Method for auxiliary pretreatment of agriculture and forestry biomass by using amino acid

Abstract

The invention discloses a method for auxiliary pretreatment of agriculture and forestry biomass by using amino acid, and belongs to the technical field of biomass refining. According to the agricultural and forestry biomass pretreatment method, four kinds of different amino acids are added in the agricultural and forestry biomass pretreatment process, generation of inhibitors in pretreatment liquid is remarkably reduced, the pretreatment efficiency is improved, the enzymolysis property of the pretreated substrate is remarkably enhanced, and the yield of the pretreated substrate is improved. And the lignin with high beta-O-4 content prepared on the basis has extremely high practical value and economic value, and an environment-friendly, economical and feasible new way is provided for resource utilization of agricultural and forestry wastes.

Description

Technical Field

[0001] The invention relates to a method for pre-treating agricultural and forestry biomass using amino acids as an auxiliary, and belongs to the technical field of biomass refining. Background Art

[0002] Utilizing agricultural and forestry biomass as renewable energy is the most promising alternative to reducing our current reliance on fossil resources. Agricultural and forestry biomass primarily consists of cellulose, hemicellulose, and lignin. Cellulose can be enzymatically hydrolyzed to produce fermentable sugars, which can then be fermented to produce industrial products such as fuel ethanol and lactic acid. However, due to its dense structure, biomass cannot be directly utilized and typically requires pretreatment to break down its structure and fractionate its components for full utilization.

[0003] Lignocellulose pretreatment conditions are typically quite harsh, often employing high temperatures, high pressures, and steam explosions. This releases compounds such as furfural, phenols, and furans during the pretreatment process. These compounds undergo a series of reactions to generate pseudolignin that adheres to the substrate surface, significantly inhibiting the enzymatic hydrolysis of cellulose and affecting its efficiency. Furthermore, in existing processes, the structure of lignin often undergoes severe degradation and condensation, particularly significantly reducing the content of its key internal structure, the β-O-4 bond. This results in significant disadvantages in the resulting lignin's reactivity, solubility, and downstream conversion and utilization.

[0004] β-O-4 bonds are the predominant ether linkage in lignin molecules, accounting for over 50% of the original lignin structure. A high content of these bonds not only signifies the structural integrity and natural activity of lignin but also directly impacts the quality of subsequent depolymerization products. Lignin with a high β-O-4 content exhibits improved controllable breakability and reaction selectivity, enabling efficient production of monomeric aromatic compounds or high-value-added intermediates in reactions such as catalytic cracking, reductive deoxygenation, and oxidative modification.

[0005] Existing studies have shown that the addition of non-ionic surfactants during the pretreatment process significantly promotes enzymatic hydrolysis. Patent CN116426585A discloses the use of surfactants to assist in the pretreatment of agricultural and forestry biomass, thereby improving the efficiency of substrate enzymatic hydrolysis and sugar production, and obtaining non-condensed, highly active lignin; Patent CN116426586A discloses that the addition of surfactants during the pretreatment process can improve the utilization rate of cellulase under mild conditions. However, chemically synthesized surfactants also have certain problems during use. On the one hand, chemically synthesized surfactants are difficult to degrade when discharged into the environment, causing certain pollution to the ecosystem. On the other hand, surfactants are also adsorbed by lignocellulose during use, which to a certain extent reduces their efficiency.

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

[0007] To solve the above problems, the present invention adds four different amino acids during the pretreatment process of agricultural and forestry biomass, which significantly reduces the generation of inhibitors in the pretreatment liquid, improves the pretreatment efficiency, and significantly enhances the enzymatic hydrolysis of the substrate after pretreatment.

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

[0009] (1) mixing dried bagasse with a dilute sulfuric acid solution, adding 2-8% w / w amino acid to the dried bagasse, and reacting; cooling, stirring, and filtering after the reaction to obtain a solid matrix and a pretreatment solution; washing and drying the solid matrix to obtain a pretreated solid matrix;

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

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

[0012] In one embodiment, the amino acids in step (1) include 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 bagasse to dilute sulfuric acid solution is 1:15-25;

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

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

[0017] In one embodiment, the buffer in step (2) is a citric acid 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 25 to 30 FPU / g of pretreated solid matrix to cellulase at 140 to 200 rpm and 40 to 50° C. for 48 to 96 hours.

