Catalytic hydrolysis process of high-concentration cellulose

Abstract

The invention discloses a catalytic hydrolysis process of high-concentration cellulose, and relates to the field of cellulose bioconversion. In order to solve the problem of hydrolysis of high-concentration cellulose, staged dynamic regulation and control, microbial synergy and pretreatment strengthening strategies are innovatively adopted, and efficient hydrolysis and cost reduction are achieved. In the pretreatment stage, a dilute acid-ammonia water coupling pretreatment technology is adopted, so that a lignin-hemicellulose network can be effectively destroyed, and the accessibility of cellulose is improved. Compared with the prior art, the method has obvious advantages. Through unique staged dynamic regulation and control, the mass transfer problem of a high-solid system is solved. The process has good compatibility with various lignocellulose raw materials, can be coupled with simultaneous saccharification and fermentation to directly produce high-concentration ethanol, simplifies the subsequent separation process, and has wide application prospects in the field of cellulose bioconversion.

Description

Technical Field

[0001] The present invention relates to the field of cellulose bioconversion, and in particular to a high-efficiency hydrolysis process of high-concentration cellulose materials under enzyme catalysis, which is suitable for the saccharification treatment of lignocellulosic biomass and provides high-concentration sugar solution for subsequent biofuel or chemical production. Background Art

[0002] In existing technologies, high-concentration enzymatic hydrolysis of cellulose faces numerous challenges: 1. Mass transfer and mixing issues: High solids content leads to high system viscosity, low contact efficiency between cellulase and substrate, and traditional continuous stirring consumes a lot of energy and can easily lead to enzyme inactivation. 2. Limited enzymatic hydrolysis efficiency: Ineffective enzyme adsorption, product inhibition, and surfactant effects of lignin are significantly affected by concentration and substrate structure, requiring precise control of reaction conditions. 3. High enzyme cost: Auxiliary enzymes such as β-glucosidase need to be added separately, increasing costs.

[0003] Existing processes, such as two-stage saccharification, reduce energy consumption but lack the integration of dynamic surfactant regulation and enzyme system optimization. Surfactant application is limited by a narrow concentration window and is easily inhibited at high concentrations. Enzyme recovery technology requires complex operations. Therefore, an integrated innovative process is urgently needed to address mass transfer, enzyme efficiency, and cost issues at high concentrations. Summary of the Invention

[0004] The purpose of the present invention is to solve the above-mentioned problems in the prior art and provide a catalytic hydrolysis process for high-concentration cellulose. By regulating the reaction parameters in stages, optimizing the enzyme system and combining the synergistic effect of pretreatment, the mass transfer problem of high-solid system is solved, efficient hydrolysis is achieved, and energy consumption and enzyme dosage are reduced.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A catalytic hydrolysis process for high-concentration cellulose comprises the following steps:

[0007] 1) Pretreatment stage: After the lignocellulose raw material is crushed, it is pretreated with dilute acid-ammonia coupling to control the solid mass of the pretreated material to account for 25%-40% of the total wet material. It is then washed with water to neutrality, dried, crushed and sieved;

[0008] 2) First enzymatic saccharification stage: The raw materials pretreated in step 1) are placed in a ribbon stirred reactor with a solid content controlled at 25% to 35%, and then complex cellulase and nonionic surfactant are added and heated and stirred for reaction;

[0009] 3) Second enzymatic saccharification stage: The material from the first enzymatic saccharification stage is transferred to a low-shear reactor, inoculated with recombinant yeast that secretes β-glucosidase to continue the reaction, and non-ionic surfactant is added according to the viscosity of the system.

[0010] The present invention also includes a product separation and enzyme recovery stage: after hydrolysis, solid-liquid separation is performed, and the residue is subjected to a secondary re-adsorption method to recover the cellulase. Specifically, fresh substrate (unhydrolyzed and saccharified lignocellulosic material) is added to the residue at a solid-liquid ratio of 1:3 to 1:5. The residue is allowed to stand at 20-30°C for 1-2 hours. After separation, the lignocellulosic material adsorbed with cellulase is recovered.

