatment such as pressurization, crushing, grinding, and pre-soaking. The process is complicated, and the maximum thickness of the processed raw materials needs to be less than 0.5mm. It will also cause lignin to be converted into lignin salts, changing the original structure of lignin, which is not in line with the green, efficient and recyclable chemical concept.

[0004] Therefore, it is an urgent problem to be solved by technicians in this field to provide a method for removing lignin with simple processing technology, environmental friendliness, low production cost and high removal efficiency. Summary of the invention

[0005] In view of this, the present invention provides a method for removing lignin from a lignocellulosic raw material. The method adds hydrogen peroxide and acetic acid to produce a synergistic effect with a composite enzyme to oxidize toxic and difficult-to-degrade organic compounds, thereby achieving the purpose of removing or separating lignin. The whiteness of the lignocellulosic raw material treated by this method can reach 76ISO, and a finished product with high whiteness and high lignin removal rate can be obtained in one step; and the thickness of the treated lignocellulosic raw material can reach 30 mm, and there is no need to crush or grind it into fine particles in advance, so the processing efficiency is greatly improved.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for removing lignin from a lignocellulosic feedstock, the method comprising the following steps:

[0008] The lignocellulose raw material is put into a mixed solution of hydrogen peroxide and glacial acetic acid, and after adding a composite enzyme for reaction, the lignocellulose raw material with lignin removed is obtained.

[0009] The present invention forms a composite enzyme by coupling perhydrolase-laccase dual enzymes, and utilizes the synergistic effect of the composite enzyme with hydrogen peroxide and acetic acid to generate hydroxyl radicals (·OH), organic free radicals (RC·), superoxide free radicals (·O 2- ) and singlet oxygen ( 1O2) and other highly active substances are used to separate lignin from lignocellulosic raw materials, solving the problem of difficult treatment of highly polluted waste liquid from lignocellulosic raw materials during pulp production.

[0010] Preferably, the concentration of the hydrogen peroxide is 20-50%, and the concentration of the glacial acetic acid is 95-99%;

[0011] The mass ratio of the hydrogen peroxide to the glacial acetic acid is 1:0.2-4.5.

[0012] Preferably, the concentration of the hydrogen peroxide is 30%, and the concentration of the glacial acetic acid is 99%.

[0013] Preferably, the mass ratio of the lignocellulosic raw material to the mixed solution is 1:1.0-3.0.

[0014] Preferably, the composite enzyme is composed of perhydrolase and laccase, and the mass ratio of effective components in the composite enzyme is perhydrolase:laccase=1:0.5.

[0015] Preferably, the mass ratio of the complex enzyme to the mixed solution is 1:50-5000.

[0016] Preferably, the thickness of the lignocellulose raw material is 0-30 mm.

[0017] Preferably, the cooking parameters are: temperature of 25-40° C., and reaction time of 4-6 h.

[0018] Preferably, the temperature is 30°C.

[0019] Preferably, the filtrate obtained after the reaction is dried to obtain lignin.

[0020] Preferably, the lignocellulosic raw material is selected from at least one of pine, eucalyptus, basswood and fir, and the delignified lignocellulosic raw material can be used for pulp production.

[0021] Preferably, the lignocellulosic raw material is any one of bamboo, coniferous wood and hardwood.

[0022] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The present invention provides a method for removing lignin from a lignocellulose raw material. In plant lignocellulose raw materials, lignin, cellulose and hemicellulose are cross-linked with each other, and lignin is rigid and not easily corroded, and usually plays a supporting role in cells. However, due to the polyhydroxy structure of lignin, when treated with an alkali method, lignin will attach new groups to become lignin salts and dissolve in water, which destroys the original structure of lignin. Under acidic conditions, lignin usually maintains its original structure. Therefore, the present invention uses a perhydrolase-laccase dual enzyme coupling to form a composite enzyme, and utilizes its synergistic effect with glacial acetic acid and hydrogen peroxide to generate hydroxyl radicals (·OH), organic free radicals (RC·), superoxide free radicals (·O 2- ) and singlet oxygen ( 1 O2) and other highly active substances can achieve a 90% lignin removal rate and an 87% hemicellulose removal rate in the lignocellulose raw material within 6 hours. This method does not produce wastewater, and the fiber whiteness after treatment can reach 76ISO. This method does not produce wastewater and does not destroy the original structure of lignin. The solution can be reused to reduce environmental pollution. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1

[0026] A method for removing lignin from a lignocellulose raw material, comprising the following steps:

