Method for inhibiting formation of pseudolignin and improving enzymatic hydrolysis of lignocellulose raw material

Abstract

The invention discloses a method for inhibiting formation of pseudolignin and improving enzymatic hydrolysis of a lignocellulose raw material, and belongs to the technical field of pretreatment and enzymatic hydrolysis of the lignocellulose raw material. The method comprises the following steps: adding ethylene glycol into an acidic aqueous solution to carry out acid-alcohol combination pretreatment on a lignocellulose raw material; carrying out solid-liquid separation after pretreatment to obtain filter residues; and washing the filter residue to be neutral, adding cellulase, and carrying out enzymolysis on the filter residue in an enzymolysis system to inhibit the formation of pseudolignin so as to realize the enzyme hydrolysis process of the lignocellulose raw material. Compared with existing research results, the method has the advantages that the formation of pseudolignin is effectively inhibited, and the enzyme hydrolysis yield is remarkably increased and reaches 74.66%.

Description

Technical Field

[0001] The invention belongs to the technical field of pretreatment and enzymatic hydrolysis of lignocellulose raw materials, and particularly relates to a method for inhibiting the formation of pseudo-lignin and improving enzymatic hydrolysis of lignocellulose raw materials. Background Art

[0002] With the increasing depletion of fossil energy and the increasing environmental pollution, the development of clean and efficient renewable biomass energy has become a key research direction around the world. Among them, lignocellulosic biomass has attracted widespread attention due to its wide sources, rich resources, strong renewability and environmental friendliness. Lignocellulose is mainly composed of three components: cellulose, hemicellulose and lignin. Cellulose and hemicellulose can be hydrolyzed to produce fermentable sugars, which are then converted into alcohol fuels such as ethanol and butanol through microbial fermentation, providing an important way to replace traditional fossil energy. However, the complex structure of lignin and the pseudo-lignin that may be formed during the pretreatment of plant fiber raw materials not only inhibit the activity of enzymes, but also significantly reduce the enzymatic hydrolysis efficiency of cellulose and hemicellulose, becoming the main technical bottleneck restricting the efficient utilization of lignocellulose. Especially in the resource utilization process of lignocellulosic raw materials such as bamboo chips, the formation of pseudo-lignin is more prominent, which seriously affects the enzymatic hydrolysis efficiency and subsequent saccharification rate. Therefore, how to effectively inhibit the formation of pseudo-lignin in the pretreatment of bamboo chips and improve the enzymatic hydrolysis efficiency of bamboo chips cellulose and hemicellulose has become a key technical problem in the current research on the efficient utilization of bamboo chips.

[0003] Bamboo chips are byproducts produced during bamboo processing. They have the advantages of wide sources, low cost, and high cellulose content. They are a lignocellulosic raw material with great development potential. Compared with other lignocellulosic raw materials, bamboo chips have the characteristics of high lignin content and complex lignin structure, and are more likely to form pseudo-lignin during pretreatment. These pseudo-lignins are deposited on the surface of lignocellulose in the form of microspheres, which hinder the binding of cellulase to cellulose through physical barriers and non-productive adsorption, thereby reducing the efficiency of enzymatic hydrolysis and becoming a problem that limits the resource utilization of bamboo chips. Therefore, before enzymatic conversion of cellulose, it is necessary to pretreat the lignocellulosic raw materials to destroy the cell wall structure of lignocellulose, thereby improving the effect of enzymatic hydrolysis. The pretreatment methods for lignocellulosic raw materials mainly include chemical pretreatment, physical pretreatment, and biological pretreatment. Among them, dilute acid high temperature pretreatment is widely used because of its simple operation and significant effect. However, traditional aqueous acid pretreatment has certain limitations in inhibiting the formation of pseudo-lignin. Therefore, developing an enzymatic hydrolysis efficiency of lignocellulosic raw materials such as bamboo chips by inhibiting the formation of pseudolignin not only has important theoretical significance, but also has broad application value in the field of biomass energy development and utilization. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for inhibiting the formation of pseudo-lignin and improving the enzymatic hydrolysis of lignocellulose raw materials. The method inhibits the formation of pseudo-lignin through a sulfuric acid-ethylene glycol combined pretreatment method, thereby promoting the enzymatic hydrolysis effect of lignocellulose raw materials.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for inhibiting the formation of pseudo-lignin and improving the enzymatic hydrolysis of lignocellulose raw materials, comprising: adding ethylene glycol to an acidic aqueous solution to perform acid-alcohol combined pretreatment on the lignocellulose raw materials; performing solid-liquid separation after the pretreatment to obtain a filter residue; then washing the filter residue to neutrality and adding cellulase to enzymatically hydrolyze the filter residue in an enzymatic hydrolysis system to inhibit the formation of pseudo-lignin and realize the enzymatic hydrolysis process of the lignocellulose raw materials.

