Methods for improving the efficiency of enzymatic hydrolysis of lignocellulose

Abstract

The present invention relates to a method for improving the efficiency of enzymatic hydrolysis of lignocellulose, comprising the steps of crushing agricultural and forestry-derived biomass feedstock to a particle size of 60 to 80 mesh, extracting it with a toluene-ethanol solution, and drying it at a constant temperature to obtain a raw material from which extracts have been removed, weighing and mixing a metal salt and glycerol in a molar ratio of 1:10 to 1:124, and obtaining a homogeneous and transparent type IV deep eutectic solvent from the mixture at a temperature of 80 to 90°C and a stirring speed of 180 rpm, weighing the raw material from which extracts have been removed and adding it to the type IV deep eutectic solvent, and reacting it at a temperature of 80 to 120°C and a stirring speed of 300 to 500 rpm for 2 to 4 hours to obtain a pretreated mixture, separating the pretreated mixture into a filter cake and a filtrate, and then drying the washed filter cake to obtain a lignocellulosic residue obtained by pretreating agricultural and forestry-derived biomass feedstock, and enzymatically hydrolyzing the lignocellulosic residue to obtain fermentable sugars. The present invention has the advantages of low cost, ease of manufacture, mild processing conditions, and short processing time.

Description

[Technical Field]

[0001] The present invention relates to the technical field of lignocellulose preparation, and in particular to a method for improving the efficiency of enzymatic hydrolysis of lignocellulose. [Background technology]

[0002] Lignocellulose is primarily composed of cellulose, hemicellulose, and lignin, and efficient saccharification of cellulose is key to the production of chemicals and liquid fuels. To achieve efficient lignocellulose conversion, it is necessary to find an efficient and environmentally friendly pretreatment solvent system that can destroy the barrier that prevents lignocellulose degradation.

[0003] The pretreatment solvent system effectively destroys the original dense structure of biomass, reduces the anti-degradation barrier, and helps convert carbohydrates into fermentable monosaccharides or fuel ethanol. Selecting an environmentally friendly, gentle, and efficient pretreatment solvent is very important for enzymatic saccharification of lignocellulose.

[0004] Deep eutectic solvents (DESs) are a new type of ionic liquid, formed by a simple physical mixture of hydrogen bond donors and hydrogen bond acceptors, which can effectively remove lignin and hemicellulose, have little effect on cellulose degradation, and are recyclable. Summary of the Invention [Problem to be solved by the invention]

[0005] The technical problem to be solved by the present invention is to provide a method for improving the efficiency of enzymatic hydrolysis of lignocellulose by using a low-cost, environmentally friendly deep eutectic solvent system to enzymatically hydrolyze lignocellulose, which has the advantages of low equipment requirements, simple operation, and effectively improving the efficiency of enzymatic hydrolysis. [Means for solving the problem]

[0006] The present invention is achieved as follows. Step 1: Crushing agricultural and forestry-derived biomass raw materials to a particle size of 60 to 80 mesh, extracting with a toluene-ethanol solution, and drying at a constant temperature to obtain raw materials from which extracts have been removed; Step 2: Weighing and mixing a metal salt and glycerol in a molar ratio of 1:10 to 1:124, and obtaining a uniform and transparent type IV deep eutectic solvent from the mixture at a temperature of 80 to 90°C and a stirring speed of 180 rpm, wherein the metal salt is one of aluminum chloride, zinc chloride, and iron chloride; Step 3: Weigh the extracted raw material prepared in step 1, add it to the type IV deep eutectic solvent prepared in step 2 in a solid-liquid ratio of 1:5 to 1:15, and react it at a temperature of 80 to 120°C and a stirring speed of 300 to 500 rpm for 2 to 4 hours to obtain a pre-treated mixture. Step 4: separating the pretreated mixture into solid and liquid by centrifugation or vacuum suction filtration to obtain a filter residue and a filtrate, and then washing the filter residue with ethanol or deionized water until metal salt components are no longer detectable in the washings to desalt it. Thereafter, the washed filter residue is dried to obtain a lignocellulosic residue obtained by pretreating agricultural and forestry-derived biomass raw materials. and step 5, adding the pretreated lignocellulose residue to an acetate buffer solution of pH 4.8 at a predetermined substrate concentration, then adding a cellulose complex enzyme, and reacting the mixture for a predetermined time in an air bath shaker at 45 to 50°C and a rotation speed of 140 to 150 r / min to obtain fermentable sugars.