[0027] (1) Preparation of compound enzyme: Perhydrolase and laccase are both freeze-dried powders stored at 4°C. Sterile water is added to the freeze-dried powders and shaken well. Perhydrolase and laccase are transferred to 50 μl of each 50 ml of culture medium containing 50 mg / ml kanamycin (kanamycin was purchased from MACKLIN-K917729), the culture medium is LB liquid culture medium, and its components are 1% (w / v) tryptone, 0.5% (w / v) yeast extract, and 1% (w / v) NaCl; when the surface of the culture medium is covered with white filamentous fungi, the surface of the culture medium is washed with sterile water, and the surface hyphae are scraped off with an inoculation loop; when the hyphae fall off, the liquid on the surface of the solid culture medium is sucked with a pipette and filtered, and the filtered liquid is placed in a centrifuge tube, centrifuged at 6000 rpm for 10 min, the supernatant is discarded, and the bacteria are collected; after washing with a buffer solution, the bacteria are centrifuged again and collected, and the bacteria are resuspended in a buffer solution in a centrifuge tube; 50% ultrasonic power is used for 2s and 2s rest, and the total time is 10min, and finally centrifuged at 12000 rpm for 10min to collect the supernatant, which is the enzyme solution;

[0028] The perhydrolase-laccase dual enzyme coupling complex enzyme was prepared according to the mass ratio of the effective components of perhydrolase and laccase being 1:0.5;

[0029] (2) Weigh 78.08 g of hydrogen peroxide (30%) and 165.09 g of glacial acetic acid (99%) and mix them to obtain a mixed solution, that is, the mass ratio of hydrogen peroxide to glacial acetic acid is 1:2.1; then take 0.243 g of the composite enzyme and mix it in a beaker with 122 g of eucalyptus chips (50*50*10 mm, with a water content of less than 55%), that is, the mass ratio of the lignocellulose raw material, the mixed solution, and the composite enzyme is 50:100:0.1, heat it in an oil bath, raise the temperature to 30°C within 20 minutes, keep the temperature constant for 4 hours, and cool it to room temperature after the reaction is completed;

[0030] The reaction solution is subjected to solid-liquid separation to obtain raw material solid and liquid, wherein the complex enzyme solution can be reused; during the reaction process, glacial acetic acid can be recycled by condensation reflux; the separated solid can be uniformly decomposed by a grinder or a pulper to prepare pulp, wherein the pulp whiteness can reach 76ISO, the lignin removal rate is 90%, and the hemicellulose removal rate is 87%;

[0031] Example 2

[0032] (1) Preparation of complex enzyme is the same as in Example 1

[0033] (2) 175.38 g of hydrogen peroxide (30%) and 62.22 g of glacial acetic acid (99%) were mixed to obtain a mixed solution, i.e., the mass ratio of hydrogen peroxide to glacial acetic acid was 1:0.35; 0.237 g of the composite enzyme and 118.8 g of eucalyptus chips (50*50*10 mm, with a moisture content of less than 55%) were then taken, i.e., the mass ratio of the lignocellulose raw material, the mixed solution, and the composite enzyme was 50:100:0.1, and the mixture was heated in an oil bath to 30° C. within 20 min, then kept at a constant temperature for 4 h, and cooled to room temperature after the reaction was completed;

[0034] The reaction solution is subjected to solid-liquid separation to obtain raw material solid and liquid, wherein the complex enzyme solution can be reused; during the reaction process, the glacial acetic acid can be recycled by condensation reflux; the separated solid can be uniformly decomposed by a grinder or a pulper to prepare pulp, wherein the pulp whiteness can reach 76ISO, the lignin removal rate is 90%, and the hemicellulose removal rate is 87%.

[0035] Example 3

[0036] (1) Preparation of complex enzyme is the same as in Example 1

[0037] (2) Weigh 78.08 g of hydrogen peroxide (30%) and 165.09 g of glacial acetic acid (99%) and mix them to obtain a mixed solution, that is, the mass ratio of hydrogen peroxide to glacial acetic acid is 1:2.1; then take 0.243 g of the composite enzyme and mix it in a beaker with 122 g of eucalyptus chips (50*50*30 mm, with a water content of less than 55%), that is, the mass ratio of the lignocellulose raw material, the mixed solution, and the composite enzyme is 50:100:0.1, heat it in an oil bath, raise the temperature to 30°C within 20 minutes, keep the temperature constant for 4 hours, and cool it to room temperature after the reaction is completed;

