A method for in-situ enzymatic hydrolysis and saccharification of straw

Abstract

This invention discloses an in-situ enzymatic hydrolysis and saccharification method for corn straw, belonging to the field of biochemical technology. The method consists of the following steps: pretreatment, activation and expansion culture, preparation of auxiliary agents, preparation of crude enzyme solution, and enzymatic hydrolysis and saccharification. The enzymatic hydrolysis and saccharification involves mixing pretreated corn straw powder, corn steep liquor powder, potassium hydroxide, ammonium sulfate, magnesium sulfate, a first portion of water, and the crude enzyme solution, stirring at 27-29℃ and 180-220 rpm for 30-40 minutes, adjusting the pH to 4.7-4.9, sterilizing, and obtaining a fermentation medium. The auxiliary agents, seed liquid, and a second portion of water are mixed, stirred at 27-29℃ and 180-220 rpm, added to the fermentation medium, and cultured with shaking at 27-29℃ and 140-160 rpm for 6-7 days. This invention can achieve high-solids enzymatic hydrolysis with a high specific sugar yield, and the specific sugar yield results show little difference when treating different batches of corn straw.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, specifically to a method for in-situ enzymatic hydrolysis of saccharified straw. Background Technology

[0002] The main component of straw is lignocellulose, a natural biomass material primarily composed of cellulose, hemicellulose, and lignin. Due to its highly lignified and crystalline structure, lignocellulose contains numerous hydrogen bonds between and within molecules, making it generally difficult to effectively degrade and utilize. Biorefining refers to the continuous processing of biomass materials into a series of products and energy sources, thereby avoiding waste. Biorefining based on a biochemical platform mainly includes pretreatment, hydrolysis, and fermentation. Biorefining can improve the economic efficiency of the entire biomass material conversion process; therefore, biorefining technology has enormous potential in degrading lignocellulose-based biomass materials.

[0003] When degrading lignocellulosic biomass materials using biorefining technology, the most common method involves first breaking the bonds between lignin, cellulose, and hemicellulose in the straw through pretreatment, then enzymatically hydrolyzing and saccharifying the cellulose with hydrolytic enzymes, followed by microbial fermentation for value enhancement. For enzymatic saccharification, the commonly used method is direct enzymatic saccharification using cellulase; however, cellulase is expensive and requires large quantities, leading to high costs. In-situ enzymatic saccharification refers to fermentation enzyme production and enzymatic saccharification occurring in the same location or container. Studies have found that liquid in-situ enzymatic saccharification can reduce the pretreatment requirements for lignocellulosic saccharification, improve saccharification efficiency, and reduce costs, representing a new approach for the efficient utilization of lignocellulosic biomass materials.

[0004] However, existing in-situ enzymatic hydrolysis saccharification methods have the following problems:

[0005] Question 1: Wen Xiaoxia. Study on protein production from rice straw by in-situ enzymatic hydrolysis and saccharification fermentation. Sichuan University of Science and Engineering. May 2022. It was published that certain inhibitory factors exist in the hydrolysate obtained from pretreatment and enzymatic hydrolysis of straw, which have toxic effects on microorganisms and inhibit their growth.

[0006] Question 2: Wen Xiaoxia, Bai Guangjian, Li Tao, Ma Yifan, Zou Wei. Optimization of in-situ enzymatic hydrolysis and saccharification process of rice straw by liquid fermentation. Food and Fermentation Industries. Published in October 2020. The paper revealed that reducing sugar levels were very low throughout the fermentation process, barely sufficient for microbial growth. Cellulase is an enzyme regulated by multiple mechanisms. During natural growth and reproduction, reducing sugars in the environment can regulate the environment through feedback, inhibiting cellulase synthesis and activity, thereby controlling the concentration of reducing sugars. Furthermore, although enzyme activity increased with fermentation time, the reducing sugars in the fermentation broth did not increase, nor did the reducing sugars after enzymatic hydrolysis increase over time; instead, they decreased, leading to a decrease in specific sugar production.