[0011] In step 1), the dilute acid-ammonia coupled pretreatment is specifically as follows: the raw material is mixed with a 1%-3% sulfuric acid solution at a solid-to-liquid ratio of 1:5-1:7 by volume, and treated at 120-130°C for 30-60 minutes. After washing, the raw material is mixed with a 10%-15% ammonia solution at a solid-to-liquid ratio of 1:5-1:7 by volume, and treated at 50-60°C for 12-24 hours. This disrupts the lignin-hemicellulose network and improves cellulose accessibility.

[0012] In the first enzymatic saccharification stage, the reaction temperature is 50-55°C, The reaction was carried out for 3 to 6 hours under the conditions of stirring at 200 to 300 rpm, so that the system was rapidly liquefied under high shear conditions, and the yield stress of the system after liquefaction was less than 10 .

[0013] The nonionic surfactant is Tween-80; in step 2), the mass proportion of the nonionic surfactant is 0.05% to 0.1% of the total system.

[0014] In step 3), the addition of the nonionic surfactant is dynamically regulated according to the viscosity of the system. When the online viscometer monitors the viscosity>10 When adding non-ionic surfactant, the mass proportion of non-ionic surfactant is 0.1%~0.2% of the total system.

[0015] In step 3), the volume of the low shear reactor is 10 times or more than that of the reactor in the first enzymatic saccharification stage, and the stirring rate is controlled to be 50-100 rpm or intermittent stirring, and the intermittent stirring is stirring for 5-10 minutes every 30 minutes, and the stirring rate is 50-100 rpm.

[0016] In step 3), the continued reaction is carried out at 37-50° C. for 48-72 hours, and the inoculation amount of the recombinant yeast secreting β-glucosidase is 5%-10% by volume.

[0017] The mass ratio of exo-enzyme, endo-enzyme and beta-glucosidase in the composite cellulase is (1-6):(1-4):1, and the total enzyme amount is 1-15 FPU / g cellulose.

[0018] The lignocellulose raw material comprises one or more of corn cobs, corn stalks, sugarcane bagasse, bamboo, bamboo shoot shells, and mushroom grass, and the particle size after pulverization and screening is 20-80 mesh.

[0019] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0020] 1. Dilute acid treatment destroys hemicellulose, and ammonia removes lignin. The two steps synergistically increase the cellulose exposure rate and provide a highly active substrate for enzymatic hydrolysis (for example, the cellulose content of corn cobs after pretreatment is 70.8%). The solid content is strictly controlled at 25% to 40% to ensure fluidity in the first stage (yield stress < 10 ) and subsequent pumping feasibility.

[0021] 2. First stage (liquefaction): High shear stirring (200-300 rpm) combined with low-concentration surfactants. High shear crushing quickly breaks up substrate agglomerates and the adsorption barrier of lignin to enzymes, reducing ineffective enzyme adsorption and increasing the enzymatic hydrolysis rate in the liquefaction stage. The glucose concentration can reach 65 g / L within 6 hours.

[0022] 3. The second stage (saccharification): Dynamically add surfactant according to viscosity (breaking the bottleneck of high concentration inhibition), stir at low speed to reduce mechanical damage to the enzyme and improve enzyme activity retention, and at the same time, introduce recombinant yeast to supplement β-glucosidase. Specifically, the second stage dynamically adds surfactant through online viscosity monitoring to solve the problem of narrow surfactant concentration window in traditional processes (high concentration surfactants tend to inhibit enzymatic hydrolysis), maintaining mass transfer efficiency while avoiding high concentration inhibition, and achieving viscosity from 20 in high solid system. Down to 8 , ensuring continued high efficiency of subsequent enzymatic hydrolysis. Endogenous β-glucosidase supplementation: Introducing recombinant yeast that secretes β-glucosidase replaces some of the exogenous β-glucosidase required (external cellulase supplementation required with existing technologies). This reduces enzyme usage and, by in situ secretion of the enzyme, relieves cellobiose inhibition, increasing total sugar yield. Furthermore, energy consumption is reduced due to segmented agitation, lowering overall costs.