[0007] Currently, deep eutectic solvents (DES) are divided into four categories. In this invention, the solvent system prepared using metal salts and glycerol is a type IV deep eutectic solvent system. Glycerol is an organic solvent commonly used in pretreatment. When used alone, lignin removal requires higher temperatures and longer times, and the removal effect can be improved in an acidic environment. The Lewis acidity provided by the metal salt increases the lignin removal rate and effectively decomposes hemicellulose, swelling cellulose rather than dissolving it. The type IV deep eutectic solvent system of this invention is highly stable, easy to prepare, and environmentally friendly, making it more applicable.

[0008] Furthermore, in step 1, the agriculture and forestry-derived biomass is bamboo.

[0009] Furthermore, in step 4, the drying conditions for the filtered residue after the washing are to dry it at 80°C until it reaches a constant gravity.

[0010] Furthermore, in step 5, the substrate concentration of the lignocellulose residue is 2 to 10 wt %, and the cellulose-conjugated enzyme is Cellic @ The cellulose complex enzyme was added in an amount of 15 to 50 FPU / g substrate, and the enzymatic hydrolysis reaction time was 3 to 48 hours. [Effects of the Invention]

[0011] Compared with conventional techniques, the method of the present invention for improving the efficiency of enzymatic hydrolysis of lignocellulose involves pretreating lignocellulose in agricultural and forestry biomass feedstocks using a type IV deep eutectic solvent prepared with metal salts and glycerol, followed by enzymatic hydrolysis of the pretreated lignocellulose residue. This method is characterized by low cost, easy preparation, mild treatment conditions, and short treatment time, as well as low equipment requirements, easy reaction control, and an environmentally friendly overall process. Meanwhile, the pretreated lignocellulose residue has improved cellulose loosening and removed large amounts of hemicellulose and lignin, which are beneficial for promoting contact between cellulase and the substrate, thereby improving the hydrolysis efficiency of cellulase. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a flow diagram of the method of the present invention for improving the efficiency of enzymatic hydrolysis of lignocellulose. [Figure 2] Scanning electron microscope images of bamboo powder before (a1, a2, a3) and after (b1, b2, b3) pretreatment with type IV deep eutectic solvent synthesized with iron chloride-glycerol. [Figure 3] FIG. 1 is a schematic diagram comparing the efficiency of enzymatic hydrolysis (i.e., glucose conversion rate) between each example of the present invention and a comparative example. [Figure 4] FIG. 1 is a schematic diagram comparing the hemicellulose removal rate and lignin removal rate of Moso bamboo residue after pretreatment in each example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] In order to clarify the technical problems, technical solutions and beneficial effects of the present invention, the present invention will be described in more detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for the purpose of illustrating the present invention and are not intended to limit the present invention.

[0014] As shown in FIG. 1, a preferred embodiment of the method for improving the efficiency of enzymatic hydrolysis of lignocellulose of the present invention includes the following steps: Step 1: The agricultural and forestry-derived biomass material is crushed to a particle size of 60 to 80 mesh, extracted with a 2:1 volumetric toluene-ethanol solution, and dried at a constant temperature of 80°C to obtain a raw material from which the extract has been removed. The agricultural and forestry-derived biomass is moso bamboo. Step 2: Weigh and mix metal salt and glycerol in a molar ratio of 1:10 to 1:124. A homogeneous, transparent Type IV deep eutectic solvent is obtained from the mixture at a temperature of 80 to 90°C and a stirring speed of 180 rpm. The metal salt is one of aluminum chloride, zinc chloride, and iron chloride. The molar ratio of metal salt to glycerol is preferably 1:124. Iron chloride is preferred as the metal salt, based on the final pretreatment effect. Step 3: Weigh the extract-removed raw material prepared in Step 1 and add it to the Type IV deep eutectic solvent prepared in Step 2 at a solid-liquid ratio of 1:5 to 1:15. Allow to react at a temperature of 80 to 120°C and a stirring speed of 300 to 500 rpm for 2 to 4 hours to obtain a pre-treated mixture. The solid-liquid ratio is preferably 1:10. Step 4: The pretreated mixture is subjected to solid-liquid separation using centrifugation or vacuum suction filtration to obtain a filter residue and a filtrate. The filter residue is then desalted by washing with ethanol or deionized water until no metal salt components are detectable in the wash liquid. The washed filter residue is then dried to obtain the lignocellulosic residue obtained by pretreating agricultural and forestry biomass feedstock. The washed filter residue is dried at 80°C until it reaches a constant gravity. Step 5: The pretreated lignocellulosic residue is added to a pH 4.8 acetate buffer solution at a predetermined substrate concentration, and then the cellulose complex enzyme is added. The mixture is reacted for a predetermined time in an air bath shaker at 45-50°C and 140-150 r / min to obtain fermentable sugars.

