Method for saccharifying lignocellulose biomass

Abstract

The invention provides a saccharification method of lignocellulose biomass, which comprises the following steps: (1) mixing and homogenizing lignocellulose biomass and alkali liquor, and carrying out microwave treatment to obtain a pretreated material; and (2) adding cellulase to carry out an enzymolysis reaction, and carrying out solid-liquid separation to obtain an enzymolysis sugar solution of the lignocellulose biomass. According to the method, alkali treatment and microwave treatment are combined, a physical barrier of lignin wrapping cellulose is destroyed by alkali liquor, the internal structure of lignocellulose biomass is rapidly heated under the assistance of microwaves, damage to a lignin connecting structure is accelerated, lignin does not need to be stripped and removed, and the method is simple and convenient. The cellulose conversion rate and the saccharification recovery rate of the lignocellulose biomass can be obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical engineering, and relates to a method for saccharifying lignocellulosic biomass, and in particular to a method for saccharifying lignocellulosic biomass in a high-solid enzymatic hydrolysis system. Background Art

[0002] Xylose residue is an acidic solid waste produced during the dilute acid hydrolysis process of xylose from agricultural and forestry waste (corn cobs, sugarcane bagasse, etc.). It is a high-quality biomass resource. However, due to insufficient dilute acid hydrolysis, it cannot completely decompose the lignocellulose and expose the cellulose. The presence of lignin creates steric hindrance, which restricts the binding of cellulase to cellulose, reducing the enzymatic saccharification efficiency of xylose residue recycling.

[0003] CN117926612A discloses a microwave-assisted deep eutectic solvent pretreatment method for reeds. The method involves pretreating pulverized reeds with a deep eutectic solvent solution composed of choline chloride and p-toluenesulfonic acid for 10-100 seconds under microwave conditions. This method effectively removes lignin in a very short time, significantly improving the saccharification efficiency of cellulose and producing a low-molecular-weight lignin product.

[0004] CN110734942A discloses a method for pretreating xylose residue to improve the effect of enzymatic saccharification, comprising the following steps: weighing xylose residue, adding a deep eutectic solvent for pretreatment, and performing solid-liquid separation after the reaction; adding a KOH solution to the separated solid for pretreatment, and performing solid-liquid separation after the reaction to collect the solid and liquid portions respectively; washing the solid portion to neutrality to obtain the pretreated xylose residue, performing enzymatic saccharification, and measuring the glucose concentration in the enzymatic hydrolysis solution. The method of efficient pretreatment of xylose residue can improve the problem of low hydrolysis efficiency of cellulase, thereby reducing the amount of cellulase used and improving the conversion efficiency of reducing sugars, providing a certain theoretical basis for the comprehensive utilization of xylose residue.

[0005] CN114317638A discloses a method for cyclic enzymatic hydrolysis and saccharification of lignocellulose using multiple enzymes and a surfactant. The method comprises the following steps: saccharifying poplar wood through four steps: pretreatment, secondary pretreatment, preenzymatic hydrolysis and enzymatic hydrolysis, and simply recovering the enzymatic hydrolysis solution for cyclic enzymatic hydrolysis; pretreating the poplar wood with sodium hydroxide, and performing a secondary pretreatment on the alkali-treated poplar wood using laccase; preenzymatic hydrolysis of the two-step pretreated poplar wood using xylanase and a surfactant; finally, enzymatic hydrolysis and saccharification of the preenzymatically hydrolyzed poplar wood using cellulase and a surfactant; and filtering and recovering the laccase, xylanase after preenzymatic hydrolysis, cellulase after enzymatic hydrolysis and the surfactant in the filtrate after the secondary pretreatment for reuse, so as to reduce costs.