[0007] Furthermore, the lignocellulose raw material is bamboo chips.

[0008] Furthermore, the temperature of the acid-alcohol combined pretreatment is 170° C. and the time is 1 h.

[0009] Furthermore, the solid-to-liquid ratio of the lignocellulosic raw material to the acid-alcohol aqueous solution is 1:20.

[0010] Furthermore, the concentration of the ethylene glycol is 0-80% by volume.

[0011] Furthermore, the acidic aqueous solution is a sulfuric acid aqueous solution with a mass fraction of 0.5%.

[0012] Furthermore, the enzymatic hydrolysis system consists of cellulase, citric acid buffer, water, tetracycline solution and filter residue, and the material substrate concentration in the enzymatic hydrolysis system is 5%.

[0013] Furthermore, the dosage of the cellulase is 20 FPU / g glucan, the concentration of the tetracycline solution is 10 g / L, and the concentration of the citric acid buffer is 1 mol / L.

[0014] Furthermore, the enzymatic hydrolysis conditions are: enzymatic hydrolysis for 48 h at 150 rpm, 50°C, and pH 4.8.

[0015] Furthermore, the method for inhibiting the formation of pseudo-lignin and improving the enzymatic hydrolysis of lignocellulose raw materials is characterized by comprising the following steps:

[0016] (1) The bamboo chips were pretreated with a 0.5% sulfuric acid aqueous solution and a 0-80% ethylene glycol solution at 170 °C for 1 h. The solid-to-liquid ratio of the bamboo chips to the acid-alcohol aqueous solution was 1:20.

[0017] (2) After acid-alcohol pretreatment, solid-liquid separation is performed to obtain a filter residue, which is then washed with distilled water until it becomes neutral;

[0018] (3) Add 1 mol / L citric acid buffer, 10 g / L tetracycline solution and 20 FPU / g glucan cellulase to the neutral filter residue, and add distilled water. Place it under the conditions of 150 rpm, 50 °C, and pH 4.8 for enzymatic hydrolysis for 48 h; the substrate concentration of the material under the enzymatic hydrolysis system is 5%.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The method of inhibiting the formation of pseudo-lignin and efficiently improving the enzymatic hydrolysis effect of lignocellulose raw materials of the present invention, adds ethylene glycol to a dilute acid aqueous solution, and performs acid-alcohol combined pretreatment on the lignocellulose raw materials, which can effectively inhibit the formation of pseudo-lignin during the pretreatment process and is conducive to promoting the enzymatic hydrolysis of cellulose. The test results show that compared with the enzymatic hydrolysis effect of bamboo chips pretreated with dilute acid alone, the enzymatic hydrolysis yield of the present invention is increased from 21.67% to 74.66%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Surface morphology of bamboo chips pretreated with dilute acid and different concentrations of ethylene glycol according to the present invention; wherein, Figure (a) is 0% by volume ethylene glycol, Figure (b) is 20% by volume ethylene glycol, Figure (c) is 40% by volume ethylene glycol, Figure (d) is 60% by volume ethylene glycol, and Figure (e) is 80% by volume ethylene glycol;

[0022] Figure 2 These are the surface morphology images of pseudo-lignin extracted with different concentrations of ethylene glycol of the present invention; wherein, Figure (a) is 0% by volume ethylene glycol, Figure (b) is 20% by volume ethylene glycol, Figure (c) is 40% by volume ethylene glycol, Figure (d) is 60% by volume ethylene glycol, and Figure (e) is 80% by volume ethylene glycol. DETAILED DESCRIPTION

[0023] The present invention is further illustrated below in conjunction with specific examples. The examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0024] In the following examples, the glucose concentration was determined by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows: chromatograph: Agillent 1200 high performance liquid chromatograph: chromatograph; Bio-Rad Aminex HPX-87H; mobile phase: 0.05 mol / L sulfuric acid solution, flow rate: 0.6 mL / min; column temperature: 55 °C; detector: differential refractive index detector; injection volume: 10 μL. Determination by external standard method. The enzymatic activity of cellulase is defined as: one international unit (FPU) of filter paper enzyme activity is equal to the amount of enzyme that generates 1 μmol of glucose per minute under standard reaction conditions.