[0023] 4. The residue is re-adsorbed by secondary re-adsorption to recover the adsorbed enzyme, which is combined with the β-glucosidase secreted by the recombinant yeast to form a circulation system of "exogenous enzyme + endogenous enzyme", with a single recovery efficiency of ≥85%.

[0024] 5. By utilizing the amphiphilicity of Tween-80, it preferentially competes for lignin adsorption sites in the first stage (non-ionic surfactants reduce ineffective adsorption). In the second stage, it supplements the solution through concentration gradient to resolve the contradiction between "insufficient mass transfer" and "surfactant inhibition" in high-solid systems. This enables the glucose yield to reach over 78% at a solid content of 30%, breaking through the application bottleneck of existing technologies (inhibition at high concentrations).

[0025] 6. Process compatibility: Applicable to a variety of lignocellulosic raw materials (corn cobs, corn straw, sugarcane bagasse, bamboo, bamboo shoots, mushroom grass, etc.), the second stage of the present invention mainly performs cellobiose hydrolysis, and has the condition to seamlessly connect with simultaneous saccharification and fermentation, further converting the glucose produced by hydrolysis into products (such as ethanol, citric acid, etc.), thereby relieving product inhibition and shortening the process flow.

[0026] In summary, the present invention proposes for the first time a staged surfactant addition process based on dynamic viscosity response, which solves the contradiction between high-concentration inhibition and insufficient mass transfer; constructs a synergistic system of recombinant yeast and complex enzyme system, reduces dependence on exogenous enzymes through endogenous β-glucosidase, and is a cross-domain innovation in biocatalysis and fermentation; integrates pretreatment-enzymatic hydrolysis-recovery full-process optimization, forming a systematic breakthrough in raw material adaptability, energy consumption control, and yield improvement, and has significant technological progress and industrial application value compared with the existing technology. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the embodiments.

[0028] A catalytic hydrolysis process for high-concentration cellulose comprises the following steps:

[0029] 1) Pretreatment stage: After the lignocellulose raw material is crushed, it is pretreated with dilute acid-ammonia coupling to control the solid mass of the pretreated material to account for 25%-40% of the total wet material. It is then washed with water to neutrality, dried, crushed and sieved;

[0030] 2) First enzymatic saccharification stage (high-speed liquefaction and initial surfactant adjustment): The raw materials pretreated in step 1) are placed in a ribbon stirred reactor with a solids content controlled at 25%-35%. Complex cellulase and a nonionic surfactant (Tween-80) are then added and heated and stirred to react. This reduces ineffective adsorption of the enzyme to lignin and increases the initial hydrolysis rate.

[0031] 3) Second enzymatic saccharification stage (low-speed saccharification and dynamic regulation): The material from the first enzymatic saccharification stage is transferred to a low-shear reactor, inoculated with recombinant yeast that secretes β-glucosidase to continue the reaction, and non-ionic surfactant is added according to the viscosity of the system;

[0032] 4) Product Separation and Enzyme Recovery: After hydrolysis, solid-liquid separation is performed, and the residue is treated with a secondary re-adsorption method to recover cellulase. Specifically, after hydrolysis, the sugar solution and the residue are separated by centrifugation or membrane filtration. Fresh substrate (unhydrolyzed and saccharified lignocellulosic material) is added to the residue at a solid-liquid ratio of 1:3 to 1:5. The mixture is allowed to stand at 20-30°C for 1-2 hours. After separation, the lignocellulosic material adsorbed with cellulase is recovered.

[0033] In step 1), the dilute acid-ammonia coupled pretreatment is specifically as follows: the raw material is mixed with a 1%-3% sulfuric acid solution at a solid-to-liquid ratio of 1:5-1:7 by volume, and treated at 120-130°C for 30-60 minutes. After washing, the raw material is mixed with a 10%-15% ammonia solution at a solid-to-liquid ratio of 1:5-1:7 by volume, and treated at 50-60°C for 12-24 hours. This disrupts the lignin-hemicellulose network and improves cellulose accessibility.

