Method for strengthening saccharification of corn stalks based on slow-release treatment of mixed solution

Abstract

In order to solve the problems of strong pH dependence, difficult reaction process control, reagent waste, high ecological risk and the like in AHP pretreatment, the invention adopts a mixed solution of potassium hydroxide and urea to treat straws, hydrogen peroxide is slowly added at a constant rate during the process, and the saccharification of corn straws is enhanced. The slow-release regulation and control on the reaction process are realized. After the corn straw is treated by the method, the corn straw is subjected to enzymolysis saccharification to produce the reducing sugar. Compared with conventional alkaline hydrogen peroxide pretreatment, the problems that alkaline hydrogen peroxide pretreatment is high in pH dependency, the reaction process is difficult to control, reagents are wasted, and the ecological risk is high are solved. The invention provides a pretreatment method which is efficient, energy-saving, easy to operate and environment-friendly, and resource utilization of lignocellulose is promoted. The invention relates to the technical field of lignocellulose pretreatment.

Description

Technical Field

[0001] This invention relates to the field of lignocellulose pretreatment technology, specifically to a method for enhancing corn straw saccharification based on slow-release treatment with a mixed liquor. Background Technology

[0002] With the acceleration of global industrialization and the over-exploitation of fossil fuels, energy shortages are becoming increasingly prominent, leading to massive greenhouse gas emissions. Addressing the energy and climate crises has become urgent. Lignocellulose, with its abundant reserves and recyclability, is considered the only sustainable biomass energy source capable of achieving emission reduction and carbon sequestration. Converting it into biomass energy or bio-based chemicals through bioconversion technology has profound significance for promoting energy structure transformation and ecological environmental protection. However, lignin in lignocellulose, such as that found in straw, forms a protective layer encapsulating cellulose and hemicellulose. This stable and stubborn structure greatly hinders the degradation and saccharification processes of cellulose and hemicellulose by microorganisms and enzymes, resulting in limited subsequent conversion efficiency. Therefore, developing an economically feasible pretreatment method to break down the stubborn structure of lignocellulose and improve fiber accessibility is crucial for promoting efficient hydrolysis and saccharification reactions.

[0003] Alkaline hydrogen peroxide (AHP) pretreatment has attracted widespread attention due to its high delignification efficiency, moderate reaction conditions, and low inhibitor yield. AHP pretreatment typically involves adjusting the hydrogen peroxide (H₂O₂) solution to alkaline conditions using NaOH. Subsequently, H₂O₂ decomposes under alkaline conditions to generate free radicals. These free radicals can selectively interact with lignin through various reaction pathways, ultimately achieving efficient delignification. However, AHP pretreatment technology has significant limitations: firstly, it is highly pH-dependent, with an optimal reaction pH range of only 11.5-11.6. Precise control of this narrow range is difficult in practice, easily leading to reduced reagent utilization and waste; secondly, the additional introduction of NaOH... + This increases the ecological risks of the pretreatment process and may have potential impacts on subsequent processes and the environment. Therefore, optimizing and improving existing AHP pretreatment processes has become a necessary measure to promote the efficient conversion of lignocellulose. Summary of the Invention

[0004] To address the problems of strong pH dependence, difficulty in controlling the reaction process, reagent waste, and high ecological risks in AHP pretreatment, this invention proposes a method for enhancing corn straw saccharification based on slow-release treatment of a mixed solution.

[0005] The technical solution adopted by the present invention to solve the above problems is as follows: This invention proposes a method for enhancing corn saccharification based on a mixed liquor slow-release treatment, comprising the following steps: Step 1: Crush, sieve, wash and dry the corn stalks; after crushing, pass the stalks through a 40-100 mesh sieve; wash the crushed stalks with clean water and then dry them at 80℃.

[0006] Step 2: Place the straw obtained in Step 1 into a mixed solution of potassium hydroxide and urea and stir. During the stirring process, add hydrogen peroxide solution at a constant rate (add hydrogen peroxide through a microfluidic injection pump or peristaltic pump). After the reaction is completed, filter, wash and dry the straw for later use. Step 3: Mix the pretreated straw from Step 2 with a buffer solution containing cellulase for enzymatic hydrolysis to obtain corn straw saccharification solution.

[0007] Furthermore, in step 2, the processing temperature is 25℃, the stirring speed is 300 rpm, and the reaction system is 100 mL.