[0006] Existing technologies typically use physical, chemical, and biological methods to remove lignin, increase cellulose porosity, and promote contact between cellulose and enzymes to improve cellulose saccharification efficiency. However, the pretreatment process requires multiple solid-liquid separations and washings, resulting in complex and water-intensive processes that require further improvement. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a saccharification method for lignocellulosic biomass. The present invention adopts an alkali method combined with microwave treatment to pretreat the lignocellulosic biomass, without the need to strip and remove lignin, which can significantly reduce the effect of lignin on enzymatic saccharification and achieve a high saccharification recovery rate and cellulose conversion rate in a high-solid enzymatic hydrolysis reaction system.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for saccharifying lignocellulosic biomass, the method comprising:

[0010] (1) mixing lignocellulosic biomass with alkali solution, homogenizing the mixture, and subjecting the mixture to microwave treatment to obtain a pretreated material;

[0011] (2) adding cellulase to carry out enzymatic hydrolysis reaction, and performing solid-liquid separation to obtain the enzymatic hydrolysis sugar solution of the lignocellulosic biomass.

[0012] The present invention adopts an alkali method combined with microwave treatment to pretreat lignocellulosic biomass, without stripping and removing lignin, which can significantly reduce the effect of lignin on enzymatic saccharification and improve the saccharification recovery rate and cellulose conversion rate under the high-solid enzymatic hydrolysis system.

[0013] Alkali can disrupt hydrogen bonds in lignocellulosic biomass, breaking down the physical barrier of lignin surrounding cellulose, causing its structure to loosen, enhancing cellulose solubility and improving accessibility to cellulase. Microwave-assisted energy transfer rapidly heats the interior of the lignocellulosic biomass, accelerating the breakdown of lignin's connecting structure and enhancing the effects of the alkali, thereby increasing saccharification recovery and cellulose conversion rates.

[0014] Preferably, in step (1), the mass concentration of the alkali solution before mixing is 0.025-0.5%, for example, it can be 0.025%, 0.03%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5%, etc.; the mass ratio of the lignocellulosic biomass to the alkali solution is 1:(0.5-6), for example, it can be 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5 or 1:6, etc.

[0015] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0016] Preferably, the alkali solution includes any one or a combination of at least two of a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a sodium carbonate aqueous solution, a potassium carbonate aqueous solution, a sodium sulfate aqueous solution or a potassium sulfate aqueous solution.

[0017] Preferably, the alkali solution comprises an aqueous sodium hydroxide solution and / or an aqueous potassium hydroxide solution.

[0018] Preferably, in step (1), the mixing and homogenizing temperature is 20-40°C, for example, it can be 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C or 40°C; the mixing and homogenizing time is 15-25min, for example, it can be 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min or 25min.

[0019] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0020] The present invention adopts an alkali method combined with microwave treatment, and the two synergistically reduce the influence of lignin on enzymatic saccharification, significantly destroy the connection structure of lignin, improve the saccharification recovery rate and cellulose conversion rate, do not need to heat at high temperature to improve the alkali treatment effect, reduce costs, and produce fewer by-products.

[0021] Preferably, the power of the microwave treatment is 100-800 W, for example, it can be 100 W, 150 W, 200 W, 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, 550 W, 600 W, 650 W, 700 W, 750 W or 800 W, etc.; the time is 30-120 s, for example, it can be 30 s, 40 s, 50 s, 60 s, 70 s, 80 s, 90 s, 100 s, 110 s or 120 s, etc.

[0022] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0023] The microwave treatment parameters determine the oscillation speed of the electromagnetic waves, affecting the frequency of molecular motion and energy transfer efficiency. The specific microwave parameter ranges used in this invention ensure that the microwave energy distribution is within a reasonable range, fully destroying the lignin connection structure while maintaining the integrity of the cellulose molecular chain, avoiding excessive degradation and ensuring the forward progress of the enzymatic hydrolysis reaction, significantly improving the saccharification recovery rate and cellulose conversion rate of lignocellulosic biomass.

[0024] Preferably, in step (1), the lignocellulosic biomass comprises any one or a combination of at least two of the solid residues of corn cobs, corn straw, wheat straw, rice straw, reed straw or sugarcane after dilute acid hydrolysis.

[0025] Preferably, in the lignocellulosic biomass, the mass percentage of cellulose on a dry basis is 40-70%, for example, 40%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68% or 70%, etc.; the mass percentage of lignin is 10-30%, for example, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30%, etc.