[0034] In the first enzymatic saccharification stage, an automatic rehydration device maintains system fluidity. The reaction is carried out at a temperature of 50-55°C and a pH of 4.8-5.2 for 3-6 hours. The reactor is stirred at a rate of 200-300 rpm to achieve rapid liquefaction under high shear conditions. After liquefaction, the yield stress of the system is less than 10 Pa·s. In step 2), the weight of the nonionic surfactant accounts for 0.05% to 0.1% of the total system.

[0035] In step 2), a ribbon-stirred reactor (diameter ≤ 50 cm) with a solids content of 25%–35% and an agitation rate of 200–300 rpm is used to create a high-shear environment, rapidly breaking up cellulose aggregates. The reactor has a pitch ratio (pitch / diameter) of 0.8–1.2, providing strong axial mixing and preventing high-solids material from settling. The ribbon width accounts for 15%–20% of the reactor diameter, enhancing shear efficiency. The ribbon and reactor inner wall roughness is ≤ 1.0 μm, minimizing material adhesion (high-solids slurries are prone to scaling, and a smooth surface makes cleaning easier).

[0036] In step 3), the volume of the low shear reactor is 10 times or more than that of the reactor in the first enzymatic saccharification stage, and the stirring rate is controlled to 50-100 rpm or intermittent stirring, and the intermittent stirring is stirring for 5-10 minutes every 30 minutes at a stirring rate of 50-100 rpm. This can reduce energy consumption and mechanical damage to the enzyme.

[0037] The low-shear reactor utilizes wide-bladed anchor-type impellers (blade width accounts for 15%-20% of the reactor diameter) or three-layer frame-type impellers. The distance between the blade edge and the vessel wall is ≤20 mm, ensuring close-to-wall mixing and preventing the deposition of high-solids materials on the wall. (Traditional ribbon impellers are prone to forming dead spots at low speeds, while anchor impellers are more suitable for laminar mixing of high-viscosity fluids.) A variable-pitch design features a larger pitch in the upper layer (pitch / diameter = 1.2-1.5) and a smaller pitch in the lower layer (1.0-1.2). This creates an axial circulation flow, improving the exchange of materials between the upper and lower layers (especially suitable for intermittent mixing). With a rated speed of 50-100 rpm and equipped with a reducer (reduction ratio of 1:50-1:100), the reactor provides stable output torque (fluctuation ≤±5%), preventing mechanical damage to the enzyme protein caused by startup shock. An integrated automated program supports a "5-minute stirring followed by a 25-minute rest" cycle. Real-time monitoring via a torque sensor automatically extends the stirring time when a sudden increase in torque (material stratification) is detected.

[0038] In step 3), the replenishment of the nonionic surfactant is dynamically controlled according to the viscosity of the system. When the online viscometer monitors a viscosity greater than 10 Pa·s, the nonionic surfactant is automatically replenished to a mass ratio of 0.1% to 0.2% of the total system. This maintains mass transfer efficiency and avoids high concentration inhibition.

[0039] In step 3), the continued reaction is carried out at 37-50° C. for 48-72 hours, and the inoculation amount of the recombinant yeast secreting β-glucosidase is 5%-10% by volume.

[0040] The mass ratio of exo-enzyme, endo-enzyme and beta-glucosidase in the composite cellulase is (1-6):(1-4):1, and the total enzyme amount is 1-15 FPU / g cellulose.

[0041] The lignocellulose raw material comprises one or more of corn cobs, corn stalks, sugarcane bagasse, bamboo, bamboo shoot shells, and mushroom grass, and the particle size after pulverization and screening is 20-80 mesh.

[0042] In the present invention, recombinant cerevisiae CEN.PK2-1C is used, and the β-glucosidase secreted by it is utilized to reduce the addition of exogenous enzymes, consume cellobiose in time, relieve product inhibition, and replace part of the exogenous β-glucosidase addition.

