A method for pretreating agroforestry biomass with amino acid

Abstract

The application discloses a method for pretreating agricultural and forestry biomass with the aid of amino acids and belongs to the technical field of biomass refining. The application develops a pretreatment method for agricultural and forestry biomass, which is natural and easy to degrade, and the method is characterized in that four different amino acids are added in the pretreatment process of the agricultural and forestry biomass. The method significantly reduces the generation of inhibitors in the pretreatment liquid, improves the pretreatment efficiency, significantly enhances the enzymatic hydrolysis of the substrate after pretreatment, and has extremely high practical value and economic value for preparing lignin with high beta-O-4 content. The method provides an environmentally friendly and economically feasible new way for the resource utilization of agricultural and forestry wastes.

Description

Technical Field

[0001] This invention relates to a method for pretreating agricultural and forestry biomass using amino acids, belonging to the field of biomass refining technology. Background Technology

[0002] Utilizing agricultural and forestry biomass as a renewable energy source is the most promising alternative to reducing current dependence on fossil resources. Agricultural and forestry biomass mainly includes cellulose, hemicellulose, and lignin. Cellulose can be enzymatically hydrolyzed to produce fermentable sugars, and the fermented sugar solution can then be used to produce industrial products such as fuel ethanol and lactic acid. Because biomass has a dense structure and cannot be used directly, it usually needs to be pretreated to break down its structure and fractionate its components to achieve full utilization.

[0003] Lignocellulose pretreatment conditions are typically quite harsh, often employing high temperature, high pressure, and steam explosion methods. This process releases compounds such as furfural, phenols, and furans. These compounds undergo a series of reactions to form pseudolignin, which adheres to the matrix surface and significantly inhibits cellulose enzymatic hydrolysis, thus affecting the efficiency of cellulose hydrolysis. Furthermore, in existing processes, the lignin structure often undergoes severe degradation and condensation, particularly a significant reduction in the content of its key internal structure—the β-O-4 bond. This results in lignin with significant disadvantages in reactivity, solubility, and downstream conversion and utilization.

[0004] The β-O-4 bond is the most prevalent ether bond in lignin molecules, accounting for over 50% of the original lignin structure. This high content not only signifies the structural integrity and natural activity of lignin but also directly affects the quality of its subsequent depolymerization products. Lignin with high β-O-4 content exhibits superior controllable cleavage and reaction selectivity, enabling efficient generation of monomeric aromatic compounds or high-value intermediates in reactions such as catalytic cracking, reductive deoxygenation, and oxidative modification.

[0005] Existing studies have shown that adding nonionic surfactants during pretreatment significantly promotes enzymatic hydrolysis. Patent CN116426585A discloses the use of surfactants to assist in the pretreatment of agricultural and forestry biomass, improving the efficiency of substrate enzymatic hydrolysis and yielding non-condensing, highly active lignin; Patent CN116426586A discloses that adding surfactants during pretreatment can improve the utilization rate of cellulase under mild conditions. However, chemically synthesized surfactants also present certain problems during use. On the one hand, chemically synthesized surfactants are difficult to degrade in the environment, causing pollution to the ecosystem. On the other hand, surfactants can also be adsorbed by lignocellulose during use, reducing their utilization efficiency to some extent.

[0006] Therefore, developing a pretreatment method for agricultural and forestry biomass with natural and easily degradable additives, and preparing lignin with high β-O-4 content based on this method, has extremely high practical and economic value, providing an environmentally friendly and economically feasible new approach for the resource utilization of agricultural and forestry waste. Summary of the Invention

[0007] To address the aforementioned issues, this invention adds four different amino acids during the pretreatment of agricultural and forestry biomass, which significantly reduces the formation of inhibitors in the pretreatment solution, improves pretreatment efficiency, and significantly enhances the enzymatic hydrolysis of the pretreated substrate.

[0008] The first objective of this invention is to provide a method for preparing lignin with high β-O-4 bond content, comprising the steps of:

[0009] (1) The dried sugarcane bagasse is mixed with dilute sulfuric acid solution, and 2-8% w / w of amino acids from the dried sugarcane bagasse is added and reacted; after the reaction, the mixture is cooled, stirred and filtered to obtain a solid matrix and a pretreatment solution; the solid matrix is ​​washed and dried to obtain a pretreated solid matrix.