[0007] Question 3: Wen Xiaoxia, Bai Guangjian, Li Tao, Ma Yifan, Zou Wei. Optimization of in-situ enzymatic hydrolysis and saccharification process of rice straw by liquid fermentation. Food and Fermentation Industries. October 2020. It was published that the pH value of the fermentation broth decreased with fermentation time, resulting in increasingly lower enzyme activity.

[0008] Question 4: Wen Xiaoxia. Study on protein production from rice straw by in-situ enzymatic hydrolysis and saccharification fermentation. Sichuan University of Science and Engineering. May 2022. The study shows that the higher the straw loading, the greater the reducing sugar content per 100 mL of saccharification liquid. For every 5 g / L loading, the sugar content increases by approximately 1 g / L. The specific sugar yield (g / g) remains constant at straw loadings of 30-40 g / L. This is because the pH of the fermentation liquid needs to be adjusted before enzymatic hydrolysis. The higher the straw loading, the thicker the fermentation liquid becomes, which is not conducive to pH adjustment. Although the reducing sugar content in the fermentation liquid increases with the increase of straw amount, the specific sugar yield (g / g) remains unchanged. The straw is not degraded into more sugar. Therefore, the amount of straw added per liter of fermentation liquid is generally controlled at 30 g / L. The low straw loading limits the accumulation of sugar content and is not conducive to large-scale utilization of straw.

[0009] The existing solutions to the above problems are as follows:

[0010] Solution 1 (for problem 1): Wen Xiaoxia. Research on protein production from rice straw by in-situ enzymatic hydrolysis and saccharification fermentation. Sichuan University of Science and Engineering. In May 2022, it was published that the pretreatment was followed by washing with water for detoxification.

[0011] Solution 2 (for Problem 2): Wen Xiaoxia, Bai Guangjian, Li Tao, Ma Yifan, Zou Wei. Optimization of in-situ enzymatic hydrolysis and saccharification process of rice straw by liquid fermentation. Food and Fermentation Industries. October 2020. This paper discloses adding crude enzyme solution during fermentation to increase cellulase in the fermentation broth, thereby increasing the specific sugar yield.

[0012] Solution 3 (for Problem 3): Wen Xiaoxia, Bai Guangjian, Li Tao, Ma Yifan, Zou Wei. Optimization of in-situ enzymatic hydrolysis and saccharification process of rice straw by liquid fermentation. Food and Fermentation Industries. October 2020. This paper discloses that adjusting the pH value during fermentation can effectively increase the accumulation of reducing sugars in the fermentation broth.

[0013] Solution 4 (addressing problem 4): Xiang Chun. Research on graded depolymerization of poplar wood and directional creation of a compound bacterial enzyme synergistic enzymatic hydrolysis system. Central South University of Forestry and Technology. May 2025. It was disclosed that batch feeding achieves high solids enzymatic hydrolysis to reduce substrate inhibition and control the viscosity of the reaction system. At the same time, it was also disclosed that surfactants were added during batch feeding to improve the enzymatic hydrolysis rate.

[0014] Following the above-mentioned solution, the applicant first pre-treated the straw and washed it with water. Then, they used *Trichoderma reesei* strains to perform in-situ enzymatic hydrolysis and saccharification of the corn straw. In the initial stage of in-situ enzymatic hydrolysis and saccharification, the straw addition was controlled at 30 g / L. During fermentation, crude enzyme solution and batch feeding were added, with surfactants added during batch feeding. The pH value was also adjusted during fermentation to improve the enzymatic hydrolysis rate and thus increase the specific sugar yield. However, the applicant found that the specific sugar yield remained low, and there were significant differences in the specific sugar yield results when treating different batches of corn straw. Analysis suggested that the reason might be as described in Chen Hongkun's "Study on Fermentation Conditions for Cellulase Production by *Trichoderma reesei*", Shandong University, April 2020: adding microcrystalline cellulose reduced the activity of filter paper enzymes. The glucose produced after cellulose degradation altered the "carbon source starvation" state of the bacteria, inducing the bacteria to transition from the enzyme production stage to the growth stage, resulting in a relative decrease in enzyme activity, massive bacterial proliferation, and a thickened fermentation broth, which in turn led to difficulties in mass transfer and affected enzyme production. The document also disclosed that pure cellulose is generally considered the most effective inducer for Trichoderma reesei. Although corn stalks underwent pretreatment such as steam explosion and water washing, the enzyme activity of the filter paper was 39.6% lower than that of the microcrystalline cellulose treatment group. Therefore, the impurity content after pretreatment varied for different batches of corn stalks, resulting in large differences in the specific sugar yield of different batches of corn stalks. Summary of the Invention