[0043] Example 1: High-concentration enzymatic hydrolysis of corncobs

[0044] Pretreatment: corn cobs were crushed to pass 40 mesh, and 2% sulfuric acid solution was added at a mass volume solid-liquid ratio of 1:6. The mixture was treated at 121°C for 60 min. After washing with water, 15% ammonia water was added and mixed at a mass volume solid-liquid ratio of 1:5. The mixture was treated at 60°C for 12 h. The solid mass after pretreatment was controlled to account for 25% of the total wet material. The material was dried, crushed, and sieved (20-80 mesh) to obtain the pretreated material (cellulose content 70.8%).

[0045] The first stage: pre-treated corn cobs (solid content 30%) were added to a 5L ribbon stirred reactor, and a complex enzyme (exo-enzyme: endo-enzyme: β-glucosidase = 5:3:2) (12 FPU / g cellulose) + 0.08% Tween-80 was added. The reaction was carried out at 250 rpm and 50 °C for 6 h. The viscosity increased from 20 Down to 8 , glucose concentration reached 65g / L.

[0046] The second stage was transferred to a 50-L low-shear reactor with a stirring rate of 80 rpm. Tween-80 was added to a concentration of 0.1% in the system. Recombinant yeast (10% inoculation) was inoculated and saccharified at 37°C for 48 hours. The final glucose concentration was 115 g / L and the yield was 78.5%.

[0047] After hydrolysis, the sugar solution and residue are separated by centrifugation or membrane filtration. The enzyme adsorbed in the residue is recovered through a secondary re-adsorption process (addition of fresh substrate, solid-to-liquid ratio of 1:3, and incubation at 20°C for 2 hours). Combined with β-glucosidase secreted by recombinant yeast, this allows for enzyme recycling, achieving a single-step recovery efficiency of 86%.

[0048] Example 2: Efficient hydrolysis of corn straw powder

[0049] Pretreatment: corn straw powder was added with 2% sulfuric acid solution at a solid-liquid ratio of 1:6, treated at 121°C for 60 min, washed with water, mixed with 15% ammonia water at a solid-liquid ratio of 1:7, and treated at 60°C for 12 h. The solid mass after pretreatment was controlled to be 35%. , dried, crushed and sieved (20-80 mesh) to obtain pretreated material (cellulose content 68%).

[0050] The first stage: pre-treated corn straw powder (solid content 30%) was added to a 5L ribbon reactor, and compound enzyme (exo-enzyme: endo-enzyme: β-glucosidase = 5:3:2) (12FPU / g cellulose) + 0.1% Tween-80 was added. The reaction was carried out at 250 rpm and 50℃ for 5 hours. The viscosity increased from 20 Down to 8 .

[0051] The second stage: transfer to a 50L low shear reactor, stir at 80 rpm, add Tween-80 to make the concentration of Tween-80 in the system 0.15% , inoculated with recombinant yeast (10% inoculation amount), saccharified at 37°C for 72 h, and the final glucose concentration was 120 g / L.

[0052] After hydrolysis, the sugar solution and residue are separated by centrifugation or membrane filtration. The enzyme adsorbed in the residue is recovered through a secondary re-adsorption process (addition of fresh substrate, solid-to-liquid ratio of 1:3, and incubation at 20°C for 2 hours). Combined with β-glucosidase secreted by recombinant yeast, this allows for enzyme recycling, achieving a single-step recovery efficiency of 86%.

[0053] Comparative Example: Traditional Two-Stage Method (Without Dynamic Control) Compared with Example 2, in this comparative example, dynamic addition of surfactant was not implemented, and other conditions were the same. The glucose yield was 65% and the energy consumption was 25% higher.

[0054] In summary, the present invention significantly improves the hydrolysis efficiency of high-concentration cellulose through staged dynamic regulation, recombinant yeast synergy and pretreatment optimization, and has significant industrial application value.