[0015] The substrate concentration of the lignocellulose residue is 2 to 10 wt %, and the cellulose complex enzyme is Cellic @The cellulose complex enzyme is CTec2, the amount of cellulose complex enzyme added is 15 to 50 FPU / g substrate, and the enzymatic hydrolysis reaction time is 3 to 48 hours. The substrate concentration of the lignocellulose reaction residue is preferably 2 wt%, and the higher the substrate concentration, the longer the time required for cellulase hydrolysis to reach a plateau phase. The cellulose complex enzyme is Cellic from Novozymes. @ The amount of cellulase added varies depending on the enzymatic hydrolysis time and effectiveness, which affects experimental and subsequent production costs. To achieve high enzymatic hydrolysis efficiency and reduce production costs, the cellulase addition amount is preferably 15 FPU / g substrate, and the enzymatic hydrolysis reaction time is 3 to 48 hours. To optimize the enzymatic hydrolysis effect of the lignocellulose pretreatment residue, substrate concentration, amount of cellulose-conjugated enzyme used, and shorten the enzymatic hydrolysis time, the enzymatic hydrolysis reaction time may be more preferably 48 hours.

[0016] In the present invention, the type IV deep eutectic solvent synthesized using metal salt-glycerol has the characteristics of being low-cost, easily available, recyclable in the use process, and environmentally friendly. Furthermore, metal salt-glycerol has a high function of removing hemicellulose and lignin, and hardly dissolves cellulose, thereby achieving efficient cellulose conversion. The method for improving the efficiency of enzymatic hydrolysis of lignocellulose according to the present invention will be further described below with reference to examples and comparative examples. Example 1

[0017] A first embodiment of the method of the present invention for improving the efficiency of enzymatic hydrolysis of lignocellulose comprises the following steps. Moso bamboo raw material was crushed and passed through a 60-80 mesh sieve. It was extracted with toluene-ethanol in a 2:1 volume ratio and then dried at 80°C until constant weight was reached, yielding a de-extracted raw material. Iron chloride and glycerol were weighed in a 1:10 ratio and stirred at 180 rpm to obtain a homogeneous, transparent liquid. A predetermined amount of the de-extracted raw material was weighed and added to an iron chloride-glycerol solvent in a 1:15 solid-liquid ratio. The reaction was allowed to proceed for 4 hours at 80°C and 300 rpm while stirring. After the reaction was complete, the solid-liquid separation was performed using vacuum suction filtration. The separated moso bamboo residue was washed with deionized water and desalted, yielding moso bamboo residue pretreated with a type IV deep eutectic solvent synthesized from iron chloride and glycerol. Moso bamboo residue was added to an acetate buffer solution at pH 4.8 to achieve a substrate concentration of 5%, and the cellulose complex enzyme (Cell @ The fermentable sugar glucose was obtained by adding CTec2) and reacting for 48 hours in an air shaker at 50°C and 140 r / min. The final measurement showed that the enzymatic hydrolysis efficiency of the pretreated moso bamboo residue was 46.63%. Comparative Example