[0008] Furthermore, in step 2, the concentration of potassium hydroxide is 2%-10% (w / v), the concentration of urea is 2%-10% (w / v), the hydrogen peroxide addition rate is 6.94-83.33 mg / min, the treatment time is 2-24 h, and the treatment load is 5-20% (w / v).

[0009] Furthermore, in step 2, the concentration of potassium hydroxide is 2% (w / v), the concentration of urea is 2% (w / v), the hydrogen peroxide addition rate is 16.67 mg / min, the treatment time is 2 hours, and the treatment load is 5% (w / v).

[0010] Furthermore, in step 2, the concentration of potassium hydroxide is 4% (w / v), the concentration of urea is 2% (w / v), the hydrogen peroxide addition rate is 41.67 mg / min, the treatment time is 2 hours, and the treatment load is 5% (w / v).

[0011] Furthermore, in step 2, the concentration of potassium hydroxide is 4% (w / v), the concentration of urea is 2% (w / v), the hydrogen peroxide addition rate is 6.94 mg / min, the treatment time is 12 hours, and the treatment load is 5% (w / v).

[0012] Furthermore, in step 2, the concentration of potassium hydroxide is 4% (w / v), the concentration of urea is 2% (w / v), the hydrogen peroxide addition rate is 6.94 mg / min, the treatment time is 12 hours, and the treatment load is 20% (w / v).

[0013] Furthermore, the straw that was washed with clean water in step 2 was then dried at 80°C.

[0014] Furthermore, in step 3, the cellulase buffer is a 50 mM citrate buffer with a pH of 4.8, the cellulase concentration is 0.3 FPU / mL, the straw treatment load is 2% (w / v), and the final amount of cellulase used is 15 FPU / g straw.

[0015] Furthermore, in step 3, the enzymatic hydrolysis temperature is 50℃, the enzymatic hydrolysis speed is 150 rpm, and the enzymatic hydrolysis time is 72 hours.

[0016] The beneficial effects of this invention are: 1. This invention proposes a method for enhancing corn straw saccharification based on a slow-release treatment using a mixed liquor. This method utilizes a microfluidic syringe pump or peristaltic pump to slowly add H2O2 to a mixture of KOH and urea. Compared to conventional AHP pretreatment, it saves reagents, increases reaction controllability, reduces ecological risks, and aligns with sustainable development principles. Based on this, the conditions for the slow-release treatment were optimized. The optimal conditions are: adding H2O2 to a KOH and urea mixture (4% KOH and 2% urea) at a flow rate of 6.94 mg / min for 12 hours.

[0017] 2. Under a 5% (w / v) treatment load, the saccharification rate of straw treated with the mixed liquor slow-release solution reached 93.88%. Under a 20% treatment load, the saccharification rate reached 75.99%, which is 3.94 times higher than that of untreated straw enzymatic hydrolysate. The mixed liquor slow-release treatment is a highly efficient, energy-saving, and green pretreatment method, which is beneficial for the commercialization and promotion of straw biomass biorefining.

[0018] 3. This invention solves the problems of strong pH dependence, difficulty in controlling the reaction process, reagent waste, and high ecological risks associated with alkaline hydrogen peroxide pretreatment. It provides a highly efficient, energy-saving, easy-to-operate, and environmentally friendly pretreatment method, which helps promote the resource utilization of lignocellulose. Detailed Implementation

[0019] The present invention will be described below with reference to specific embodiments. Unless otherwise specified, the methods, reagents, and equipment used in the present invention are all conventional methods, reagents, and equipment.

[0020] In the following examples, neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), and ash (A) were detected using a semi-automatic cellulose analyzer. The contents of cellulose, hemicellulose, and lignin were calculated using formulas (1)-(3):

[0021]

[0022]

[0023] In the following examples, the contents of reducing sugars, glucose, and xylose were detected using the DNS colorimetric method, a glucose kit, and a xylose kit, respectively. The saccharification rate (RSR), saccharification yield (RY), and saccharification yield (ARY) of reducing sugars were calculated using formulas (4)-(6):

[0024]

[0025]

[0026] Example 1: A method for enhancing corn saccharification based on mixed liquor slow-release treatment includes the following steps: (1) Straw preparation: Crush the corn stalks and pass them through a 40-100 mesh sieve. Wash and dry them before use.