Claims

1. A catalytic hydrolysis process for high-concentration cellulose, characterized in that: The following steps are involved: 1) Pretreatment stage: After the lignocellulose raw material is crushed, it is pretreated with dilute acid-ammonia coupling to control the solid mass of the pretreated material to account for 25%-40% of the total wet material. It is then washed with water to neutrality, dried, crushed and sieved; 2) First enzymatic saccharification stage: The raw materials pretreated in step 1) are placed in a ribbon stirred reactor with a solid content controlled at 25% to 35%, and then complex cellulase and nonionic surfactant are added and heated and stirred for reaction; 3) Second enzymatic saccharification stage: The material from the first enzymatic saccharification stage is transferred to a low-shear reactor, inoculated with recombinant yeast that secretes β-glucosidase to continue the reaction, and non-ionic surfactant is added according to the viscosity of the system.

2. The catalytic hydrolysis process for high-concentration cellulose according to claim 1, characterized in that: It also includes product separation and enzyme recovery stages: solid-liquid separation is performed after the hydrolysis is completed, and the cellulase is recovered from the residue using a secondary re-adsorption method.

3. The catalytic hydrolysis process for high-concentration cellulose according to claim 1, wherein: In step 1), the dilute acid-ammonia coupling pretreatment is specifically as follows: the raw material is mixed with a sulfuric acid solution having a concentration of 1% to 3% at a mass volume solid-liquid ratio of 1:5 to 1:7, and treated at 120 to 130° C. for 30 to 60 minutes. After washing with water, the raw material is mixed with ammonia water having a concentration of 10% to 15% at a mass volume solid-liquid ratio of 1:5 to 1:7, and treated at 50 to 60° C. for 12 to 24 hours.

4. The catalytic hydrolysis process for high-concentration cellulose according to claim 1, wherein: In the first enzymatic saccharification stage, the reaction is carried out for 3 to 6 hours at a reaction temperature of 50 to 55°C and a pH of 4.8 to 5.

2. The stirring rate of the reactor is 200 to 300 rpm, so that the system is rapidly liquefied under a high shear environment. After liquefaction, the yield stress of the system is less than 10 Pa·s.

5. The catalytic hydrolysis process for high-concentration cellulose according to claim 1, wherein: The nonionic surfactant is Tween-80; in step 2), the mass proportion of the nonionic surfactant is 0.05% to 0.1% of the total system.

6. The catalytic hydrolysis process for high-concentration cellulose according to claim 1, wherein: In step 3), the amount of the nonionic surfactant added is dynamically controlled according to the viscosity of the system. When the online viscometer detects a viscosity greater than 10 Pa·s, the nonionic surfactant is added to make the mass proportion of the nonionic surfactant in the total system 0.1% to 0.2%.

7. The catalytic hydrolysis process for high-concentration cellulose according to claim 1, wherein: In step 3), the volume of the low shear reactor is 10 times or more than that of the reactor in the first enzymatic saccharification stage, and the stirring rate is controlled to be 50-100 rpm or intermittent stirring, and the intermittent stirring is stirring for 5-10 minutes every 30 minutes, and the stirring rate is 50-100 rpm.

8. The process for catalytic hydrolysis of high-concentration cellulose according to claim 1, wherein: In step 3), the continued reaction is carried out at 37-50° C. for 48-72 hours, and the inoculation amount of the recombinant yeast secreting β-glucosidase is 5%-10% by volume.

9. The catalytic hydrolysis process for high-concentration cellulose according to claim 1, wherein: The mass ratio of exo-enzyme, endo-enzyme and beta-glucosidase in the composite cellulase is (1-6):(1-4):1, and the total enzyme amount is 1-15 FPU / g cellulose.

10. The catalytic hydrolysis process for high-concentration cellulose according to claim 1, characterized in that: The lignocellulose raw material comprises one or more of corn cobs, corn stalks, sugarcane bagasse, bamboo, bamboo shoot shells, and mushroom grass, and the particle size after pulverization and screening is 20-80 mesh.