[0010] (2) The pretreated solid matrix is ​​mixed with buffer, enzymatically hydrolyzed, and centrifuged to obtain the enzymatically hydrolyzed solid matrix; the enzymatically hydrolyzed solid matrix is ​​washed, centrifuged, mixed with dioxane, and reacted; after the reaction, centrifuged to obtain lignin extract, concentrated, acidified to precipitate, and dried to obtain lignin with high β-O-4 bond content.

[0011] In one embodiment, the sugarcane bagasse dried in step (1) is prepared by crushing the sugarcane bagasse, passing it through a 20-mesh sieve, and drying it at 50-60°C.

[0012] In one embodiment, the amino acid in step (1) includes any one or more of histidine, aspartic acid, arginine, and glutamine.

[0013] Optionally, the amino acids in step (1) include histidine, aspartic acid, and arginine.

[0014] In one embodiment, the concentration of dilute sulfuric acid in step (1) is 1-3% v / v; the mass ratio of dried sugarcane bagasse to dilute sulfuric acid solution is 1:15-25;

[0015] Optionally, the concentration of dilute sulfuric acid is 1% v / v, and the ratio of dried sugarcane bagasse to dilute sulfuric acid is 1:20.

[0016] In one embodiment, cooling in step (1) is to reduce the temperature to 50±5°C.

[0017] In one embodiment, the buffer in step (2) is a citrate buffer (20% w / v, g / mL) with a pH of 4.8 to 6.

[0018] In one embodiment, the ratio of the pretreated solid matrix to the buffer solution in step (2) is 2-30 g: 100 mL.

[0019] In one embodiment, the enzymatic hydrolysis in step (2) is performed by adding cellulase to a pretreated solid matrix of 25-30 FPU / g and hydrolyzing at 140-200 rpm and 40-50°C for 48-96 h.

[0020] In one embodiment, the ratio of the solid matrix after enzymatic hydrolysis to dioxane in step (2) is 1g: 15-25mL; the reaction is carried out at 180-200rpm and 40-50℃ for 18-30h.

[0021] Optionally, in step (2), the ratio of the solid matrix after enzymatic hydrolysis to dioxane is 1g:20mL; the reaction is carried out at 180-200rpm and 45-50℃ for 20-24h.

[0022] A second object of the present invention is to provide lignin with high β-O-4 bond content prepared by any of the above methods.

[0023] A third objective of this invention is to provide a novel material, the product of which is prepared from the above-mentioned lignin with high β-O-4 bond content; the product comprises a hydrogel.

[0024] A fourth objective of this invention is to provide a method for reducing the formation of inhibitors and increasing the β-O-4 bond content during the pretreatment of biomass raw materials, wherein amino acids are added during the pretreatment process, and the method includes the following steps:

[0025] (1) The dried sugarcane bagasse is mixed with dilute sulfuric acid solution, and 2-8% w / w amino acids are added and reacted; after the reaction, the mixture is cooled, stirred and filtered to obtain a solid matrix and a pretreatment solution; the solid matrix is ​​washed and dried to obtain a pretreated solid matrix.

[0026] (2) The pretreated solid matrix is ​​mixed with buffer solution, enzymatically hydrolyzed, and centrifuged to obtain the enzymatically hydrolyzed solid matrix; the enzymatically hydrolyzed solid matrix is ​​washed, centrifuged, mixed with dioxane, and reacted; after the reaction, it is centrifuged to obtain lignin extract, which is concentrated, acidified to precipitate, and dried to obtain lignin.

[0027] In one embodiment, the sugarcane bagasse dried in step (1) is prepared by crushing the sugarcane bagasse, passing it through a 20-mesh sieve, and drying it at 50-60°C.

[0028] In one embodiment, the amino acid in step (1) includes any one or more of histidine, aspartic acid, arginine, and glutamine.

[0029] In one embodiment, the concentration of dilute sulfuric acid in step (1) is 1-3% v / v; the mass ratio of dried sugarcane bagasse to dilute sulfuric acid solution is 1:15-25;

[0030] Optionally, the concentration of the dilute sulfuric acid is 1% v / v, and the mass ratio of the dried sugarcane bagasse to the dilute sulfuric acid solution is 1:20.

[0031] In one embodiment, cooling in step (1) is to reduce the temperature to 50±5°C.

[0032] In one embodiment, the buffer in step (2) is a citrate buffer (20% w / v, g / mL) with a pH of 4.8 to 6.

[0033] In one embodiment, the ratio of the pretreated solid matrix to the buffer solution in step (2) is 2-30 g: 100 mL.