[0027] (2) Straw pretreatment: Prepare a potassium hydroxide and urea mixture containing 2% KOH and 2% urea. Then, mix 100 mL of the potassium hydroxide and urea mixture with 5 g of corn straw. Add H2O2 to the reaction system using a microfluidic syringe pump at a flow rate of 16.67 mg / min. Continue the treatment at 25℃ and 300 rpm for 2 hours. After the reaction, filter, wash, dry, and weigh the straw, and detect the cellulose, hemicellulose, and lignin content in the pretreated straw.

[0028] (3) Pre-treatment of straw hydrolysis: 1 g of the pre-treated straw from step (2) and 50 mL of cellulase buffer were placed in... Mix well in a 100 mL Erlenmeyer flask and enzymatically hydrolyze at 50 °C and 150 rpm for 72 hours. After the reaction, measure the concentrations of reducing sugar, glucose, and xylose in the hydrolysate.

[0029] Example 2: The difference between Example 2 and Example 1 lies in the straw pretreatment: a potassium hydroxide and urea mixture containing 4% KOH and 2% urea was prepared. Then, 100 mL of the potassium hydroxide and urea mixture was mixed with 5 g of corn straw. H₂O₂ was added to the reaction system using a microfluidic syringe pump at a flow rate of 41.67 mg / min, and the treatment was continued for 2 hours at 25°C and 300 rpm. All other reaction conditions and operating procedures were the same.

[0030] Example 3: The difference between Example 3 and Example 2 lies in the straw pretreatment: a potassium hydroxide and urea mixture containing 4% KOH and 2% urea was prepared. Then, 100 mL of the potassium hydroxide and urea mixture was mixed with 5 g of corn straw. H₂O₂ was added to the reaction system using a microfluidic syringe pump at a flow rate of 6.94 mg / min. The treatment was continued for 12 hours at 25°C and 300 rpm. All other reaction conditions and operating procedures were the same.

[0031] Example 4: The difference between Example 4 and Example 3 lies in the straw pretreatment: a potassium hydroxide and urea mixture containing 4% KOH and 2% urea was prepared. Then, 100 mL of the potassium hydroxide and urea mixture was mixed with 20 g of corn straw. H2O2 was added to the reaction system using a microfluidic syringe pump at a flow rate of 6.94 mg / min, and the treatment was continued for 12 hours at 25°C and 300 rpm. All other reaction conditions and operating procedures were the same.

[0032] Comparative example: (1) Straw preparation: Crush the corn stalks and pass them through a 40-100 mesh sieve. Wash and dry them before use.

[0033] (2) Straw hydrolysis: Mix 1 g of untreated straw and 50 mL of cellulase buffer in a 100 mL Erlenmeyer flask. The mixture was homogenized and enzymatically hydrolyzed at 50℃ and 150 rpm for 72 hours. After the reaction, the concentrations of reducing sugar, glucose, and xylose in the hydrolysate were measured.

[0034] The composition of the pretreated straw in Examples 1-4 and the comparative straw is shown in Table 1.

[0035] Table 1. Summary of the composition of pretreated straw in Examples 1-4 and comparative straw:

[0036] The composition of the pretreated straw and the comparative straw enzymatic hydrolysate of Examples 1-4, as well as the RSR, RY, and ARY values, are shown in Table 2.

[0037] Table 2. Summary of composition, reducing sugar saccharification rate (RSR), reducing sugar yield (RY), and actual reducing sugar yield (ARY) of pretreated straw and comparative straw enzymatic hydrolysate from Examples 1-4:

[0038] The results from Examples 1-4 and the comparative examples show that the saccharification rate of reducing sugars in the slow-release treatment of straw reached a maximum of 93.37 ± 1.47% (Example 3), and even when the treatment load was increased to 20%, it still reached 75.99 ± 0.55% (Example 4). Under a high treatment load of 20%, the RSR, RY, and ARY of the enzymatic hydrolysis of pretreated straw were 75.99 ± 0.55%, 63.58 ± 0.55%, and 40.32 ± 0.90%, respectively, which were 4.94, 5.91, and 3.75 times that of untreated straw.