[0026] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0027] Preferably, in step (2), the amount of the cellulase used is 5-15 FPU / g substrate, for example, it can be 5 FPU / g substrate, 6 FPU / g substrate, 7 FPU / g substrate, 8 FPU / g substrate, 9 FPU / g substrate, 10 FPU / g substrate, 11 FPU / g substrate, 12 FPU / g substrate, 13 FPU / g substrate, 14 FPU / g substrate or 15 FPU / g substrate, etc.

[0028] Preferably, the temperature of the enzymatic hydrolysis reaction is 45-55°C, for example, it can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C.

[0029] Further preferably, the temperature of the enzymatic hydrolysis reaction is 48-50°C, for example, it can be 48°C, 48.2°C, 48.4°C, 48.6°C, 48.8°C, 49°C, 49.2°C, 49.4°C, 49.6°C, 49.8°C or 50°C.

[0030] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0031] Maintaining the temperature of the enzymatic hydrolysis reaction at the optimum temperature of cellulase (48-50° C.) has higher reaction efficiency.

[0032] Preferably, in step (2), the enzymatic hydrolysis reaction is carried out in a stepwise enzymatic hydrolysis manner, specifically comprising: adding materials and / or water, adjusting the solid content of the pretreated material, and then performing four enzymatic hydrolysis reactions;

[0033] Adjusting the solid content of the pretreated material to 13-17% (e.g., 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, or 17%) before performing a first enzymatic hydrolysis reaction;

[0034] Adjusting the solid content of the pretreated material to 18-22% (e.g., 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, or 22%) before performing a second enzymatic hydrolysis reaction;

[0035] Adjusting the solid content of the pretreated material to 23-27% (e.g., 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, or 27%) before performing a third enzymatic hydrolysis reaction;

[0036] The solid content of the pretreated material is adjusted to 28-32% (for example, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5% or 32%) and then the fourth enzymatic hydrolysis reaction is performed.

[0037] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0038] Lignocellulosic biomass is porous, heterogeneous, and has poor fluidity. In traditional lignocellulosic biomass saccharification systems, limited water is tightly bound by the substrate, creating a localized high-viscosity environment. Cellulase must overcome greater diffusion resistance to reach the substrate, resulting in a decreased saccharification rate. Furthermore, increasing sugar concentration induces feedback inhibition in the limited water phase, inhibiting the forward enzymatic hydrolysis reaction.

[0039] In this high-solid enzymatic hydrolysis system, the present invention designs a step-by-step enzymatic hydrolysis reaction, and adjusts the solid content of the material through four gradients to affect the rheological properties of the saccharification system, reduce the viscosity of the system to within an appropriate range, and improve the reaction efficiency of cellulase to achieve a higher cellulose conversion rate and saccharification recovery rate.

[0040] Preferably, the time of the first enzymatic hydrolysis reaction is 2-8 h, for example, it can be 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h or 8 h, etc.; the pH value is 5-5.5, for example, it can be 5, 5.05, 5.1, 5.15, 5.2, 5.25, 5.3, 5.35, 5.4, 5.45 or 5.5, etc.

[0041] Preferably, the second enzymatic hydrolysis reaction time is 15-20h, for example, it can be 15h, 15.5h, 16h, 16.5h, 17h, 17.5h, 18h, 18.5h, 19h, 19.5h or 20h, etc.; the pH value is 5-5.5, for example, it can be 5, 5.05, 5.1, 5.15, 5.2, 5.25, 5.3, 5.35, 5.4, 5.45 or 5.5, etc.

[0042] Preferably, the time of the third enzymatic hydrolysis reaction is 20-48h, for example, it can be 20h, 22h, 25h, 28h, 30h, 32h, 35h, 38h, 40h, 42h, 45h or 48h; the pH value is 4.5-5.2, for example, it can be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 or 5.2.

[0043] Preferably, the fourth enzymatic hydrolysis reaction time is 48-96h, for example, it can be 48h, 50h, 55h, 60h, 65h, 70h, 75h, 80h, 85h, 90h, 95h or 96h; the pH value is 4.5-5.2, for example, it can be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 or 5.2.