[0025] The surface morphology of the acid-alcohol combined pretreated bamboo chips was determined using a scanning electron microscope (400-I, Hitachi, Japan) at an accelerating voltage of 15 kV.

[0026] The surface morphology of the extracted pseudolignin was determined by cryo-electron microscopy.

[0027] Example 1

[0028] A method for inhibiting the formation of pseudolignin and improving enzymatic hydrolysis of lignocellulose raw materials comprises the following steps:

[0029] (1) Weigh 5 g of bamboo chips and add 0.5% (m / v) sulfuric acid aqueous solution and different concentrations of ethylene glycol solution (volume fractions of 0%, 20%, 40%, 60% and 80%, respectively) at a solid-liquid ratio of 1:20 (w / v) to the chips. Treat at 170 ℃ for 1 h.

[0030] (2) After acid-alcohol pretreatment, solid-liquid separation is performed, and the surface morphology of the solid residue is as follows: Figure 1 The obtained solid residue was washed with distilled water until neutral, and filtered to obtain the bamboo chips after acid-alcohol pretreatment, and the main component analysis thereof is shown in Table 1.

[0031] (3) Weigh 1.000 g of the above acid-alcohol pretreated materials in 125 mL enzyme hydrolysis bottles, add 1 mL of 1 mol / L citric acid buffer, 0.2 mL of 10 g / L tetracycline solution and 20 FPU / g glucan cellulase to each enzyme hydrolysis bottle, and add appropriate amount of distilled water to make the total volume of water in the adsorption system 20 mL. After the system is fully mixed, cover the bottle cap tightly and place it in a constant temperature shaker at 150 rpm, pH 4.8, and 50 ° C for enzymatic hydrolysis for 48 h. The enzymatic hydrolysis yield of bamboo chips is shown in Table 2.

[0032] (4) After the enzymatic hydrolysis is completed, the hydrolyzate is centrifuged at 10,000 rpm / min for 5 min, and the supernatant is taken to determine the glucose content. Among them, the cellulase hydrolysis yield (%) = (glucose concentration in the hydrolyzate g / L × 0.9) × 0.02 ÷ (absolute dry weight of substrate g × glucan content) × 100%. In the formula: 0.9 is the conversion coefficient of glucose to cellulose; 0.05 is the volume of the hydrolyzate, L.

[0033] (5) Use 1,4-dioxane / water (96:4, v / v) solution to extract the acid-alcohol combined pretreated material to prepare pseudo-lignin. Weigh 5 g of the pretreated red material into a conical flask, add 50 mL of solvent according to the solid-liquid ratio of 1:10, and extract at room temperature for 1 day with magnetic stirring at a speed of 150 r / min. After the end, the solid and liquid are separated, and fresh organic extraction solution is added to the extracted solid again to continue the extraction. The above process is repeated three times to ensure that the pseudo-lignin is extracted to the maximum extent. The supernatants collected three times are mixed together, and the extraction solvent is removed by rotary evaporation and vacuum freeze drying to obtain pseudo-lignin solid. The surface morphology of pseudo-lignin is as follows Figure 2 The results of pseudolignin yield before and after extraction are shown in Table 3.

[0034] Table 1 Analysis of the changes in the main components of bamboo chips after acid-alcohol combined pretreatment

[0035]

[0036] The results in Table 1 show that the acid-alcohol combined pretreatment hydrolyzes most of the xylan in the bamboo chips into monosaccharides and xylo-oligosaccharides, dissolves a small amount of glucan, and removes most of the lignin.

[0037] Table 2 Enzymatic hydrolysis yield of bamboo chips pretreated with acid and alcohol (%)

[0038]

[0039] Table 2 shows the enzymatic hydrolysis yield of bamboo chips pretreated with acid-alcohol combination. As the concentration of ethylene glycol increases, the enzymatic hydrolysis yield increases. When the concentration of ethylene glycol is 80%, the enzymatic hydrolysis yield is the highest, reaching 74.66%. The increase in the enzymatic hydrolysis yield may be due to the enhanced ability of the acid-alcohol combination pretreatment system to remove pseudo-lignin under more stringent pretreatment conditions. The removal of pseudo-lignin in bamboo chips reduces the hindrance and nonspecific adsorption of lignin to cellulase.