[0018] Using moso bamboo as a control, the efficiency of direct enzymatic hydrolysis of moso bamboo was analyzed by enzymatically hydrolyzing the moso bamboo without pretreatment with a type IV deep eutectic solvent synthesized from metal salts and glycerol. Moso bamboo raw material was crushed to 60-80 mesh, extracted with toluene-ethanol, and then dried at a constant temperature of 80°C. A predetermined amount of the extracted moso bamboo raw material was added to an acetate buffer solution at pH 4.8 with a substrate concentration of 2%. The cellulose complex enzyme (Cellic @ CTec2) was added and the reaction was carried out for 48 hours in an air bath shaker at 50°C and 140 r / min, and the enzymatic hydrolysis efficiency was 17.78%. The enzymatic hydrolysis efficiency of Example 1 was 2.6 times that of the comparative example, which indicates that the pretreatment method using the deep eutectic solvent of the present invention can significantly improve the enzymatic hydrolysis efficiency of moso bamboo. Example 2

[0019] A second embodiment of the method of the present invention for improving the efficiency of enzymatic hydrolysis of lignocellulose comprises the following steps. Moso bamboo raw material was crushed and passed through a 60-80 mesh sieve. It was extracted with toluene-ethanol in a 2:1 volume ratio and then dried at 80°C until constant weight was reached, yielding a de-extracted raw material. Iron chloride and glycerol were weighed out in a 1:124 ratio and stirred at 180 rpm to obtain a homogeneous, transparent liquid. A predetermined amount of the de-extracted raw material was weighed out and added to an iron chloride-glycerol solvent in a 1:10 solid-liquid ratio. The mixture was stirred at 100°C and 300 rpm for 3 hours. After the reaction was complete, the solid-liquid separation was performed using vacuum suction filtration. The separated moso bamboo residue was washed with deionized water and desalted to obtain moso bamboo residue pretreated with a type IV deep eutectic solvent synthesized from iron chloride and glycerol. Moso bamboo residue was added to a 2% substrate concentration in a pH 4.8 acetate buffer, and a cellulose complex enzyme (Cellic@CTec2) with 15 FPU / g of moso bamboo residue substrate was added. The reaction was carried out in an air shaker at 50°C and 140 r / min for 48 hours to obtain the fermentable sugar glucose. The final measured enzymatic hydrolysis efficiency of the pretreated moso bamboo residue was 67.87%, 3.8 times that of the untreated moso bamboo in the comparative example. Example 3

[0020] A third embodiment of the method of the present invention for improving the efficiency of enzymatic hydrolysis of lignocellulose comprises the following steps. Moso bamboo raw material was crushed and passed through a 60-80 mesh sieve. It was extracted with toluene-ethanol in a 2:1 volume ratio and then dried at 80°C until constant weight was obtained, yielding a de-extracted raw material. Iron chloride and glycerol were weighed out in a 1:124 ratio and stirred at 180 rpm to obtain a homogeneous, transparent liquid. A predetermined amount of the de-extracted raw material was weighed out and added to an iron chloride-glycerol solvent in a 1:10 solid-liquid ratio. The reaction was allowed to proceed for 2 hours, stirring at 300 rpm at 120°C. After the reaction was complete, the solid-liquid separation was performed using vacuum suction filtration. The separated moso bamboo residue was washed with deionized water and desalted, yielding moso bamboo residue pretreated with a type IV deep eutectic solvent synthesized from iron chloride and glycerol. Moso bamboo residue was added to a 2% substrate concentration in a pH 4.8 acetate buffer, and a cellulose complex enzyme (Cellic@CTec2) with 15 FPU / g of moso bamboo residue substrate was added. The reaction was carried out in an air shaker at 50°C and 140 r / min for 48 hours to obtain the fermentable sugar glucose. The final measurement showed that the enzymatic hydrolysis efficiency of the pretreated moso bamboo residue was 91.13%, 5.1 times that of the unpretreated moso bamboo used in the comparison. Example 4

