A mechanical and biochemical synchronous pretreatment method and a hydrolysis method therewith

Abstract

The present invention discloses a pretreatment method for mechanical and biochemical synchronization, comprising the following steps: 1) adding a slurry of a mixture of a lignocellulose raw material and a sodium citrate buffer solution to a ball mill, then adding laccase and 1-hydroxybenzotriazole, and then placing the mixture in a ball mill for mechanical and biochemical synchronous pretreatment. After the synchronous pretreatment is completed, centrifugation, washing and drying are performed to obtain pretreated lignocellulose. The present invention also discloses a hydrolysis method for lignocellulose comprising the pretreatment method. The mechanical and biochemical synchronous pretreatment method provided by the present invention significantly improves the hydrolysis efficiency of the lignocellulose raw material under the synergistic effect of mechanical and biochemical reactions. The conversion rates of cellulose and hemicellulose in the lignocellulose raw material reach 99.2% and 75.3%, respectively, substantially achieving complete hydrolysis of cellulose.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a mechanical and biochemical synchronous pretreatment method and a hydrolysis method having the same. Background Art

[0002] Lignocellulosic waste biomass has attracted widespread attention as a potential source of clean and efficient biofuel due to its widespread distribution and vast reserves. However, the complex and stable cross-linked structure between lignocellulosic components makes it difficult for microorganisms to directly utilize it, necessitating pretreatment to improve its accessibility.

[0003] Currently, the main methods for pretreatment of lignocellulose include physical and chemical methods, such as acid / alkaline treatment or hydrothermal treatment, and biological methods, such as treatment with fungi and their secreted enzymes. In recent years, the combination of two or more pretreatment techniques, either sequentially or simultaneously, has emerged as an effective approach to intensify the pretreatment process, thereby reducing the number of treatment steps, lowering chemical and energy requirements, and minimizing the production of unwanted inhibitors.

[0004] Physical and chemical methods can destroy the protective layer structure of lignocellulose and achieve the degradation of hemicellulose and lignin, but they will also produce derivative by-products and inhibitors such as furans and phenols that are difficult to be effectively utilized by microorganisms. In addition, the residual chemicals from the acid / alkali treatment will significantly inhibit the microbial and enzyme conversion process. Biological pretreatment has the advantages of mild conditions, less energy requirements, and environmental friendliness, and the by-products produced usually do not inhibit subsequent hydrolysis, but there are also some disadvantages, such as low conversion efficiency, need to be carried out under sterile conditions, require a long incubation period, and are accompanied by degradation of some cellulose and hemicellulose. Most of the existing combined pretreatments combine acid, alkali, or hydrothermal, steam explosion and other methods that require high energy input. The by-products produced also inhibit the subsequent enzymatic hydrolysis and fermentation process, and the acid-base wastewater produced will further increase costs. Summary of the Invention

[0005] The present invention aims to solve, at least to some extent, one of the technical problems in the related art. To this end, the main purpose of the present invention is to provide a pretreatment method that synchronizes mechanical and biochemical processes and a hydrolysis method therewith, aiming to solve the technical problem of low hydrolysis products of lignocellulose in the prior art.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A mechanical and biochemical synchronous pretreatment method comprises the following steps:

[0008] A slurry of a mixture of a lignocellulose raw material and a sodium citrate buffer solution is added to a ball mill, and then laccase and 1-hydroxybenzotriazole are added. The lignocellulose is then placed in a ball mill for mechanical and biochemical simultaneous pretreatment. After the simultaneous pretreatment, the lignocellulose is centrifuged, washed and dried to obtain pretreated lignocellulose.

[0009] In certain embodiments, the sodium citrate buffer solution has a molar concentration of 0.05-5 mol / L and a pH of 4-6.

[0010] In some embodiments, the process further comprises pre-treating the lignocellulose raw material: crushing the lignocellulose raw material, sieving the lignocellulose raw material to 35-60 mesh, and drying the lignocellulose raw material at 100-110°C.