[0039] In summary, this invention abandons the conventional AHP pretreatment mode and proposes a novel approach: gradually adding H2O2 to a mixture of potassium hydroxide and urea using a microfluidic injection pump, while optimizing the treatment conditions for the slow-release treatment of the mixture. This method solves the problems of strong pH dependence, difficulty in controlling reaction conditions, reagent waste, and ecological risks associated with conventional AHP pretreatment, making it a novel, efficient, energy-saving, easy-to-operate, and environmentally friendly pretreatment method. Furthermore, the waste liquid from the slow-release treatment of the mixture contains a large amount of C, N, and K elements, which can be used as agricultural liquid fertilizer after simple treatment, realizing the resource utilization of the waste liquid. Applying the slow-release treatment technology to various treatment scenarios for lignocellulose will help promote the resource utilization of lignocellulose.

[0040] This invention provides a novel mode for the slow-release treatment of mixed solutions. The above description is merely a preferred embodiment of the invention and is not intended to limit the invention in any way. Although the invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the invention, based on the technical essence of the invention, and within the spirit and principles of the invention, shall still fall within the protection scope of the invention.

Claims

1. A method for enhancing corn straw saccharification based on mixed liquor slow-release treatment, characterized in that, Includes the following steps: Step 1: Crush, sieve, wash and dry the corn stalks; Step 2: Place the straw obtained in Step 1 into a mixed solution of potassium hydroxide and urea and stir. During the stirring process, add hydrogen peroxide solution at a constant rate. After the reaction is complete, filter, wash and dry the straw for later use. Step 3: Mix the pretreated straw from Step 2 with a buffer solution containing cellulase for enzymatic hydrolysis to obtain corn straw saccharification solution.

2. The method for enhancing corn straw saccharification based on mixed liquor slow-release treatment according to claim 1, characterized in that, In step 2, the processing temperature is 25℃, the stirring speed is 300 rpm, and the reaction system is 100 mL.

3. The method for enhancing corn straw saccharification based on mixed liquor slow-release treatment according to claim 2, characterized in that, In step 2, the concentration of potassium hydroxide is 2%-10% (w / v), the concentration of urea is 2%-10% (w / v), the hydrogen peroxide addition rate is 6.94-83.33 mg / min, the treatment time is 2-24 h, and the treatment load is 5-20% (w / v).

4. The method for enhancing corn straw saccharification based on slow-release treatment of a mixed liquor according to claim 3, characterized in that, In step 2, the concentration of potassium hydroxide was 2% (w / v), the concentration of urea was 2% (w / v), the hydrogen peroxide addition rate was 16.67 mg / min, the treatment time was 2 hours, and the treatment load was 5% (w / v).

5. The method for enhancing corn straw saccharification based on slow-release treatment of a mixed liquor according to claim 3, characterized in that, In step 2, the concentration of potassium hydroxide was 4% (w / v), the concentration of urea was 2% (w / v), the hydrogen peroxide addition rate was 41.67 mg / min, the treatment time was 2 hours, and the treatment load was 5% (w / v).

6. The method for enhancing corn straw saccharification based on slow-release treatment of a mixed liquor according to claim 3, characterized in that, In step 2, the concentration of potassium hydroxide was 4% (w / v), the concentration of urea was 2% (w / v), the hydrogen peroxide addition rate was 6.94 mg / min, the treatment time was 12 hours, and the treatment load was 5% (w / v).

7. The method for enhancing corn saccharification based on slow-release treatment of mixed liquor according to claim 3, characterized in that, In step 2, the concentration of potassium hydroxide was 4% (w / v), the concentration of urea was 2% (w / v), the hydrogen peroxide addition rate was 6.94 mg / min, the treatment time was 12 hours, and the treatment load was 20% (w / v).

8. The method for enhancing corn straw saccharification based on slow-release treatment of a mixed liquor according to claim 1, characterized in that, The straw that was washed with clean water in step 2 was then dried at 80°C.

9. The method for enhancing corn straw saccharification based on mixed liquor slow-release treatment according to claim 1, characterized in that, In step 3, the cellulase buffer was a 50 mM citrate buffer with a pH of 4.8, the cellulase concentration was 0.3 FPU / mL, the straw treatment load was 2% (w / v), and the final amount of cellulase used was 15 FPU / g straw.

10. A method for enhancing corn saccharification based on slow-release treatment of a mixed liquor according to claim 9, characterized in that, In step 3, the enzymatic hydrolysis temperature is 50℃, the enzymatic hydrolysis speed is 150 rpm, and the enzymatic hydrolysis time is 72 hours.

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