[0044] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0045] The gradient control of time and pH value in the four enzymatic hydrolysis reactions of the present invention can improve the reaction efficiency of cellulase, further adapt to the rheological properties under the high-solid enzymatic hydrolysis system, and significantly improve the cellulose conversion rate and saccharification recovery rate of lignocellulosic biomass.

[0046] Preferably, in step (2), the enzymatic hydrolysis reaction further comprises adding an enzymatic hydrolysis aid.

[0047] Preferably, the mass percentage of the enzymatic hydrolysis aid in the enzymatic hydrolysis reaction system is 0.1-5%, for example, it can be 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0048] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0049] Preferably, the enzymatic hydrolysis aid comprises any one or a combination of at least two of alkyl glycoside, polyethylene glycol, magnesium chloride, ferric chloride, glyceryl monostearate or manganese chloride.

[0050] Preferably, the enzymatic hydrolysis aid comprises alkyl glycoside, polyethylene glycol, magnesium chloride and ferric chloride.

[0051] Driven by dispersive interactions, alkyl glycosides and polyethylene glycol rapidly bind to lignin, enhancing the surface hydrophilicity of lignin and inhibiting nonproductive adsorption. Magnesium chloride and ferric chloride adsorb on cellulase to form a complex, increasing steric hindrance and controlling competitive adsorption. The synergistic combination of alkyl glycosides, polyethylene glycol, magnesium chloride, and ferric chloride in the present invention solves the problems of nonproductive adsorption between residual lignin and cellulase, as well as cellulase inactivation. This extends the action time of cellulase and significantly improves the saccharification recovery rate and cellulose conversion rate of lignocellulosic biomass.

[0052] Preferably, the mass ratio of the alkyl glycoside, polyethylene glycol, magnesium chloride and ferric chloride is (0.3-0.8):(0.3-0.8):(0.04-0.2):(0.04-0.2).

[0053] The specific point values ​​in the first one (0.3-0.8) can be 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75 or 0.8, etc.

[0054] The specific point values ​​in the second one (0.3-0.8) can be 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75 or 0.8, etc.

[0055] The specific point values ​​in the first one (0.04-0.2) can be 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2, etc.

[0056] The specific point values ​​in the second one (0.04-0.2) can be 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2, etc.

[0057] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0058] Preferably, the number of carbon atoms in the alkyl glycoside is 8-18, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0059] Preferably, the number average molecular weight of the polyethylene glycol is 1000-6000, for example, it can be 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500 or 6000.

[0060] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0061] Preferably, in step (2), the solid-liquid separation method includes filtration and / or centrifugation.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The present invention combines alkali treatment and microwave treatment, using alkali solution to destroy the physical barrier of lignin wrapped around cellulose, and microwaves to rapidly heat up the internal structure of lignocellulosic biomass, accelerating the destruction of the lignin connection structure. Without the need to strip off and remove lignin, the cellulose conversion rate and saccharification recovery rate of lignocellulosic biomass can be significantly improved.

[0064] In the high-solid enzymatic hydrolysis system of lignocellulosic biomass, four enzymatic hydrolysis reactions were designed, and the solid content of the material, reaction temperature, reaction time and pH value were adjusted in a step-by-step manner to regulate the substrate and enzyme to always maintain an adaptive reaction system and achieve the optimal degree of saccharification reaction. DETAILED DESCRIPTION

[0065] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0066] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0067] The sources of materials used in the following specific embodiments are as follows:

[0068] Xylose residue was purchased from Jinan Shengquan Group Co., Ltd., and on a dry basis, the mass percentage of cellulose was 69.49% and the mass percentage of lignin was 21.13%. Cellulase was purchased from Novozymes (China) Biotechnology Co., Ltd.