[0011] In certain embodiments, in step 1), the mass ratio of the lignocellulosic raw material to the sodium citrate buffer solution is (3-8):100, the amount of laccase added is 40-60 U of laccase per gram of lignocellulosic raw material, and the amount of 1-hydroxybenzotriazole added is 0.03-0.08 g of 1-hydroxybenzotriazole per gram of lignocellulosic raw material.

[0012] Furthermore, the conditions for the simultaneous mechanical and biochemical pretreatment in step 1) are: simultaneous pretreatment at a rotation speed of 200-500 rpm and a temperature of 35-45° C. for 10-15 hours.

[0013] In certain embodiments, the centrifugation, washing and drying in step 1) are specifically as follows: after the synchronous pretreatment is completed, centrifugation is performed at 8000-12000 rpm at room temperature for 10-20 minutes, the supernatant is poured out, and then the supernatant is washed with deionized water 3-5 times, and then dried in a freeze dryer at -55°C for 12-36 hours.

[0014] A method for hydrolyzing pretreated lignocellulose prepared using the aforementioned pretreatment method comprises the following steps: preparing a slurry of the pretreated lignocellulose with a sodium citrate buffer solution, adding cellulase to the slurry for a hydrolysis reaction, and filtering the slurry after the hydrolysis reaction is complete to obtain a hydrolyzed solution. Furthermore, the hydrolysis reaction is carried out at a rotation speed of 150-200 rpm and a temperature of 40-60°C for 60-84 hours.

[0015] In certain embodiments, the mass ratio of the simultaneously pretreated lignocellulosic raw material to the sodium citrate buffer solution is (3-8):100, and the amount of cellulase added is 20-40 FPU of cellulase per gram of simultaneously pretreated lignocellulosic raw material.

[0016] Compared with the prior art, the present invention has at least the following advantages:

[0017] The present invention provides a synchronous mechanical and biochemical pretreatment method that pretreats lignocellulosic raw materials by combining mechanical (ball milling) and biochemical (laccase) processes. Ball milling promotes slurry mixing and reduces the particle size of the lignocellulosic raw materials, increasing the specific surface area and facilitating laccase attachment. Laccase can degrade lignin, creating cracks and holes on the cellulose surface, which further facilitates ball milling. Through the synergistic effect of the two, the synchronous pretreatment effectively degrades lignin in wheat straw, reduces the particle size of wheat straw, increases the specific surface area, reduces cellulose crystallinity, and destroys the surface structure of wheat straw. This method has the advantages of mild conditions and environmental friendliness, significantly improving the hydrolysis efficiency of lignocellulosic raw materials. Experimental verification shows that after using the hydrolysis method of the present invention, the conversion rates of cellulose and hemicellulose in the lignocellulosic raw materials reach 99.2% and 75.3%, respectively, essentially achieving complete cellulose hydrolysis. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below with reference to specific examples. The following examples are merely illustrative and non-restrictive, and should not be used to limit the scope of protection of the present invention.

[0019] When expressing a certain amount, concentration or other value or parameter in the form of a range, preferred range, or preferred upper and lower numerical limits, it should be understood that it is equivalent to specifically disclosing any range by combining any pair of upper range limits or preferred numerical values with any lower range limit or preferred numerical value, without considering whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values listed herein include the endpoints of the range and all integers and fractions within the range.

[0020] Unless otherwise indicated, all percentages, parts, ratios, etc. herein are by weight.

[0021] The materials, methods, and examples herein are illustrative and, unless otherwise indicated, are not to be construed as limiting.

[0022] In the following embodiments, the lignocellulose raw materials used may be wheat straw, corn straw, sugarcane bagasse, rice straw, sawdust or bamboo powder;

[0023] The sodium citrate buffer solution used is prepared by dissolving anhydrous citric acid and sodium citrate in water, wherein the concentration of anhydrous citric acid is 8.838 g / L and the concentration of sodium citrate is 13.936 g / L, and the solution is prepared and used immediately.