[0034] In one embodiment, the enzymatic hydrolysis in step (2) is performed by adding cellulase to a pretreated solid matrix of 25-30 FPU / g and hydrolyzing at 140-200 rpm and 40-50°C for 48-96 h.

[0035] In one embodiment, the ratio of the solid matrix after enzymatic hydrolysis to dioxane in step (2) is 1g: 15-25mL; the reaction is carried out at 180-200rpm and 40-50℃ for 18-30h.

[0036] Optionally, in step (2), the ratio of the solid matrix after enzymatic hydrolysis to dioxane is 1g:20mL; the reaction is carried out at 180-200rpm and 45-50℃ for 20-24h.

[0037] This invention also provides a method for amino acid-assisted pretreatment of agricultural and forestry biomass, the method comprising the following steps:

[0038] (1) The dried agricultural and forestry biomass was mixed with dilute sulfuric acid solution, and 1-3% v / v amino acids were added and reacted; after the reaction, the mixture was cooled, stirred and filtered to obtain a solid matrix and a pretreatment solution; the solid matrix was washed and dried to obtain a pretreated solid matrix.

[0039] (2) The pretreated solid matrix is ​​mixed with buffer solution, enzymatically hydrolyzed, and centrifuged to obtain the enzymatically hydrolyzed solid matrix; the enzymatically hydrolyzed solid matrix is ​​washed, centrifuged, mixed with dioxane, and reacted; after the reaction, it is centrifuged to obtain lignin extract, which is concentrated, acidified to precipitate, and dried to obtain lignin.

[0040] In one embodiment, the agricultural and forestry biomass is sugarcane bagasse.

[0041] In one embodiment, the amino acid in step (1) includes any one or more of histidine, aspartic acid, arginine, and glutamine.

[0042] A fifth object of the present invention is to provide an application of any of the above-described methods in the field of biomass refining, the application including pretreatment of agricultural and forestry biomass and preparation of lignin.

[0043] The beneficial effects of this invention are:

[0044] The pretreatment method provided by this invention adds amino acids during the pretreatment process, causing the amino acids to react with the inhibitors generated during the pretreatment process, aiming to reduce the formation of inhibitors and improve the efficiency of subsequent substrate enzymatic hydrolysis for sugar production.

[0045] Specifically, the pretreatment method of the present invention achieves a glucose concentration of 5 g / L, a glucose hydrolysis rate of over 49.54%, and a high inhibitor removal rate and β-O-4 content. Detailed Implementation

[0046] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0047] Raw materials used in the examples:

[0048] The bagasse was taken from a sugar factory in Guangxi Zhuang Autonomous Region and consisted of 39.1% cellulose, 22.0% hemicellulose and 25.2% lignin.

[0049] Cellic Ctec3 cellulase (purchased from Novozymes (China) Investment Co., Ltd.);

[0050] Histidine, arginine, lysine, aspartic acid, glutamine, and glutamic acid were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0051] Test method:

[0052] 1. Two-dimensional NMR detection method:

[0053] 50 mg of lignin sample was dissolved in 0.5 mL of DMSO, and the HSQC spectrum correspondence program was used to acquire the signal of the sample. 1 H and 13 The spectral widths of the two dimensions of C are 5000Hz and 18000Hz, respectively. 1 The number of sampling points in dimension H is 1024, the relaxation time is 1.5s, and the summation is 64 times. 13 The number of sampling points in dimension C is 256. The carbon-hydrogen coupling constant is 145 Hz. Zeroing is performed before Fourier transform. Calculations are performed based on semi-quantitative analysis of the integral intensity of the relevant signal.

[0054] 2. GPC (gel permeation chromatography) testing was performed at Hangzhou Yanqu Information Technology Co., Ltd., and the specific parameters are as follows:

[0055] The detection was performed using an Agilent 1260 column, consisting of three columns in series: an Agilent PLgel 5μm Mixed-c column, a 10μm Mixed-b column (300×7.5mm), and a PLgel 5μm Guard column (50×7.5mm). The column temperature was 35℃, the mobile phase was THF, and the flow rate was 1 ml / min.

[0056] 3. Glucose concentration:

[0057] Detection was performed by high-performance liquid chromatography (Hitachi, Japan). The chromatographic column was a Bio-Rad Aminex HPX-87H, the column temperature was 60℃, the mobile phase was 5mM sulfuric acid, and the flow rate was 0.6mL / min.

