A method for promoting hydrolysis of lignocellulose to fermentable sugars

Abstract

This invention discloses a method for promoting the hydrolysis of lignocellulose into fermentable sugars, belonging to the field of biorefining technology. The method includes the following steps: (1) adding ammonium sulfate to the lignocellulose raw material, then mixing it with a glycerol-water mixture, and pretreating it at a certain temperature and time to destroy the dense structure of the lignocellulose; (2) adding ammonium sulfate and cellulase to the pretreated system, and carrying out a saccharification reaction under suitable conditions to convert it into fermentable sugars. This invention solves the problems of high energy consumption, high pollution, and low saccharification and enzymatic hydrolysis efficiency of traditional pretreatment by leveraging the solubilizing, crystallinity-reducing, and biocatalyst-protecting effects of glycerol and the synergistic effect of ammonium sulfate. Compared with traditional hot water pretreatment, the yield of fermentable sugars is significantly increased, and the pretreatment process does not require strong acids or bases; glycerol can be recycled and reused, significantly reducing production costs and possessing significant industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biorefining technology, specifically relating to a method for promoting the hydrolysis of lignocellulose into fermentable sugars. Background Technology

[0002] Lignocellulose is the most abundant renewable biomass resource on Earth, and its main components include cellulose, hemicellulose, and lignin. Due to the dense crystalline structure of lignocellulose and the protective effect of lignin, its direct saccharification efficiency is extremely low. Therefore, pretreatment is required to disrupt its structure in order to improve the efficiency of subsequent saccharification reactions.

[0003] Existing lignocellulose pretreatment technologies mainly include physical, chemical, biological, and combined methods. Physical methods are energy-intensive and can only partially destroy the raw material structure; chemical methods, while effectively removing lignin, produce inhibitory byproducts, polluting the environment and increasing subsequent processing costs; biological methods have long reaction cycles and low efficiency. Furthermore, during the saccharification process after traditional pretreatment, cellulase is easily affected by substrate crystallinity, lignin residue, and product inhibition, resulting in low enzymatic hydrolysis efficiency and high enzyme consumption, further increasing production costs.

[0004] In existing technologies, glycerol is used only in small quantities in the liquefaction reaction of biomass, or as an auxiliary agent for enzymatic reactions. This invention proposes a method for the synergistic pretreatment of lignocellulose with the pyrolysis catalyst ammonium sulfate, aiming to promote the saccharification of lignocellulose. Summary of the Invention

[0005] To address the problems of high energy consumption, significant pollution, low saccharification and enzymatic hydrolysis efficiency, and high cost in existing lignocellulose pretreatment methods, this invention provides a method for promoting the hydrolysis of lignocellulose into fermentable sugars. Through the synergistic effect of glycerol and ammonium sulfate in the pretreatment and saccharification processes, the efficient conversion of lignocellulose is achieved.

[0006] The technical solution of this invention is as follows: A glycerol-water mixture was used to pretreat lignocellulose. Glycerol's solubilizing effect disrupted the binding of lignin and carbohydrates, reducing cellulose crystallinity. Simultaneously, glycerol protected cellulase activity during saccharification, reducing product inhibition and significantly improving saccharification efficiency. Furthermore, ammonium sulfate enhanced the lignin removal and hemicellulose degradation effects of glycerol, allowing for lower pretreatment temperature / time and improved degradation efficiency. The specific steps are as follows: (1) Pulverize the lignocellulose raw material to 40-80 mesh, dry it to a moisture content ≤10%, add 0-2.0% (w / w) ammonium sulfate as an auxiliary agent, and mix the raw material with a 30-50% (v / v) glycerol-water mixture at a solid-liquid ratio of 1:5-1:15 (g / mL). Pretreat the mixture for 60-120 min at 100-140℃ and 0.1-0.3 MPa. After pretreatment, cool to room temperature, filter to obtain the pretreated product, and recover the glycerol by vacuum filtration for recycling.

[0007] (2) Mix the pretreated product with the buffer solution at a solid-liquid ratio of 1:10-1:20 (g / mL), adjust the pH to 4.0-6.0, add 0-2.0% (w / w) ammonium sulfate as an auxiliary agent and 2-10 FPU / g raw material of compound cellulase, add 0.05-0.2 g / L Tween 80, and saccharify for 24-72 h at 30-55℃ and 100-200 rpm. Take samples to detect the concentration of fermentable sugars.