[0021] A fourth embodiment of the method of the present invention for improving the efficiency of enzymatic hydrolysis of lignocellulose comprises the following steps. Moso bamboo raw material was crushed and passed through a 60-80 mesh sieve. It was extracted with toluene-ethanol in a 2:1 volume ratio and then dried at 80°C until constant weight was reached, yielding a de-extracted raw material. Iron chloride and glycerol were weighed out in a 1:15 ratio and stirred at 180 rpm to obtain a homogeneous, transparent liquid. A predetermined amount of the de-extracted raw material was weighed out and added to an iron chloride-glycerol solvent in a 1:5 solid-liquid ratio. The mixture was stirred at 100°C and 300 rpm for 3 hours. After the reaction was complete, the solid-liquid separation was performed using vacuum suction filtration. The separated moso bamboo residue was washed with deionized water and desalted to obtain moso bamboo residue pretreated with a type IV deep eutectic solvent synthesized from iron chloride and glycerol. Moso bamboo residue was added to an acetate buffer solution at pH 4.8 at a substrate concentration of 10%, and the cellulose complex enzyme (Cellic @The fermentable sugar glucose was obtained by adding CTec2) and reacting for 48 hours in an air shaker at 50°C and 140 r / min. The final measured enzymatic hydrolysis efficiency of the pretreated moso bamboo residue was 69.41%, which was 3.9 times higher than the enzymatic hydrolysis efficiency of the unpretreated moso bamboo in the comparative example. Example 5

[0022] A fifth embodiment of the method of the present invention for improving the efficiency of enzymatic hydrolysis of lignocellulose comprises the following steps. Moso bamboo raw material was crushed and passed through a 60-80 mesh sieve. It was extracted with toluene-ethanol in a 2:1 volume ratio and then dried at 80°C until constant weight was reached, yielding a de-extracted raw material. Zinc chloride and glycerol were weighed out in a 1:124 ratio and stirred at 180 rpm to obtain a homogeneous, transparent liquid. A predetermined amount of the de-extracted raw material was weighed and added to a zinc chloride-glycerol solvent in a 1:10 solid-liquid ratio. The mixture was stirred at 100°C and 300 rpm for 3 hours. After the reaction was complete, the solid-liquid mixture was separated by vacuum suction filtration. The separated moso bamboo residue was washed with deionized water and desalted to obtain moso bamboo residue pretreated with a type IV deep eutectic solvent synthesized from zinc chloride and glycerol. Moso bamboo residue was added to a 2% substrate concentration in a pH 4.8 acetate buffer, and a cellulose complex enzyme (Cellic@CTec2) with 15 FPU / g of moso bamboo residue substrate was added. The reaction was carried out in an air shaker at 50°C and 140 r / min for 48 hours to obtain the fermentable sugar glucose. The final measurement showed that the enzymatic hydrolysis efficiency of the pretreated moso bamboo residue was 24.75%, 1.4 times that of the unpretreated moso bamboo in the comparative example. Example 6

[0023] A sixth embodiment of the method of the present invention for improving the efficiency of enzymatic hydrolysis of lignocellulose comprises the following steps. Moso bamboo raw material was crushed and passed through a 60-80 mesh sieve. It was extracted with toluene-ethanol in a 2:1 volume ratio and then dried at 80°C until constant weight was reached, yielding a de-extracted raw material. Aluminum chloride and glycerol were weighed out in a 1:124 ratio and stirred at 180 rpm to obtain a homogeneous, transparent liquid. A predetermined amount of the de-extracted raw material was weighed out and added to an aluminum chloride-glycerol solvent in a 1:10 solid-liquid ratio. The reaction was allowed to proceed for 3 hours, stirring at 100°C and 300 rpm. After the reaction was complete, the solid-liquid separation was performed using vacuum suction filtration. The separated moso bamboo residue was washed with deionized water and desalted, yielding a moso bamboo residue pretreated with a type IV deep eutectic solvent synthesized from aluminum chloride and glycerol. Moso bamboo residue was added to a 2% substrate concentration in a pH 4.8 acetate buffer, and a cellulose complex enzyme (Cellic@CTec2) with 15 FPU / g of moso bamboo residue substrate was added. The reaction was carried out in an air shaker at 50°C and 140 r / min for 48 hours to obtain the fermentable sugar glucose. The final measurement showed that the enzymatic hydrolysis efficiency of the pretreated moso bamboo residue was 59.78%, 3.4 times that of the unpretreated moso bamboo used in the comparison.