[0069] Example 1

[0070] This embodiment provides a method for saccharifying lignocellulosic biomass, comprising:

[0071] (1) 200 g of xylose residue was mixed with 168 g of 0.25% sodium hydroxide aqueous solution, homogenized at 30° C. for 20 min, and then microwaved at 500 W for 60 s to obtain a pretreated material, which was then divided into four equal portions for later use;

[0072] (2) Maintaining the cellulase activity at 10 FPU / g substrate, a stepwise enzymatic hydrolysis reaction was performed at 49°C: ① First enzymatic hydrolysis reaction: take 1 portion of pretreated material, add water to adjust the solid content to 15%, react for 5 hours, and maintain the pH value at 5.3; ② Second enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 20%, react for 18 hours, and maintain the pH value at 5.5; ③ Third enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 25%, react for 36 hours, and maintain the pH value at 4.8; ④ Fourth enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 30%, react for 72 hours, and maintain the pH value at 4.9;

[0073] The enzymatic hydrolysis reaction system contains 0.5% alkyl glycoside (APG1218), 0.5% polyethylene glycol 2000, 0.05% magnesium chloride, and 0.15% ferric chloride as enzymatic hydrolysis aids. After four enzymatic hydrolysis reactions, the mixture is centrifuged at 8000 g for 15 minutes, and the liquid is collected to obtain an enzymatic hydrolyzed sugar solution.

[0074] Example 2

[0075] This embodiment provides a method for saccharifying lignocellulosic biomass, comprising:

[0076] (1) 200 g of xylose residue was mixed with 100 g of 0.1% potassium hydroxide aqueous solution, homogenized at 40° C. for 15 min, and then microwaved at 800 W for 30 s to obtain a pretreated material, which was then divided into four equal portions for later use;

[0077] (2) Maintaining the cellulase activity at 15 FPU / g substrate, a stepwise enzymatic hydrolysis reaction was performed at 48°C: ① First enzymatic hydrolysis reaction: take 1 portion of pretreated material, add water to adjust the solid content to 13%, react for 2 hours, and maintain the pH value at 5.5; ② Second enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 22%, react for 20 hours, and maintain the pH value at 5.2; ③ Third enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 23%, react for 20 hours, and maintain the pH value at 4.5; ④ Fourth enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 32%, react for 96 hours, and maintain the pH value at 5.2;

[0078] The enzymatic hydrolysis reaction system contains 0.3% alkyl glycoside (APG1214), 0.8% polyethylene glycol 3000, 0.04% magnesium chloride, and 0.2% ferric chloride as enzymatic hydrolysis aids. After four enzymatic hydrolysis reactions, the mixture is centrifuged at 8000 g for 15 minutes, and the liquid is collected to obtain an enzymatic hydrolyzed sugar solution.

[0079] Example 3

[0080] This embodiment provides a method for saccharifying lignocellulosic biomass, comprising:

[0081] (1) 200 g of xylose residue was mixed with 300 g of 0.5% sodium hydroxide aqueous solution, homogenized at 20° C. for 25 min, and then microwaved at a power of 100 W for 120 s to obtain a pretreated material, which was divided into 4 equal portions for later use;

[0082] (2) Maintaining the cellulase activity at 5 FPU / g substrate, a stepwise enzymatic hydrolysis reaction was performed at 50°C: ① First enzymatic hydrolysis reaction: take 1 portion of pretreated material, add water to adjust the solid content to 17%, react for 8 hours, and maintain the pH value at 5; ② Second enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 18%, react for 15 hours, and maintain the pH value at 5; ③ Third enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 27%, react for 48 hours, and maintain the pH value at 5.2; ④ Fourth enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 28%, react for 48 hours, and maintain the pH value at 4.5;

[0083] The enzymatic hydrolysis reaction system contains 0.8% alkyl glycoside (APG0810), 0.3% polyethylene glycol 4000, 0.2% magnesium chloride, and 0.04% ferric chloride as enzymatic hydrolysis aids. After four enzymatic hydrolysis reactions, the mixture is centrifuged at 8000 g for 15 minutes, and the liquid is collected to obtain an enzymatic hydrolyzed sugar solution.