[0008] The cellulase is a complex enzyme system of cellulase, hemicellulase and β-glucosidase, with a total enzyme addition of 2-10 FPU / g raw material.

[0009] The ammonium sulfate concentration is preferably 1.0-2.0% (w / w); more preferably, the ammonium sulfate concentration is 1.5% (w / w).

[0010] In step 1), the ammonium sulfate concentration is preferably 1.0-2.0% (w / w), and more preferably, the ammonium sulfate concentration is 2.0% (w / w).

[0011] In step 2), the ammonium sulfate concentration is preferably 1.0-2.0% (w / w), and more preferably, the ammonium sulfate concentration is 1.5% (w / w).

[0012] More preferably, the ammonium sulfate concentration in step 1) is 2.0% (w / w), and the ammonium sulfate concentration in step 2) is 1.5% (w / w).

[0013] The ammonium sulfate in the pretreatment and saccharification stages have different targets (pretreatment destroys the lignocellulose structure, while saccharification enhances enzyme activity and stability). By controlling the concentration in stages, the synergistic effect of maximizing the structural destruction and maximizing enzyme activation can be achieved, avoiding the conflict between the effects of a single concentration in the two stages.

[0014] Preferably, the amount of the compound enzyme system added is 4 FPU / g raw material.

[0015] A glycerol concentration of 30-50% creates a suitable solvent environment that effectively breaks down the dense structure of lignocellulose without causing excessive dehydration of the raw material or inhibition of enzyme activity due to excessive concentration. A glycerol concentration of 40% is preferred.

[0016] The combination of temperature (100-140℃) and pretreatment time (60-120 min) can achieve partial removal of lignin and reduction of cellulose crystallinity with low energy consumption, while avoiding excessive degradation and the generation of inhibitory byproducts.

[0017] Preferably, the pretreatment temperature is 120-140℃ and the pretreatment time is 90-120 min. More preferably, the pretreatment temperature is 140℃ and the pretreatment time is 90 min.

[0018] The optimal temperature range for saccharifying enzymes is 30-55℃. Adding Tween 80 can further improve the binding efficiency between the enzyme and the substrate. Residual glycerol can protect enzyme activity and reduce feedback inhibition of β-glucosidase by glucose.

[0019] Preferably, the adjuvant is added during both the pretreatment and degradation of lignocellulose. Wheat straw is pulverized to 40-80 mesh, dried to a moisture content ≤10%, and 2.0% (w / w) ammonium sulfate is added as an adjuvant. The raw material is mixed with a 40% (v / v) glycerol-water mixture at a solid-liquid ratio of 1:5-1:15 (g / mL), and pretreated at 140℃ and 0.1-0.3 MPa for 90 min. After pretreatment, the mixture is cooled to room temperature, filtered to obtain the pretreated product, and the glycerol is recovered and recycled by vacuum filtration.

[0020] (2) Mix the pretreated product with the buffer solution at a solid-liquid ratio of 1:10-1:20 (g / mL), adjust the pH to 4.0-6.0, add 1.5% (w / w) ammonium sulfate as an auxiliary agent and 2-10 FPU / g raw material of compound cellulase, add 0.05-0.2 g / L Tween 80, and saccharify for 48 h at 30-55℃ and 100-200 rpm. Take a sample to detect the concentration of fermentable sugars.

[0021] Preferably, the amount of Tween 80 added during the saccharification process is 0.1 g / L.

[0022] The total concentration of glucose and xylose in the fermentable sugar solution is ≥25 g / L, preferably ≥30 g / L, and more preferably ≥40 g / L.

[0023] Glycerol, as a green, non-toxic, and renewable polyol solvent, possesses excellent solubilizing and hydrogen bonding capabilities. Ammonium sulfate can enhance the effect of glycerol on lignin removal and hemicellulose degradation, reducing pretreatment temperature / time; simultaneously, it can regulate the ionic strength of the system, stabilize the cellulase conformation, and enhance enzyme catalytic activity. The synergistic effect of these two substances can further reduce substrate mass transfer resistance and mitigate enzyme inactivation and ineffective binding, thereby significantly improving the enzymatic hydrolysis efficiency of lignocellulose and the yield of reducing sugars.