[0024] As shown in Figure 2, the surface of the bamboo fibers in untreated bamboo powder is relatively smooth, complete, and densely arranged, as shown in Figures 2-a1, 2-a2, and 2-a3. In contrast, the surface of the bamboo fibers in bamboo powder pretreated with the iron chloride-glycerol type IV deep eutectic solvent of the present invention is rough and has deep grooves. As shown in Figures 2-b1, 2-b2, and 2-b3, the fibers are broken in many places, and the degree of damage to the fracture surfaces is high. This significantly increases the contact area between cellulase and cellulose, improving the adsorption capacity of cellulose to cellulase and further improving the efficiency of enzymatic hydrolysis of cellulose.

[0025] As shown in Figure 3, compared with the comparative example, the enzymatic hydrolysis efficiency of lignocellulosic biomass was improved after pretreatment with the type IV deep eutectic solvent synthesized with iron chloride-glycerol of the present invention. In particular, the iron chloride-based DES and aluminum chloride-based DES are closely related to the properties of the metal salts, and the pretreatment process conditions have different effects on the final enzymatic hydrolysis efficiency.

[0026] As shown in Figure 4, the pretreatment process conditions and pretreatment solvent system affect the removal of lignin and hemicellulose components to varying degrees. The removal rate can reflect the destruction of the structural compactness of lignocellulosic biomass to some extent. Therefore, removing lignin and hemicellulose can promote the enzymatic hydrolysis of lignocellulosic biomass and improve the efficiency of enzymatic hydrolysis.

[0027] The above description is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. 1. A method for improving the efficiency of enzymatic hydrolysis of lignocellulose, comprising: Step 1: Crushing agricultural and forestry-derived biomass raw materials to a particle size of 60 to 80 mesh, extracting with a toluene-ethanol solution, and drying at a constant temperature to obtain raw materials from which extracts have been removed; Step 2: Weighing and mixing a metal salt and glycerol in a molar ratio of 1:10 to 1:124, and obtaining a uniform and transparent Type IV deep eutectic solvent from the mixture at a temperature of 80 to 90°C and a stirring speed of 180 rpm, wherein the metal salt is any one of aluminum chloride, zinc chloride, and iron chloride; Step 3: Weigh the extract-removed raw material prepared in step 1, add it to the type IV deep eutectic solvent prepared in step 2 in a solid-liquid ratio of 1:5 to 1:15, and react it at a temperature of 80 to 120°C and a stirring speed of 300 to 500 rpm for 2 to 4 hours to obtain a pre-treated mixture; Step 4: subjecting the pretreated mixture to solid-liquid separation by centrifugation or vacuum suction filtration to obtain a filter residue and a filtrate, respectively; washing the filter residue with ethanol or deionized water until metal salt components are no longer detectable in the washings to desalt it; and then drying the washed filter residue to obtain a lignocellulose residue obtained by pretreating agricultural and forestry-derived biomass raw materials; and step 5, adding the pretreated lignocellulose residue at a predetermined substrate concentration to an acetate buffer solution at pH 4.8, then adding the cellulose complex enzyme, and reacting for a predetermined time in an air bath shaker at 45 to 50°C and 140 to 150 r / min to obtain fermentable sugars.

2. 2. The method for improving the efficiency of enzymatic hydrolysis of lignocellulose according to claim 1, wherein in step 1, the agricultural and forestry-derived biomass is bamboo.

3. 2. The method for improving the efficiency of enzymatic hydrolysis of lignocellulose according to claim 1, wherein in step 4, the drying conditions for the filtered residue after washing are drying at 80°C until it reaches a constant gravity.

4. In step 5, the substrate concentration of the lignocellulose residue is 2 to 10 wt %, and the cellulose-conjugated enzyme is Cellic (CellCell) from Novozymes. ＠ 2. The method for improving the efficiency of enzymatic hydrolysis of lignocellulose according to claim 1, wherein the enzyme is CTec2, the amount of cellulose complex enzyme added is 15 to 50 FPU / g substrate, and the enzymatic hydrolysis reaction time is 3 to 48 hours.

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