[0058] 4. Inhibitor concentration:

[0059] GC / MS analysis was performed by first extracting the pretreated solution with an equal volume of dichloromethane (DCM), then removing residual water with anhydrous sodium sulfate, and finally drying the sample with nitrogen. Gas chromatography was performed using an Agilent 7890B Ultra Alloy-5 column (Frontier Laboratories), with a length of 30 meters, an inner diameter of 0.25 mm, and a thickness of 0.25 μm. The electron ionization source was maintained at 250 °C and 70 eV. The mass spectrometry scan range was 30–700. The oven temperature was maintained at 60 °C for 4 min to delay solvent removal, then increased to 105 °C (12 °C min). -1 Heat up and hold for 2 minutes; then heat to 160℃ (15℃ min). –1 Heat up and hold for 1 minute; until 250℃ (10℃ min). -1 Heat up and hold for 2 minutes; finally raise to 315℃ (10℃ min). -1 Heat up and maintain the temperature for 8 minutes. The total running time is 40 minutes.

[0060] Inhibition rate calculation formula:

[0061]

[0062] Among them, S 对照组 For the liquid chromatography-mass spectrometry (LC-MS) detection area of ​​the group without amino acid treatment, S 实验组 The area for liquid chromatography-mass spectrometry (LC-MS) analysis of the amino acid-treated group.

[0063] Example 1: Histidine-assisted pretreatment of agricultural and forestry biomass

[0064] A method for pretreating agricultural and forestry biomass using amino acids includes the following steps:

[0065] (1) Sugarcane bagasse is crushed, passed through a 20-mesh sieve, and dried in an oven at 60°C until constant weight is obtained to obtain dried sugarcane bagasse;

[0066] (2) 5g of dried sugarcane bagasse was mixed with 100g of 1% (v / v) dilute sulfuric acid solution, and 0.2g of histidine (4% w / w dried sugarcane bagasse) was added. The mixture was then mixed in a high-pressure reactor and heated to 160℃ at 300rpm for 60min. After the reaction was completed, the temperature was lowered to 50±5℃, and 150mL of boiling water (100℃) was added and stirred for 10min. The mixture was then filtered to obtain a solid matrix and a pretreated solution. The solid matrix was washed twice with 150mL of tap water, filtered, and dried at 60℃ to obtain the pretreated solid matrix.

[0067] (3) Take 8g of the pretreated solid matrix and mix it with 40mL of pH 4.8 citrate buffer (20% w / v, g / mL), add 30FPU / g of Cellic Ctec3 cellulase of the pretreated solid matrix, and enzymatically hydrolyze at 180rpm and 50℃ for 72 hours.

[0068] After enzymatic hydrolysis, centrifugation was performed to collect the solid matrix. The solid was washed twice with pure water to remove residual sugars on the matrix surface, and then centrifuged to collect the solid. Dioxane (solid-liquid ratio 1g:20mL) was added to the solid, and the reaction was carried out at 180rpm and 50℃ for 24h to extract lignin. After the reaction was completed, the extract was centrifuged, concentrated, and then added dropwise to 10 times the volume of acidic water (pH=2.0, dilute hydrochloric acid) to obtain lignin precipitate. The precipitate was freeze-dried to obtain enzymatically hydrolyzed lignin (EHL).

[0069] Example 2: Aspartic acid-assisted pretreatment of agricultural and forestry biomass

[0070] Based on Example 1, histidine was replaced with aspartic acid, and the remaining steps were the same as in Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolysate.

[0071] Example 3: Arginine-assisted pretreatment of agricultural and forestry biomass

[0072] Based on Example 1, histidine was replaced with arginine, and the remaining steps were the same as in Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolysate.

[0073] Example 4: Glutamine-assisted pretreatment of agricultural and forestry biomass

[0074] Based on Example 1, histidine was replaced with glutamine, the drying temperature was 60°C, and the remaining steps were the same as in Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolysate.

[0075] Comparative Example 1: No amino acids added

[0076] Based on Example 1, without adding histidine, the remaining steps are the same as in Example 1, and lignin samples and sugarcane bagasse enzymatic hydrolysate are prepared.

[0077] Comparative Example 2: Glutamic acid-assisted pretreatment of agricultural and forestry biomass

[0078] Based on Example 1, histidine was replaced with glutamic acid, and the remaining steps were the same as in Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolysate.

[0079] Example 6: Enzymatic hydrolysis effect detection

[0080] The lignin samples prepared in Examples 1-5 and Comparative Examples 1-4 were compared with sugarcane bagasse enzymatic hydrolysate for testing.