[0024] Beneficial effects:

[0025] Compared with the prior art, the technical advantages of the present invention are as follows: Synergistic enhancement of pretreatment and saccharification: Glycerol disrupts the structure of lignocellulose during the pretreatment stage and protects enzyme activity during the saccharification stage, thus solving the problem of disconnect between pretreatment and saccharification in traditional processes.

[0026] Synergistic degradation: Glycerol and ammonium sulfate work synergistically to reduce mass transfer resistance, mitigate enzyme inactivation and ineffective adsorption, and significantly improve the enzymatic hydrolysis efficiency of lignocellulose and the yield of reducing sugars.

[0027] Green and environmentally friendly: The pretreatment process does not require strong acids or alkalis, but only uses a glycerol-water mixture system. Furthermore, the glycerol can be recycled and reused, resulting in no secondary pollution and reducing environmental protection costs.

[0028] High conversion efficiency: The enzymatic hydrolysis efficiency of lignocellulose is improved after pretreatment, which is significantly better than traditional pretreatment technology.

[0029] Low cost: Glycerin is widely available, and its recycling and reuse further reduce raw material costs. Attached Figure Description

[0030] Figure 1 The effect of different adjuvants on the saccharification yield of lignocellulose; Figure 2 The effect of different ammonium sulfate addition methods on the saccharification yield of lignocellulose. Detailed Implementation

[0031] Example 1

[0032] Wheat straw was pulverized to 60 mesh and dried to a moisture content of 8%. 10 g of wheat straw was mixed with a glycerol-water mixture at a solid-liquid ratio of 1:10 (g / mL), resulting in a glycerol volume concentration of 30%. Then, ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium nitrate, and sodium sulfate were added at 1% (w / w, ammonium sulfate mass / wheat straw mass) respectively. The mixture was pretreated at 120℃ and 0.2 MPa for 60 min. After cooling, the mixture was filtered, and the glycerol was recovered by vacuum filtration.

[0033] 3 g of pretreated wheat straw was mixed with citrate-sodium citrate buffer (pH 4.5) at a solid-liquid ratio of 1:15 (g / mL), and 4 FPU / g of raw material composite cellulase (Cellic® CTec3 HS purchased from Novozymes) and 0.1 g / L Tween 80 were added. The mixture was hydrolyzed at 50℃ and 180 rpm for 48 h, and the concentrations of glucose and xylose were measured.

[0034] The results showed that the addition of different adjuvants was effective in pretreating lignocellulose, increasing the fermentable sugar content after cellulase hydrolysis. Among them, the addition of ammonium sulfate had the most significant effect, with a fermentable sugar concentration of 30.5 g / L, which was 19.14% higher than the control group without adjuvants.

[0035] Example 2: Effect of the method of adding adjuvants on saccharification effect The adjuvant is added only during the lignocellulose pretreatment process, as follows: Wheat straw was pulverized to 60 mesh and dried to a moisture content of 8%. 10 g of wheat straw was mixed with a glycerol-water mixture at a solid-liquid ratio of 1:10 (g / mL), resulting in a glycerol volume concentration of 30%. 1% (w / w) ammonium sulfate was added, and the mixture was pretreated at 120℃ and 0.2 MPa for 60 min. After cooling, the mixture was filtered, and the glycerol was recovered by vacuum filtration.

[0036] 3 g of pretreated wheat straw was mixed with citrate-sodium citrate buffer (pH 4.5) at a solid-liquid ratio of 1:15 (g / mL), and 4 FPU / g of raw material composite cellulase and 0.1 g / L Tween-80 were added. The mixture was hydrolyzed at 50℃ and 180 rpm for 48 h, and the concentrations of glucose and xylose were measured.

[0037] The adjuvant is added only during the degradation of lignocellulose, and the specific process is as follows: Wheat straw was pulverized to 60 mesh and dried to a moisture content of 8%. 10 g of wheat straw was mixed with a glycerol-water mixture at a solid-liquid ratio of 1:10 (g / mL), resulting in a glycerol volume concentration of 30%. The mixture was pretreated at 120℃ and 0.2 MPa for 60 min. After cooling, the mixture was filtered, and the glycerol was recovered by vacuum filtration.