[0024] In the following examples, the glucose concentration and xylose concentration in the hydrolyzate were measured by high performance liquid chromatography (HPLC), and the proportions of cellulose, hemicellulose, and lignin in the wheat straw were measured according to the "Determination of Structural Carbohydrates and Lignin in Biomass" method published by the National Renewable Energy Laboratory (NREL) in 2008. The conversion rates of cellulose and hemicellulose were calculated as follows:

[0025]

[0026]

[0027] The weight of lignin is the sum of the weights of acid-soluble lignin and acid-insoluble lignin. Acid-soluble lignin is measured by using deionized water or 4% sulfuric acid as a blank sample and measuring the absorbance at 205 nm in a UV-visible spectrophotometer. The sample is diluted as needed to keep the absorbance within the range of 0.2 to 1.0. The mass of acid-soluble lignin can be calculated using the following formula:

[0028]

[0029] Where m ASL is the mass of acid-soluble lignin, OD is the absorbance, and N is the dilution multiple.

[0030] Example 1

[0031] The hydrolysis method provided by the present invention includes a pretreatment step of mechanical and biochemical synchronization, comprising the following steps:

[0032] 1) After the lignocellulose raw material is crushed, the lignocellulose raw material is sieved to 35-60 mesh, and dried at 110°C;

[0033] 2) adding a lignocellulosic raw material and a sodium citrate buffer solution to a ball mill to prepare a mixed slurry with a mass concentration of 3 wt%, then adding 40 U of laccase and 0.03 g of 1-hydroxybenzotriazole per gram of lignocellulosic raw material, and then placing the laccase and 1-hydroxybenzotriazole in a ball mill for simultaneous pretreatment at a speed of 500 rpm and a temperature of 45°C for 10 hours; after the simultaneous pretreatment, centrifuging at room temperature and 8000 rpm for 20 minutes, decanting the supernatant, and then washing three times with deionized water, and then drying in a freeze dryer at -55°C for 36 hours.

[0034] 3) The lignocellulosic raw material pretreated in step 2) was mixed with a sodium citrate buffer solution to form a slurry with a mass concentration of 3 wt%, and then 20 FPU of cellulase was added to the slurry per gram of the pretreated lignocellulosic raw material. The hydrolysis reaction was carried out at a speed of 200 rpm and a temperature of 60° C. for 60 h. After the hydrolysis reaction was completed, the hydrolyzate was filtered to obtain a hydrolyzate.

[0035] Example 2

[0036] The present invention provides a hydrolysis method with a synchronous mechanical and biochemical pretreatment step, comprising the following steps:

[0037] 1) After the lignocellulose raw material is crushed, the lignocellulose raw material is sieved to 35-60 mesh, and dried at 105°C;

[0038] 2) adding a lignocellulosic raw material and a sodium citrate buffer solution to a ball mill to prepare a mixed slurry with a mass concentration of 5 wt%, then adding 50 U of laccase and 0.05 g of 1-hydroxybenzotriazole per gram of lignocellulosic raw material, adding laccase and 1-hydroxybenzotriazole, and then placing the slurry in a ball mill at a speed of 300 rpm and a temperature of 40°C for simultaneous pretreatment for 12 hours; after the simultaneous pretreatment, centrifuging at room temperature and 10,000 rpm for 15 minutes, decanting the supernatant, and then washing three times with deionized water, and drying in a freeze dryer at -55°C for 24 hours.

[0039] 3) The lignocellulosic raw material pretreated in step 2) was mixed with a sodium citrate buffer solution to form a slurry with a mass concentration of 5 wt%, and then 30 FPU of cellulase was added to the slurry per gram of the pretreated lignocellulosic raw material. The slurry was hydrolyzed at a speed of 170 rpm and a temperature of 50° C. for 72 h. After the hydrolysis reaction was completed, the hydrolyzate was filtered to obtain a hydrolyzate.

[0040] Example 3

[0041] The present invention provides a hydrolysis method comprising a pretreatment step of mechanical and biochemical synchronization, comprising the following steps:

[0042] 1) After the lignocellulose raw material is crushed, the lignocellulose raw material is sieved to 35-60 mesh, and dried at 100°C;

[0043] 2) adding a lignocellulosic raw material and a sodium citrate buffer solution to a ball mill to prepare a mixed slurry with a mass concentration of 8 wt%, then adding 60 U of laccase and 0.08 g of 1-hydroxybenzotriazole per gram of lignocellulosic raw material, and then placing the laccase and 1-hydroxybenzotriazole in a ball mill for simultaneous pretreatment at a speed of 200 rpm and a temperature of 35°C for 15 hours; after the simultaneous pretreatment, centrifuging at room temperature and 12,000 rpm for 10 minutes, decanting the supernatant, and then washing with deionized water five times, and then drying in a freeze dryer at -55°C for 12 hours.