[0040] Table 3 Lignin content, removal rate and pseudo-lignin yield of bamboo chips before and after extraction

[0041]

[0042] The results in Table 3 show that the acid-alcohol combined pretreatment can effectively destroy the lignin structure in bamboo chips and significantly improve the lignin removal rate. At the same time, pseudo-lignin is effectively removed.

[0043] Figure 1 This is the surface morphology of bamboo chips pretreated with acid-alcohol combination. After acid-alcohol combination pretreatment, obvious corrosion and fracture were observed on the surface of bamboo chips, and many pseudo-lignin balls appeared. With the increase of ethylene glycol concentration, the number of surface lignin balls decreased, and the surface pseudo-lignin was removed.

[0044] Figure 2 This is the surface morphology of the extracted pseudo-lignin. When ethylene glycol is not added, the presence of pseudo-lignin round balls can be clearly observed. After adding ethylene glycol, the pseudo-lignin balls are gradually degraded into fragments, which is more conducive to improving the efficiency of enzymatic hydrolysis.

[0045] The pseudo lignin described in the present application is the same as the relevant definition and explanation of pseudo lignin in the art, that is, during the dilute acid pretreatment of cellulose, carbohydrates form a series of lignin-like structural substances through reactions such as degradation, dehydration, and condensation. These substances are deposited on the surface of cellulose or dissolved in the solution in the form of droplets and microspheres, and increase the Klason lignin content (K value) in the pretreated cellulose. These lignin-like structural substances are called pseudo lignin.

[0046] During the dilute acid pretreatment process, cellulose and hemicellulose in the wood fiber raw materials will form pseudo-lignin globules through degradation, dehydration, and condensation processes, which not only hinder the hydrolysis of cellulase in the form of a physical barrier, but also reduce the efficiency of enzymatic hydrolysis through ineffective adsorption with cellulase. However, the mechanism of action of pseudo-lignin and cellulase and its influence on the hydrolysis of cellulase are still unclear.

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for inhibiting the formation of pseudolignin and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: Ethylene glycol is added to an acidic aqueous solution to perform an acid-alcohol combined pretreatment on the lignocellulose raw material; after the pretreatment, the solid and liquid are separated to obtain a filter residue; then the filter residue is washed to neutrality and cellulase is added to enzymatically hydrolyze the filter residue in an enzymatic hydrolysis system to inhibit the formation of pseudolignin and realize the enzymatic hydrolysis process of the lignocellulose raw material.

2. The method of claim 1 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The lignocellulose raw material is bamboo chips.

3. The method of claim 1 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The acid-alcohol combination pretreatment is carried out at a temperature of 170° C. and for a time of 1 h.

4. The method of claim 1 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The solid-to-liquid ratio of the lignocellulose raw material to the acid-alcohol aqueous solution is 1:

20.

5. The method of claim 1 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The concentration of the ethylene glycol is 0-80% by volume.

6. The method of claim 1 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The acidic aqueous solution is a sulfuric acid aqueous solution with a mass fraction of 0.5%.

7. The method of claim 1 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The enzymatic hydrolysis system consists of cellulase, citric acid buffer, water, tetracycline solution and filter residue, and the material substrate concentration in the enzymatic hydrolysis system is 5%.

8. The method of claim 7 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The dosage of the cellulase is 20 FPU / g dextran, the concentration of the tetracycline solution is 10 g / L, and the concentration of the citric acid buffer is 1 mol / L.

9. The method of claim 1 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The enzymatic hydrolysis conditions are: 150 rpm, 50°C, pH 4.8 for 48 h.

10. The method of claim 1 for inhibiting pseudolignin formation and improving enzymatic hydrolysis of lignocellulose raw materials, characterized in that: The following steps are involved: (1) The bamboo chips were pretreated with a 0.5% sulfuric acid aqueous solution and a 0-80% ethylene glycol solution at 170 °C for 1 h. The solid-to-liquid ratio of the bamboo chips to the acid-alcohol aqueous solution was 1:

20. (2) After acid-alcohol pretreatment, solid-liquid separation is performed to obtain a filter residue, which is then washed with distilled water until it becomes neutral; (3) Add 1 mol / L citric acid buffer, 10 g / L tetracycline solution and 20 FPU / g glucan cellulase to the neutral filter residue, and add distilled water. Place it under the conditions of 150 rpm, 50 °C, and pH 4.8 for enzymatic hydrolysis for 48 h; the substrate concentration of the material under the enzymatic hydrolysis system is 5%.

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