[0038] 3 g of pretreated wheat straw was mixed with citrate-sodium citrate buffer (pH 4.5) at a solid-liquid ratio of 1:15 (g / mL), 1% (w / w) ammonium sulfate was added, along with 4 FPU / g of raw material composite cellulase and 0.1 g / L Tween-80. The mixture was hydrolyzed at 50℃ and 180 rpm for 48 h, and the concentrations of glucose and xylose were measured.

[0039] The adjuvant is added during both the pretreatment and degradation of lignocellulose, and the specific process is as follows: Wheat straw was pulverized to 60 mesh and dried to a moisture content of 8%. 10 g of wheat straw was mixed with a glycerol-water mixture at a solid-liquid ratio of 1:10 (g / mL), resulting in a glycerol volume concentration of 30%. 1% (w / w) ammonium sulfate was added, and the mixture was pretreated at 120℃ and 0.2 MPa for 60 min. After cooling, the mixture was filtered, and the glycerol was recovered by vacuum filtration.

[0040] 3 g of pretreated wheat straw was mixed with citrate-sodium citrate buffer (pH 4.5) at a solid-liquid ratio of 1:15 (g / mL), 1% (w / w) ammonium sulfate was added, along with 4 FPU / g of raw material compound cellulase and 0.1 g / L Tween 80. The mixture was hydrolyzed at 50℃ and 180 rpm for 48 h, and the concentrations of glucose and xylose were measured.

[0041] The results showed that when ammonium sulfate was added during both the pretreatment and degradation of lignocellulose, the synergistic effect of glycerol and ammonium sulfate promoted the removal of lignin and maintained the activity of cellulase, thus improving the saccharification effect. The final total concentration of fermentable sugars obtained was 31.6 g / L.

[0042] Example 3: Effect of different concentrations of ammonium sulfate as an auxiliary agent on saccharification efficiency The method was the same as in Example 2, except that the adjuvant was added during both the pretreatment and degradation of lignocellulose. The difference was that the ammonium sulfate concentrations were 0, 0.5, 1.0, 1.5, and 2.0% (w / w), respectively, and the pretreatment was carried out at 120°C and 0.2 MPa for 60 min. After cooling, the mixture was filtered, and glycerol was recovered by vacuum filtration.

[0043] The results showed that when the ammonium sulfate concentration was 1.5% (w / w), the total concentration of fermentable sugars after saccharification was 32.5 g / L, which was the highest concentration of fermentable sugars obtained compared with other single concentration groups. The experimental results are shown in Table 1.

[0044] Then, a staged strategy was adopted to control the concentration of ammonium sulfate: 1.5% (w / w) was maintained during the saccharification stage, and the concentrations during the pretreatment stage were 0.5%, 1.0%, and 2.0% (w / w); the concentration was maintained at 1.5% (w / w) during the pretreatment stage, and 0.5%, 1.0%, and 2.0% (w / w) during the saccharification stage. Pretreatment was carried out at 120℃ and 0.2 MPa for 60 min. After cooling, the mixture was filtered, and glycerol was recovered by vacuum filtration.

[0045] The results showed that when the ammonium sulfate concentration in the pretreatment stage was 2% (w / w) and the concentration in the saccharification stage was 1.5% (w / w), the yield of fermentable sugars was the highest at 34.2 g / L, which was significantly higher than that of pretreatment without ammonium sulfate or saccharification with a single concentration (Table 1). The phased addition of ammonium sulfate allows for precise control of the lignocellulose saccharification process. The pretreatment stage focuses on the structural disruption of lignocellulose, while the saccharification stage focuses on enzyme stability. Phased concentration addition avoids the problems of "over-catalysis in the pretreatment stage and insufficient enzyme activity in the saccharification stage" or "insufficient catalysis in the pretreatment stage and excessive addition in the saccharification stage leading to salting out."

[0046] Example 4: Effect of different glycerol concentrations on glycation efficiency The method was the same as in Example 3, except that the ammonium sulfate concentration was 1.5% (w / w), and the glycerol concentrations were 30%, 40%, and 50%, respectively, with other conditions remaining unchanged.