[0084] Example 4

[0085] This embodiment provides a method for saccharifying lignocellulosic biomass, comprising:

[0086] (1) 200 g of xylose residue was mixed with 168 g of 0.25% sodium hydroxide aqueous solution, homogenized at 30° C. for 20 min, and then microwaved at 500 W for 60 s to obtain a pretreated material, which was then divided into four equal portions for later use;

[0087] (2) Maintaining the cellulase activity at 10 FPU / g substrate, a stepwise enzymatic hydrolysis reaction was performed at 49°C: ① First enzymatic hydrolysis reaction: take 1 portion of pretreated material, add water to adjust the solid content to 20%, react for 5 hours, and maintain the pH value at 5.3; ② Second enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 15%, react for 18 hours, and maintain the pH value at 5.5; ③ Third enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 30%, react for 36 hours, and maintain the pH value at 4.8; ④ Fourth enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 25%, react for 72 hours, and maintain the pH value at 4.9;

[0088] The enzymatic hydrolysis reaction system contains 0.5% alkyl glycoside (APG1218), 0.5% polyethylene glycol 2000, 0.05% magnesium chloride, and 0.15% ferric chloride as enzymatic hydrolysis aids. After four enzymatic hydrolysis reactions, the mixture is centrifuged at 8000 g for 15 minutes, and the liquid is collected to obtain an enzymatic hydrolyzed sugar solution.

[0089] Example 5

[0090] This embodiment provides a method for saccharifying lignocellulosic biomass, comprising:

[0091] (1) 200 g of xylose residue was mixed with 168 g of 0.25% sodium hydroxide aqueous solution, homogenized at 30° C. for 20 min, and then microwaved at 500 W for 60 s to obtain a pretreated material, which was then divided into four equal portions for later use;

[0092] (2) Maintaining the cellulase activity at 10 FPU / g substrate, a stepwise enzymatic hydrolysis reaction was performed at 49°C: ① First enzymatic hydrolysis reaction: take 1 portion of pretreated material, add water to adjust the solid content to 15%, react for 72 hours, and maintain the pH value at 4.9; ② Second enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 20%, react for 36 hours, and maintain the pH value at 4.8; ③ Third enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 25%, react for 18 hours, and maintain the pH value at 5.5; ④ Fourth enzymatic hydrolysis reaction: add 1 portion of pretreated material, add water to adjust the solid content to 30%, react for 5 hours, and maintain the pH value at 5.3;

[0093] The enzymatic hydrolysis reaction system contains 0.5% alkyl glycoside (APG1218), 0.5% polyethylene glycol 2000, 0.05% magnesium chloride, and 0.15% ferric chloride as enzymatic hydrolysis aids. After four enzymatic hydrolysis reactions, the mixture is centrifuged at 8000 g for 15 minutes, and the liquid is collected to obtain an enzymatic hydrolyzed sugar solution.

[0094] Example 6

[0095] This embodiment provides a method for saccharifying lignocellulosic biomass, comprising:

[0096] (1) 200 g of xylose residue was mixed with 168 g of 0.25% sodium hydroxide aqueous solution, homogenized at 30° C. for 20 min, and then microwaved at a power of 500 W for 60 s to obtain a pretreated material;

[0097] (2) Maintaining the cellulase activity at 10 FPU / g substrate, the enzymatic hydrolysis reaction was carried out at 49°C: after adding water to adjust the solid content to 20%, the reaction was continued for 131 h, and the pH was maintained at 5.2;

[0098] The enzymatic hydrolysis reaction system contains 0.5% alkyl glycoside (APG1218), 0.5% polyethylene glycol 2000, 0.05% magnesium chloride, and 0.15% ferric chloride as enzymatic hydrolysis aids. After the enzymatic hydrolysis reaction is completed, the mixture is centrifuged at 8000g for 15 minutes, and the liquid is collected to obtain an enzymatic hydrolyzed sugar solution.

[0099] Example 7

[0100] This embodiment provides a saccharification method for lignocellulosic biomass, which differs from Example 1 only in that: in step (1), "microwave treatment at a power of 500 W for 60 s" is replaced by "microwave treatment at a power of 1000 W for 20 s", and the other steps remain unchanged.

[0101] Example 8

[0102] This embodiment provides a saccharification method for lignocellulosic biomass, which differs from Example 1 only in that: in step (1), "microwave treatment at a power of 500 W for 60 s" is replaced by "microwave treatment at a power of 50 W for 150 s", and the other steps remain unchanged.