[0044] 3) The lignocellulosic raw material pretreated in step 2) was mixed with a sodium citrate buffer solution to form a slurry with a mass concentration of 8 wt%, and then 40 FPU of cellulase was added to the slurry per gram of the pretreated lignocellulosic raw material. The hydrolysis reaction was carried out at a speed of 200 rpm and a temperature of 60° C. for 84 hours. After the hydrolysis reaction was completed, the hydrolyzate was filtered to obtain a hydrolyzate.

[0045] Comparative Example 1:

[0046] The method in this comparative example is basically the same as the hydrolysis method in Example 2 including the simultaneous mechanical and biochemical pretreatment steps, except that the simultaneous pretreatment step in step 2) is omitted.

[0047] Comparative Example 2

[0048] The method in this comparative example is basically the same as the hydrolysis method in Example 2 including the simultaneous mechanical and biochemical pretreatment steps, except that laccase is not added in step 2), and only single ball milling pretreatment is performed.

[0049] Comparative Example 3

[0050] The method in this comparative example is basically the same as the hydrolysis method in Example 2 including the simultaneous mechanical and biochemical pretreatment steps, except that in step 2), no single ball milling pretreatment is performed, and only laccase is added for pretreatment.

[0051] Comparative Example 4

[0052] The method in this comparative example is basically the same as the hydrolysis method in Example 2 including the simultaneous mechanical and biochemical pretreatment steps, except that in step 2), single ball milling pretreatment is first performed for 3 hours, and then laccase is added for pretreatment for 12 hours.

[0053] Comparative Example 5

[0054] The method in this comparative example is basically the same as the hydrolysis method in Example 2 including the simultaneous mechanical and biochemical pretreatment steps, except that in step 2), laccase is first added for pretreatment for 12 hours, and then single ball milling pretreatment is performed for 3 hours.

[0055] Comparative Example 6

[0056] The method in this comparative example is basically the same as the hydrolysis method including the simultaneous mechanical and biochemical pretreatment steps in Example 2, except that step 1) is the same, laccase and 1-hydroxybenzotriazole are not added in step 2), and laccase and 1-hydroxybenzotriazole are added in step 3), specifically:

[0057] 1) After the lignocellulose raw material is crushed, the lignocellulose raw material is sieved to 35-60 mesh, and dried at 105°C;

[0058] 2) adding lignocellulosic raw material and sodium citrate buffer solution to a ball mill to prepare a mixed slurry with a mass concentration of 5 wt%, and pretreating the mixture in a ball mill at a speed of 300 rpm and a temperature of 40°C for 12 h; after the pretreatment, centrifuging at room temperature and 10,000 rpm for 15 min, discarding the supernatant, and washing the mixture three times with deionized water, and then drying the mixture in a freeze dryer at -55°C for 24 h.

[0059] 3) The lignocellulosic raw material pretreated in step 2) was mixed with a sodium citrate buffer solution to form a slurry with a mass concentration of 5 wt%, and then 30 FPU of cellulase was added to the slurry per gram of the pretreated lignocellulosic raw material. Laccase 50 U and 0.05 g of 1-hydroxybenzotriazole were added to the slurry per gram of the lignocellulosic raw material. Laccase and 1-hydroxybenzotriazole were added. The mixture was hydrolyzed at a speed of 170 rpm and a temperature of 50° C. for 72 h. After the hydrolysis reaction was completed, the hydrolyzate was filtered to obtain a hydrolyzate.