[0047] The results showed that when the glycerol concentration was 40%, the total concentration of fermentable sugars after saccharification was 37.2 g / L. When the glycerol concentration was below 40%, the effect of destroying the lignocellulose structure was insufficient; when it was above 40%, the residual glycerol was too high, which led to a slight inhibition of enzyme activity and a decrease in the yield of fermentable sugars. The experimental results are shown in Table 1.

[0048] Example 5: Effect of pretreatment temperature on saccharification efficiency The method is the same as in Example 4, except that the pretreatment temperature is set to 100℃, 120℃, and 140℃, and other conditions remain unchanged.

[0049] The results showed that the highest yield of fermentable sugars (38.2 g / L) was achieved at a pretreatment temperature of 140℃. Below 140℃, the lignocellulose structure was not sufficiently disrupted; above 140℃, a small amount of inhibitors (such as furfural) were produced, inhibiting the hydrolytic activity of cellulase. The experimental results are shown in Table 1.

[0050] Example 6: Effect of pretreatment time on saccharification effect The method is the same as in Example 5, except that the pretreatment time is set to 60 min, 90 min, and 120 min, and other conditions remain unchanged.

[0051] The results showed that the highest yield of fermentable sugars, reaching 41.8 g / L, was achieved with a pretreatment time of 120 min. Although extending the pretreatment time could increase the yield of fermentable sugars, extending it from 90 min to 120 min resulted in almost no increase in yield, while energy consumption increased significantly. Therefore, a pretreatment time of 90 min was selected. The experimental results are shown in Table 1.

[0052] Example 7 Effect of cellulase hydrolysis time on saccharification efficiency The method is the same as in Example 6, except that the cellulase hydrolysis time is set to 24 h, 48 h, and 72 h, while other conditions remain unchanged.

[0053] The results showed that the highest fermentable sugar yield (42.8 g / L) was achieved when cellulase hydrolysis time was 72 h. After 48 h of cellulase hydrolysis, the fermentable sugar yield had already reached 41.4 g / L, and extending the hydrolysis time by 24 h did not significantly increase the yield. Therefore, high concentrations of fermentable sugars may have inhibited cellulase activity. The experimental results are shown in Table 1.

[0054] Table 1 Effects of different conditions on lignocellulose saccharification

Claims

1. A method for promoting the hydrolysis of lignocellulose into fermentable sugars, characterized in that, Includes the following steps: (1) Crush the lignocellulose raw material and mix it with the glycerol-water mixture at a solid-liquid ratio of 1:5-1:15 (g / mL). Pre-treat the mixture at 100-140℃ for 60-120 min to obtain the pre-treated product. (2) Cool the pretreated product to room temperature, adjust the pH of the system to 4.0-6.0, add cellulase, and saccharify at 30-55℃ and 100-200 rpm for 24-72 h to obtain a fermentable sugar solution.

2. The method according to claim 1, characterized in that, In step (1), the volume concentration of glycerol in the glycerol-water mixture is 30-50%.

3. The method according to claim 1, characterized in that, During the pretreatment process in step (1), the system pressure is 0.1-0.3 MPa.

4. The method according to claim 1, characterized in that, Ammonium sulfate is added as an auxiliary agent in steps (1) and (2), and the amount added is 0-2% (w / w) of the mass of the lignocellulose raw material.

5. The method according to claim 1, characterized in that, The cellulase mentioned in step (2) is a complex enzyme system of cellulase, hemicellulase and β-glucosidase, with a total enzyme addition of 2-10 FPU / g raw material.

6. The method according to claim 1, characterized in that, In step (2), 0.05-0.2 g / L of Tween 80 is added as an enzyme-catalyzing agent during the saccharification process.

7. The method according to claim 4, characterized in that: Wheat straw was mixed with a glycerol-water mixture with a glycerol volume concentration of 40%, and ammonium sulfate with a concentration of 1-2% (w / w) was added. The mixture was pretreated at 140℃ for 90 min to obtain the pretreated product.

8. The method according to claim 4, characterized in that: Ammonium sulfate is added as an auxiliary agent in steps (1) and (2). The concentration of ammonium sulfate in step (1) is 2% (w / w) and the concentration of ammonium sulfate in step (2) is 1.5% (w / w).

9. The method according to claim 1, characterized in that, The total concentration of glucose and xylose in the fermentable sugar solution is ≥25 g / L.

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