[0038] The reaction solution is subjected to solid-liquid separation to obtain raw material solid and liquid, wherein the complex enzyme solution can be reused; during the reaction process, glacial acetic acid can be recycled by condensation reflux; the separated solid can be uniformly decomposed by a grinder or a pulper to prepare pulp, wherein the pulp whiteness can reach 76ISO, the lignin removal rate is 90%, and the hemicellulose removal rate is 87%;

[0039] Example 4

[0040] (1) Preparation of complex enzyme is the same as in Example 1

[0041] (2) 200.0 g of hydrogen peroxide (30%) and 40.0 g of glacial acetic acid (99%) were mixed to obtain a mixed solution, i.e., the mass ratio of hydrogen peroxide to glacial acetic acid was 1:0.2; 0.240 g of the composite enzyme and 120.0 g of eucalyptus chips (50*50*30 mm, with a water content of less than 55%) were then taken, i.e., the mass ratio of the lignocellulose raw material, the mixed solution, and the composite enzyme was 50:100:0.1, and the mixture was heated in an oil bath to 30° C. within 20 min, then kept at a constant temperature for 4.5 h, and cooled to room temperature after the reaction was completed;

[0042] The reaction solution is subjected to solid-liquid separation to obtain raw material solid and liquid, wherein the complex enzyme solution can be reused; during the reaction process, the glacial acetic acid can be recycled by condensation reflux; the separated solid can be uniformly decomposed by a grinder or a pulper to prepare pulp, wherein the pulp whiteness is 76ISO, the lignin removal rate is 90%, and the hemicellulose removal rate is 87%;

[0043] Example 5

[0044] (1) Preparation of complex enzyme is the same as in Example 1

[0045] (2) 43.64 g of hydrogen peroxide (30%) and 196.36 g of glacial acetic acid (99%) were mixed to obtain a mixed solution, i.e., the mass ratio of hydrogen peroxide to glacial acetic acid was 1:4.5; 0.240 g of the composite enzyme and 120.0 g of eucalyptus chips (50*50*30 mm, with a moisture content of less than 55%) were then taken, i.e., the mass ratio of the lignocellulose raw material, the mixed solution, and the composite enzyme was 50:100:0.1, and the mixture was heated in an oil bath to 40° C. within 20 min, then kept at a constant temperature for 4.5 h, and cooled to room temperature after the reaction was completed;

[0046] The reaction solution is subjected to solid-liquid separation to obtain raw material solid and liquid, wherein the complex enzyme solution can be reused; during the reaction process, the glacial acetic acid can be recycled by condensation reflux; the separated solid can be uniformly decomposed by a grinder or a pulper to prepare pulp, wherein the pulp whiteness is 76ISO, the lignin removal rate is 90%, and the hemicellulose removal rate is 87%;

[0047] Example 6

[0048] (1) Preparation of complex enzyme is the same as in Example 1

[0049] (2) Weigh 78.08 g of hydrogen peroxide (30%) and 165.09 g of glacial acetic acid (99%) and mix them to obtain a mixed solution, that is, the mass ratio of hydrogen peroxide to glacial acetic acid is 1:2.1; then take 0.049 g of the complex enzyme and mix them in a beaker with 122 g of eucalyptus chips (50*50*30 mm, with a water content of less than 55%), that is, the mass ratio of the lignocellulose raw material, the mixed solution, and the complex enzyme is 50:100:0.02, and the mass ratio of the complex enzyme to the mixed solution is 1:5000. Heat in an oil bath, raise the temperature to 30°C within 20 minutes, keep the temperature constant for 6 hours, and cool to room temperature after the reaction is completed;

[0050] The reaction solution is subjected to solid-liquid separation to obtain raw material solid and liquid, wherein the complex enzyme solution can be reused; during the reaction process, glacial acetic acid can be recycled by condensation reflux; the separated solid can be uniformly decomposed by a grinder or a pulper to prepare pulp, wherein the pulp whiteness can reach 76ISO, the lignin removal rate is 90%, and the hemicellulose removal rate is 87%;

[0051] Example 7

[0052] (1) Preparation of complex enzyme is the same as in Example 1

[0053] (2) Weigh 78.08 g of hydrogen peroxide (30%) and 165.09 g of glacial acetic acid (99%) and mix them to obtain a mixed solution, that is, the mass ratio of hydrogen peroxide to glacial acetic acid is 1:2.1; 0.243 g of the complex enzyme is mixed in a beaker, and 244 g of eucalyptus chips (50*50*30 mm, with a water content of less than 55%), that is, the mass ratio of the lignocellulose raw material, the mixed solution, and the complex enzyme is 100:100:0.1, and the mass ratio of the lignocellulose raw material to the mixed solution is 1:1, and the temperature is raised by heating in an oil bath, and the temperature is raised to 40°C within 20 minutes, and then the temperature is maintained for 6 hours. After the reaction is completed, it is cooled to room temperature;