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

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

[0022] The 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] The third object of the present invention is to provide a novel material, wherein the product is prepared from the above-mentioned lignin with high β-O-4 bond content; the product includes a hydrogel.

[0024] A fourth object of the present invention is to provide a method for reducing inhibitor formation and increasing β-O-4 bond content during pretreatment of biomass feedstock, wherein amino acids are added during the pretreatment process, the method comprising the steps of:

[0025] (1) mixing dried sugarcane bagasse with a dilute sulfuric acid solution, adding 2-8% w / w amino acid, and reacting; cooling, stirring, and filtering after the reaction to obtain a solid matrix and a pretreatment solution; washing and drying the solid matrix to obtain a pretreated solid matrix;

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

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

[0028] In one embodiment, the amino acids in step (1) include 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 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 bagasse to the dilute sulfuric acid solution is 1:20.

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

[0032] In one embodiment, the buffer in step (2) is a citric acid 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 25 to 30 FPU / g of pretreated solid matrix to cellulase at 140 to 200 rpm and 40 to 50° C. for 48 to 96 hours.

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

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

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

[0038] (1) Mixing dried agricultural and forestry biomass with a dilute sulfuric acid solution, adding 1-3% v / v amino acid, and reacting; cooling, stirring, and filtering after the reaction to obtain a solid matrix and a pretreatment solution; washing and drying the solid matrix to obtain a pretreated solid matrix;

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

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

[0041] In one embodiment, the amino acids in step (1) include 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 methods in the field of biomass refining, wherein the application includes pre-treating agricultural and forestry biomass and preparing lignin.

[0043] Beneficial effects of the present invention:

[0044] The pretreatment method provided by the present invention aims to reduce the generation of inhibitors and improve the sugar production efficiency of subsequent substrate enzymatic hydrolysis by adding amino acids during the pretreatment process so that the amino acids react with inhibitors generated during the pretreatment process.

[0045] Specifically, the glucose concentration obtained by the pretreatment method of the present invention reaches 5 g / L, the glucose enzymatic hydrolysis rate reaches more than 49.54%, and has a high inhibitor removal rate and β-O-4 content. DETAILED DESCRIPTION

[0046] The following describes preferred embodiments of the present invention. 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] The raw materials used in the embodiment are:

[0048] Bagasse was obtained from a sugar factory in Guangxi Zhuang Autonomous Region and consists 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 nuclear magnetic resonance detection method:

[0053] 50 mg of lignin sample was dissolved in 0.5 mL of DMSO, and the signal of the sample was collected using the HSQC spectrum corresponding program. 1 H and 13 The spectral widths of the two dimensions of C are 5000 Hz and 18000 Hz respectively. 1 The number of sampling points in the H dimension is 1024, the relaxation time is 1.5s, and the accumulation is 64 times. 13 The number of sampling points in the C dimension is 256. The carbon-hydrogen coupling constant is 145 Hz. Zero purging is performed before Fourier transform. Calculations are based on semiquantitative analysis of the integrated intensity of the correlation signal.

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

[0055] Agilent 1260 was used for detection. The chromatographic columns were Agilent PLgel 5μm Mixed-c, 10μm Mixed-b, 300*7.5mm, and PLgel 5μm Guard 50×7.5mm, connected in series. The column temperature was 35°C, 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) using a Bio-Rad Aminex HPX-87H column, a column temperature of 60°C, a mobile phase of 5 mM sulfuric acid, and a flow rate of 0.6 mL / min.