[0103] Example 9

[0104] This embodiment provides a method for saccharifying lignocellulosic biomass, which differs from Example 1 only in that: in step (2), alkyl glycoside (APG1218) is not added, and its reduced amount is proportionally distributed to polyethylene glycol 2000, magnesium chloride and ferric chloride, and other steps remain unchanged.

[0105] Example 10

[0106] This embodiment provides a method for saccharifying lignocellulosic biomass, which differs from Example 1 only in that: in step (2), polyethylene glycol 2000 is not added, and its reduced amount is proportionally distributed to alkyl glycoside (APG1218), magnesium chloride and ferric chloride, and the other steps remain unchanged.

[0107] Example 11

[0108] This embodiment provides a method for saccharifying lignocellulosic biomass, which differs from Example 1 only in that: in step (2), magnesium chloride is not added, and its reduced amount is proportionally distributed to alkyl glycoside (APG1218), polyethylene glycol 2000 and ferric chloride, and the other steps remain unchanged.

[0109] Example 12

[0110] This embodiment provides a method for saccharifying lignocellulosic biomass, which differs from Example 1 only in that: in step (2), ferric chloride is not added, and its reduced amount is proportionally distributed to alkyl glycoside (APG1218), polyethylene glycol 2000 and magnesium chloride, and the other steps remain unchanged.

[0111] Comparative Example 1

[0112] This comparative example provides a method for saccharifying lignocellulosic biomass, which differs from Example 1 only in that, in step (1), microwave treatment at a power of 500 W for 60 seconds is not performed, and other steps remain unchanged.

[0113] Comparative Example 2

[0114] This comparative example provides a saccharification method for lignocellulosic biomass, which differs from Example 1 only in that: in step (1), "microwave treatment at a power of 500 W for 60 s" is replaced by "ultrasonic treatment at a power of 500 W for 60 s".

[0115] Test Case

[0116] The cellulose conversion rate and fermentable sugar quality corresponding to Examples 1-12 and Comparative Examples 1-2 were tested respectively:

[0117] (1) The solids after biomass enzymatic hydrolysis were washed and centrifuged multiple times to remove soluble solids, dried, and weighed to obtain the insoluble solid mass; the cellulose content was determined using the National Renewable Energy Laboratory (NREL) method;

[0118]

[0119] (2) Detecting the glucose concentration (g / 100g) and cellobiose concentration (g / 100g) in the enzymatic hydrolyzed sugar solution by high performance liquid chromatography;

[0120] Fermentable sugar mass = enzymatic sugar solution mass × (glucose concentration + cellobiose concentration)

[0121] The test results are shown in Table 1. The present invention combines alkali treatment and microwave treatment to significantly reduce the interference of lignin on the saccharification of lignocellulosic biomass, and improve the saccharification recovery rate and cellulose conversion rate under the high-solid enzymatic hydrolysis system. The specific parameter control of the microwave treatment ensures that the microwave energy distribution is within a reasonable range, which can not only fully destroy the lignin connection structure, but also maintain the integrity of the cellulose molecular chain, avoid excessive degradation, and promote the forward progress of the enzymatic hydrolysis reaction. The present invention designs a step-by-step enzymatic hydrolysis reaction, adjusts the solid content of the material, affects the rheological properties of the high-solid enzymatic hydrolysis system, regulates the viscosity of the system within a suitable range, and significantly improves the reaction efficiency of cellulase; the time and pH value of the enzymatic hydrolysis reaction assist in gradient adaptation to achieve a higher cellulose conversion rate and saccharification recovery rate. Using alkyl glycosides, polyethylene glycol, magnesium chloride and ferric chloride as enzymatic hydrolysis aids can synergistically reduce non-productive adsorption and improve enzymatic hydrolysis efficiency.

[0122] Table 1

[0123]

[0124]

[0125] While the present invention illustrates a method for saccharifying lignocellulosic biomass through the aforementioned embodiments, the present invention is not limited to these embodiments, nor does it necessarily rely on these embodiments for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0126] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0127] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for saccharifying lignocellulosic biomass, characterized in that: The method comprises: (1) mixing lignocellulosic biomass with alkali solution, homogenizing the mixture, and subjecting the mixture to microwave treatment to obtain a pretreated material; (2) adding cellulase to carry out enzymatic hydrolysis reaction, and performing solid-liquid separation to obtain the enzymatic hydrolysis sugar solution of the lignocellulosic biomass.