[0081] (1) Lignin

[0082] The lignins prepared in Examples 1-2 and Comparative Examples 1 and 4 were analyzed using 2D-HSQC and GPC to determine the lignin-related linkages (β-O-4) and molecular weight. The results are shown in Table 1.

[0083] Table 1. Lignin-related bonds and molecular weights

[0084]

[0085] The dispersion coefficient is an important parameter characterizing the molecular weight distribution width of a polymer. Polymers with smaller dispersion coefficients generally have more controllable melt flowability, making them suitable for precision processing. The β-O-4 bond, as the most important linking unit in lignin, is considered to be the first structure to be broken during the oxidative degradation of lignin. High β-O-4 lignin has more free phenolic hydroxyl groups, which facilitates subsequent chemical modifications (such as esterification and etherification) to prepare functional materials, giving it a significant advantage.

[0086] The results show that the dispersion coefficients of the samples in Examples 1 and 2 are both smaller than those of the comparative example, confirming that the samples have the potential to prepare advanced materials.

[0087] (2) Sugarcane bagasse enzymatic hydrolysate

[0088] In Examples 1-5, 0.4 mL of sugarcane bagasse enzymatic hydrolysate prepared in Comparative Examples 1-4 was taken, centrifuged at 8000 rpm, and the supernatant was diluted 10 times to determine the glucose concentration. The results are shown in Table 2.

[0089] Table 2 Comparison results of glucose hydrolysis rate

[0090]

[0091] (3) Inhibitor removal rate

[0092] The pretreatment solution prepared in step (2) of Example 1 was used to test the inhibitor removal rate. The results are shown in Table 3.

[0093] Table 3 Inhibitor Removal Rate

[0094]

[0095] Note: " / " indicates a removal rate of 0%.

[0096] The results in summary indicate that the introduction of exogenous amino acids not only promotes the efficiency of cellulose enzymatic hydrolysis through lignin structure modification, but more importantly, it effectively inhibits the formation of fermentation inhibitors. The pretreatment method provided by this invention is simple to operate and requires no additional steps. It significantly improves the enzymatic hydrolysis properties of the pretreatment matrix and yields highly active, non-condensed lignin, which is beneficial for achieving full utilization of biomass components.

[0097] Example 7: Effect of Amino Acid Addition Amount on Enzymatic Hydrolysis

[0098] Based on Examples 1-4, the amount of histidine added was changed to 2%, 6%, 8%, and 10%, respectively, while the other steps remained the same. The effect of amino acid dosage on the enzymatic hydrolysis effect was detected, and the results are shown in Table 4. The results show that the enzymatic hydrolysis effect was significantly better when the amount of histidine added was 6%–8% than when it was 2%.

[0099] Table 4. Effect of Amino Acid Dosage on Enzymatic Hydrolysis

[0100]

[0101] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing lignin with high β-O-4 bond content, characterized in that, Including the following steps: (1) The dried sugarcane bagasse is mixed with dilute sulfuric acid solution, and 2-8% w / w of aspartic acid in the dried sugarcane bagasse is added and reacted; after the reaction, the mixture is cooled, stirred and filtered to obtain a solid matrix and a pretreatment solution; the solid matrix is ​​washed and dried to obtain a pretreated solid matrix; the concentration of dilute sulfuric acid is 1-3% v / v; the mass ratio of dried sugarcane bagasse to dilute sulfuric acid solution is 1:15-25. (2) The pretreated solid matrix is ​​mixed with buffer, enzymatically hydrolyzed, and centrifuged to obtain the enzymatically hydrolyzed solid matrix; the enzymatically hydrolyzed solid matrix is ​​washed, centrifuged, mixed with dioxane, and reacted; after the reaction, centrifuged to obtain lignin extract, concentrated, acidified to precipitate, and dried to obtain lignin with high β-O-4 bonds.

2. The method according to claim 1, characterized in that, In step (2), the ratio of the pretreated solid matrix to the buffer solution is 2~30 g: 100 mL.

3. The method according to claim 1, characterized in that, In step (2), enzymatic hydrolysis is performed by adding cellulase to a pretreated solid matrix of 25-30 FPU / g and hydrolyzing at 140-200 rpm and 40-50℃ for 48-96 h.

4. The method according to claim 1, characterized in that, In step (2), the ratio of the solid matrix after enzymatic hydrolysis to dioxane is 1 g: 15~25 mL; the reaction is carried out at 180~200 rpm and 40~50℃ for 18~30 h.

5. The lignin with high β-O-4 bond content prepared by the method according to any one of claims 1 to 4.

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