[0058] 4. Inhibitor concentration:

[0059] By GC / MS detection, the pretreatment liquid was first extracted with an equal volume of dichloromethane (DCM), and then the residual water was removed with anhydrous sodium sulfate, and finally the sample was blown dry by a nitrogen blower. The gas phase detection used an Agilent 7890B, and the sample was chromatographed using an Ultra Alloy-5 column (Frontier Laboratory) with a length of 30 meters, an inner diameter of 0.25 mm, and a thickness of 0.25 μm. The electron ionization ion source was maintained at 250°C and 70 eV. The mass spectrometer scan range was 30 to 700. The oven temperature was maintained at 60°C for 4 minutes to delay the solvent, and then increased to 105°C (12°C min -1 Heat up, hold for 2 min); to 160 °C (15 °C min –1 Heating, hold for 1 minute); to 250℃ (10℃ min -1 Heating, hold for 2 minutes); finally raised to 315℃ (10℃ min -1 Heating and keeping warm for 8 minutes). The cumulative running time is 40 minutes.

[0060] Inhibition rate calculation formula:

[0061]

[0062] Among them, S 对照组 is the liquid quality detection area of ​​the group without adding amino acids, S 实验组 This is the liquid quality detection area of ​​the group treated with amino acids.

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

[0064] A method for pretreating agricultural and forestry biomass using amino acids as an aid, comprising the steps of:

[0065] (1) crushing the bagasse, passing it through a 20-mesh sieve, and drying it in an oven at 60° C. to a constant weight to obtain dried bagasse;

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

[0067] (3) 8 g of the pretreated solid matrix was mixed with 40 mL of pH 4.8 citric acid buffer (20% w / v, g / mL), and 30 FPU / g of pretreated solid matrix of Cellic Ctec3 cellulase was added. The enzyme was hydrolyzed at 180 rpm and 50°C for 72 h.

[0068] After enzymatic hydrolysis, the solid matrix after enzymatic hydrolysis was collected by centrifugation, and the solid was washed twice with pure water to remove residual sugar on the surface of the matrix, and the solid was collected by centrifugation; 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 to obtain the extract, concentrated, and then dropped into 10 times the volume of acid water (pH=2.0, dilute hydrochloric acid) to obtain lignin precipitate, which was freeze-dried to obtain enzymatic hydrolyzed lignin (EHL).

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

[0070] On the basis of Example 1, histidine was replaced by aspartic acid, and the remaining steps were consistent with Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolyzate.

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

[0072] On the basis of Example 1, histidine was replaced by arginine, and the remaining steps were consistent with Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolyzate.

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

[0074] On the basis of Example 1, histidine was replaced by glutamine, the drying temperature was 60° C., and the remaining steps were consistent with Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolyzate.

[0075] Comparative Example 1: No amino acid added

[0076] On the basis of Example 1, histidine was not added, and the remaining steps were consistent with Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolyzate.

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

[0078] On the basis of Example 1, glutamic acid was used to replace histidine, and the remaining steps were consistent with Example 1 to prepare lignin samples and sugarcane bagasse enzymatic hydrolysate.

[0079] Example 6: Enzyme hydrolysis effect detection

[0080] The lignin samples and sugarcane bagasse enzymatic hydrolysates prepared in Examples 1 to 5 and Comparative Examples 1 to 4 were taken for testing.

[0081] (1) Lignin

[0082] The lignin prepared in Examples 1-2 and Comparative Examples 1 and 4 was 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 linkages and molecular weights

[0084]

[0085] The dispersion coefficient is an important parameter that characterizes the width of a polymer's molecular weight distribution. Polymers with smaller dispersion coefficients generally have more controllable melt flow properties and are therefore suitable for precision processing. The β-O-4 bond, the primary linking unit in lignin, is considered the first structure to be broken during lignin's oxidative degradation. High-β-O-4 lignin has a higher number of free phenolic hydroxyl groups, making it easier to subsequently chemically modify (such as esterification and etherification) to prepare functional materials, offering significant advantages.

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

[0087] (2) Bagasse enzymatic hydrolysate

[0088] 0.4 mL of the bagasse hydrolyzate prepared in Examples 1 to 5 and Comparative Examples 1 to 4 was sampled and centrifuged at 8000 rpm. The supernatant was diluted 10 times and the glucose concentration was determined. The results are shown in Table 2.