[0060] Performance testing:

[0061] The present invention provides a method for promoting the hydrolysis of wood fiber. The hydrolyzate prepared by the method of Example 2 is used as an example. The hydrolyzate and the hydrolyzates prepared by the methods of Comparative Examples 1-6 are tested for performance. The results are shown in Table 2:

[0062] Table 2 Performance test of hydrolyzates prepared by the methods of Example 2 and Comparative Examples 1-6

[0063] Cellulose conversion rate Hemicellulose conversion rate Example 1 86.7％ 62.9％ Example 2 99.2％ 75.3％ Example 3 89.1％ 67.3％ Comparative Example 1 28.3％ 14.6％ Comparative Example 2 59.2％ 56.8％ Comparative Example 3 45.8％ 20.4％ Comparative Example 4 53.4％ 29.8％ Comparative Example 5 55.6％ 34.5％ Comparative Example 6 57.8％ 31.7％

[0064] As can be seen from the above table, the hydrolyzate of the lignocellulosic raw material prepared by the method of the present application has a cellulose conversion rate increased by 70.9% and a hemifiber conversion rate increased by 60.7% compared with the lignocellulosic raw material that has not been subjected to simultaneous ball milling and laccase treatment; relative to the lignocellulosic raw material that has only been subjected to ball milling treatment, the cellulose conversion rate has been increased by 40%, and the hemifiber conversion rate has been increased by 18.5%; relative to the lignocellulosic raw material that has only been pretreated with laccase, the cellulose conversion rate has been increased by 53.4%, and the hemifiber conversion rate has been increased by 54.9%; relative to the lignocellulosic raw material that has been pretreated in cascade, the cellulose conversion rate has been increased by about 44%, and the hemifiber conversion rate has been more than doubled; in addition, although the laccase is not added in the pretreatment stage but in the biochemical reaction stage, which can also improve the cellulose conversion rate, the conversion effect is not significantly improved.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A mechanical and biochemical synchronous pretreatment method, characterized in that: The method comprises the following steps: adding a slurry of a mixture of a lignocellulose raw material and a sodium citrate buffer solution into a ball mill, adding laccase and 1-hydroxybenzotriazole, and then placing the mixture into a ball mill for mechanical and biochemical synchronous pretreatment. After the synchronous pretreatment, the mixture is centrifuged, washed, and dried to obtain pretreated lignocellulose; the mass ratio of the lignocellulose raw material to the sodium citrate buffer solution is (3-8):100, the amount of the laccase added is 40-60 U per gram of the lignocellulose raw material, and the amount of the 1-hydroxybenzotriazole added is 0.03-0.08 g per gram of the lignocellulose raw material; and the conditions for the mechanical and biochemical synchronous pretreatment in step 1) are: synchronous pretreatment at a rotation speed of 200-500 rpm and a temperature of 35-45° C. for 10-15 hours.

2. The pretreatment method according to claim 1, characterized in that The molar concentration of the sodium citrate buffer solution is 0.05-5 mol / L, and the pH value is 4-6.

3. The pretreatment method according to claim 1, characterized in that The method also includes pre-processing the lignocellulose raw material: crushing the lignocellulose raw material, sieving the lignocellulose raw material to 35-60 mesh, and drying it at 100-110°C.

4. The pretreatment method according to claim 3, characterized in that The centrifugation, washing and drying in step 1) are specifically as follows: after the synchronous pretreatment is completed, centrifuge at 8000-12000 rpm at room temperature for 10-20 minutes, pour off the supernatant, wash with deionized water 3-5 times, and dry in a freeze dryer at -55°C for 12-36 hours.

5. A method for hydrolyzing pretreated lignocellulose using the pretreatment method according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: preparing the pretreated lignocellulose and sodium citrate buffer solution into slurry, adding cellulase into the slurry to carry out hydrolysis reaction, and filtering to obtain hydrolyzed liquid after the hydrolysis reaction is completed.

6. The hydrolysis method according to claim 5, characterized in that The conditions of the hydrolysis reaction are: a rotation speed of 150-200 rpm and a temperature of 40-60° C. maintained for 60-84 hours.

7. The hydrolysis method according to claim 5, characterized in that The mass ratio of the pretreated lignocellulose to the sodium citrate buffer solution is (3-8):100, and the amount of the cellulase added is 20-40 FPU of cellulase per gram of pretreated lignocellulose.

Images