2. The method according to claim 1, characterized in that In step (1), the mass concentration of the alkali solution before mixing is 0.025-0.5%, and the mass ratio of the lignocellulosic biomass to the alkali solution is 1:(0.5-6); Preferably, the alkali solution comprises any one or a combination of at least two of a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a sodium carbonate aqueous solution, a potassium carbonate aqueous solution, a sodium sulfate aqueous solution or a potassium sulfate aqueous solution; Preferably, the alkali solution comprises an aqueous sodium hydroxide solution and / or an aqueous potassium hydroxide solution.

3. The method according to claim 1 or 2, characterized in that In step (1), the mixing and homogenizing temperature is 20-40°C and the time is 15-25 minutes; Preferably, the power of the microwave treatment is 100-800 W, and the time is 30-120 s.

4. The method according to any one of claims 1 to 3, characterized in that In step (1), the lignocellulosic biomass comprises any one or a combination of at least two of the solid residues of corn cobs, corn straw, wheat straw, rice straw, reed straw or sugarcane after dilute acid hydrolysis; Preferably, in the lignocellulosic biomass, the mass percentage of cellulose is 40-70%, and the mass percentage of lignin is 10-30%, calculated on a dry basis.

5. The method according to any one of claims 1 to 4, characterized in that In step (2), the amount of the cellulase is 5-15 FPU / g substrate; Preferably, the temperature of the enzymatic hydrolysis reaction is 45-55°C.

6. The method according to any one of claims 1 to 5, characterized in that In step (2), the enzymatic hydrolysis reaction is carried out in a stepwise enzymatic hydrolysis manner, specifically comprising: adding materials and / or water, adjusting the solid content of the pretreated material, and then performing four enzymatic hydrolysis reactions; The solid content of the pretreated material was adjusted to 13-17% before the first enzymatic hydrolysis reaction; The solid content of the pretreated material is adjusted to 18-22% and then a second enzymatic hydrolysis reaction is performed; The solid content of the pretreated material is adjusted to 23-27% and then a third enzymatic hydrolysis reaction is performed; The solid content of the pretreated material was adjusted to 28-32% and then the fourth enzymatic hydrolysis reaction was performed.

7. The method according to claim 6, characterized in that The first enzymatic hydrolysis reaction lasts for 2-8 hours and has a pH of 5-5.5; Preferably, the second enzymatic hydrolysis reaction lasts for 15-20 hours and has a pH value of 5-5.5; Preferably, the third enzymatic hydrolysis reaction lasts for 20-48 hours and has a pH value of 4.5-5.2; Preferably, the fourth enzymatic hydrolysis reaction lasts for 48-96 hours and has a pH value of 4.5-5.

2.

8. The method according to any one of claims 1 to 7, characterized in that In step (2), the enzymatic hydrolysis reaction further comprises adding an enzymatic hydrolysis aid; Preferably, the mass percentage of the enzymatic hydrolysis aid in the enzymatic hydrolysis reaction system is 0.1-5%.

9. The method according to claim 8, characterized in that The enzymatic hydrolysis aid comprises any one or a combination of at least two of alkyl glycoside, polyethylene glycol, magnesium chloride, ferric chloride, glyceryl monostearate or manganese chloride; Preferably, the enzymatic hydrolysis aid comprises alkyl glycoside, polyethylene glycol, magnesium chloride and ferric chloride; Preferably, the mass ratio of the alkyl glycoside, polyethylene glycol, magnesium chloride and ferric chloride is (0.3-0.8):(0.3-0.8):(0.04-0.2):(0.04-0.2); Preferably, the number of carbon atoms in the alkyl glycoside is 8-18; Preferably, the number average molecular weight of the polyethylene glycol is 1000-6000.

10. The method according to any one of claims 1 to 9, characterized in that In step (2), the solid-liquid separation method includes filtration and / or centrifugation.

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