[0089] Table 2 Comparison of glucose enzymolysis rates

[0090]

[0091] (3) Inhibitor removal rate

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

[0093] Table 3 Inhibitor removal rate

[0094]

[0095] Note: “ / ” means the removal rate is 0%.

[0096] These results demonstrate that the introduction of exogenous amino acids not only improves the efficiency of cellulose enzymatic hydrolysis by modifying the lignin structure but, more importantly, effectively suppresses the formation of fermentation inhibitors. The pretreatment method provided by this invention is simple to operate, requiring no additional steps. It significantly improves the enzymatic hydrolysis of the pretreated substrate and produces highly active, non-condensed lignin, facilitating the full utilization of biomass components.

[0097] Example 7: Effect of amino acid addition on enzymatic hydrolysis

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

[0099] Table 4 Effect of amino acid dosage on enzymatic hydrolysis

[0100]

[0101] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing lignin with a high β-O-4 bond content, characterized in that: Including steps: (1) mixing dried bagasse with a dilute sulfuric acid solution, adding 2-8% w / w amino acid to the dried bagasse, and reacting; cooling, stirring, and filtering after the reaction to obtain a solid matrix and a pretreatment solution; washing and drying the solid matrix to obtain a pretreated solid matrix; (2) The pretreated solid matrix is ​​mixed with a buffer solution, enzymatically hydrolyzed, and centrifuged to obtain an enzymatically hydrolyzed solid matrix; the enzymatically hydrolyzed solid matrix is ​​washed and centrifuged, mixed with dioxane, and reacted; after the reaction, centrifuged to obtain a lignin extract, which is concentrated, acid-precipitated, and dried to obtain a lignin with a high β-O-4 bond content.

2. The method according to claim 1, characterized in that The amino acids in step (1) include any one or more of histidine, aspartic acid, arginine, and glutamine.

3. The method according to claim 1, characterized in that The concentration of the dilute sulfuric acid in step (1) is 1-3% v / v; the mass ratio of the dried bagasse to the dilute sulfuric acid solution is 1:15-25.

4. The method according to claim 1, wherein The ratio of the pretreated solid matrix to the buffer solution in step (2) is 2-30 g:100 mL.

5. The method according to claim 1, wherein In step (2), the enzymatic hydrolysis is carried out by adding 25 to 30 FPU / g of pretreated solid matrix to cellulase at 140 to 200 rpm and 40 to 50° C. for 48 to 96 hours.

6. The method according to claim 1, characterized in that In step (2), the ratio of the enzymatically hydrolyzed solid matrix to dioxane is 1 g:15-25 mL; the reaction is carried out at 180-200 rpm and 40-50° C. for 18-30 h.

7. Lignin with high β-O-4 bond content prepared by the method according to any one of claims 1 to 6.

8. A new material, characterized in that: The product is prepared from the high-β-O-4 bond content lignin according to claim 6; and the novel material includes a hydrogel.

9. A method for reducing the formation of inhibitors and increasing the content of β-O-4 bonds during the pretreatment of biomass raw materials, characterized in that: Amino acids are added during the pretreatment process, the method comprising the steps of: (1) mixing dried bagasse with a dilute sulfuric acid solution, adding 2-8% w / w amino acid to the dried bagasse, and reacting; cooling, stirring, and filtering after the reaction to obtain a solid matrix and a pretreatment solution; washing and drying the solid matrix to obtain a pretreated solid matrix; (2) The pretreated solid matrix is ​​mixed with a buffer solution, enzymatically hydrolyzed, and centrifuged to obtain an enzymatically hydrolyzed solid matrix; the enzymatically hydrolyzed solid matrix is ​​washed and centrifuged, mixed with dioxane, and reacted; after the reaction, centrifuged to obtain a lignin extract, which is concentrated, acid-precipitated, and dried to obtain lignin.

10. Use of the method according to any one of claims 1 to 6 or the method according to claim 9 in the field of biomass refining, characterized in that: The applications include pre-treating agricultural and forestry biomass and preparing lignin.

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