[0054] The reaction solution is subjected to solid-liquid separation to obtain raw material solid and liquid, wherein the complex enzyme solution can be reused; during the reaction process, glacial acetic acid can be recycled by condensation reflux; the separated solid can be uniformly decomposed by a grinder or a pulper to prepare pulp, wherein the pulp whiteness can reach 76ISO, the lignin removal rate is 90%, and the hemicellulose removal rate is 87%;

[0055] Comparative Example 1

[0056] The difference between Comparative Example 1 and Example 1 is that the solution for removing lignocellulose is prepared by a traditional method, that is, the raw materials are sodium hydroxide and sodium sulfite. 26.0 g of sodium hydroxide and 13.1 g of sodium sulfite are mixed in 260 g of deionized water to form a 2.5 mol / L sodium hydroxide and 0.4 mol / L sodium sulfite solution, and the reaction is carried out at 103° C. for 6 hours. The lignin removal rate is 43%. This method requires a high temperature, and a large amount of sodium ions will remain in the solution, and the solution cannot be recycled. The relevant data results are shown in Table 1.

[0057] The difference between the relevant comparative examples 2-9 and Example 1 is that the use and proportion of the lignocellulose raw material, the mixed solution and the complex enzyme are different. The relevant specific parameters and results are shown in Table 1.

[0058] Table 1 Raw material ratios and results of Examples 1-7 and Comparative Examples 1-9

[0059]

[0060]

[0061] As can be seen from the above table, the present invention utilizes glacial acetic acid, hydrogen peroxide and perhydrolase-laccase dual enzyme coupling to form a synergistic effect of complex enzyme production to generate hydroxyl radicals (·OH), organic free radicals (RC·), superoxide free radicals (·O 2- ) and singlet oxygen ( 1 O2) and other highly active substances, which can achieve a 90% lignin removal rate and an 87% hemicellulose removal rate in the lignocellulose raw material within 6 hours, and this method does not produce wastewater. The fiber whiteness after treatment can reach 76ISO; this method does not produce wastewater and does not destroy the original structure of lignin. The solution can be reused to reduce environmental pollution. There is no need to crush and grind the raw materials. The raw material thickness can be up to 30mm, which is a significant improvement.

[0062] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for removing lignin from a lignocellulosic raw material, characterized in that The method comprises the following steps: The lignocellulose raw material is placed in a mixed solution of hydrogen peroxide and glacial acetic acid, and a composite enzyme is added to react to obtain the lignocellulose raw material with lignin removed; The composite enzyme is composed of perhydrolase and laccase, wherein the mass ratio of the effective components in the composite enzyme is perhydrolase:laccase=1:0.

5.

2. A method for removing lignin from a lignocellulosic raw material according to claim 1, characterized in that: The concentration of the hydrogen peroxide is 20-50%, and the concentration of the glacial acetic acid is 95-99%; The mass ratio of the hydrogen peroxide to the glacial acetic acid is 1:0.2-4.

5.

3. A method for removing lignin from a lignocellulosic raw material according to claim 1, characterized in that: The mass ratio of the lignocellulose raw material to the mixed solution is 1:1.0-3.

0.

4. A method for removing lignin from a lignocellulosic raw material according to claim 1, characterized in that: The mass ratio of the complex enzyme to the mixed solution is 1:50-5000.

5. A method for removing lignin from a lignocellulosic raw material according to claim 1, characterized in that: The thickness of the lignocellulose raw material is 0-30 mm.

6. A method for removing lignin from a lignocellulosic raw material according to claim 1, characterized in that: The reaction parameters are: temperature of 25-40°C and reaction time of 4-6h.

7. A method for removing lignin from a lignocellulose raw material according to claim 6, characterized in that: The temperature was 30°C.

8. A method for removing lignin from a lignocellulosic raw material according to claim 1, characterized in that: The filtrate obtained after the reaction is dried to obtain lignin.

9. A method for removing lignin from a lignocellulosic raw material according to claim 1, characterized in that: The lignocellulose raw material is any one of bamboo, coniferous wood and broadleaved wood.

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