[0015] To address the shortcomings of existing technologies, this invention provides a method for in-situ enzymatic hydrolysis and saccharification of straw, which can achieve high-solids enzymatic hydrolysis, high specific sugar yield, and small differences in specific sugar yield when processing different batches of corn straw.

[0016] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0017] A method for in-situ enzymatic hydrolysis and saccharification of straw comprises the following steps: pretreatment, activation and expansion culture, preparation of adjuvants, preparation of crude enzyme solution, and enzymatic hydrolysis and saccharification.

[0018] The pretreatment involves adding corn stalk powder and sodium hydroxide aqueous solution into a container, covering the mouth of the container, keeping it at 55-60℃ for 30-35 hours, filtering, taking the filter residue, washing it with water until the pH value is neutral, and drying it to obtain pretreated corn stalk powder.

[0019] In the pretreatment, the ratio of corn stalk powder to sodium hydroxide aqueous solution is 200g:2000-2500mL;

[0020] The sodium hydroxide aqueous solution has a mass fraction of 1.3-1.5%;

[0021] The activation and expansion culture involved selecting a loopful of Trichoderma reesei strain, spreading it evenly on 60 mL of activation medium, and culturing it at 28°C for 7 days. A single colony was then picked and inoculated into 150 mL of expansion medium, and cultured at 28°C and 200 rpm for 36 hours with shaking. The viable cell count was then adjusted to obtain the seed culture.

[0022] In the activation and expansion culture, the Trichoderma reesei strain was donated by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, with accession number CGMCC No. 21470;

[0023] The components of the activation culture medium are: 25 g / L glucose, 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, 20 g / L agar powder, and pH 4.8.

[0024] The expanded culture medium consists of: 25 g / L glucose, 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, and a pH of 4.8.

[0025] The viable bacteria count in the seed liquid was 1×10⁻⁶. 8 CFU / mL;

[0026] The preparation of the auxiliary agent involves mixing sodium hydroxide, urea, and the first portion of water, stirring until completely dissolved, keeping it at -12°C for 2 hours, transferring it to a cold water bath, mixing it with microcrystalline cellulose and calcium nitrate, stirring for 2-2.5 hours, continuously bubbling carbon dioxide into it, filtering, taking the filter residue, washing it with water, mixing it with polyethylene glycol 1000 and the second portion of water, ball milling it, and drying it to obtain the auxiliary agent.

[0027] In the preparation aids, the ratio of sodium hydroxide, urea, first part water, microcrystalline cellulose, calcium nitrate, polyethylene glycol 1000, and second part water is 233g:400g:2700mL:100-120g:45-50g:15-18g:60-70mL.

[0028] When continuously introducing carbon dioxide, the introduction rate is 60-70 L / h, and the continuous introduction time is 2.5-3 h.

[0029] In the ball milling process, alumina grinding balls with a diameter of 10 mm are used;

[0030] In the ball milling process, the ball-to-material ratio is 3-4:1, the ball milling speed is 300-350 rpm, and the ball milling time is 20-30 min.

[0031] To prepare the crude enzyme solution, pretreated corn stalk powder and nutrient solution are mixed and stirred at 27-29℃ and 180-220 rpm for 30-40 min. The pH value is adjusted to 4.7-4.9, sterilized, and seed solution is added. The mixture is then cultured at 27-29℃ for 7-7.5 days. Acetate-sodium acetate buffer is added, and the mixture is shaken and cultured at 27-29℃ and 180-220 rpm for 1-1.5 h. The supernatant is then collected to obtain the crude enzyme solution.

[0032] In the preparation of the crude enzyme solution, the ratio of pretreated corn stalk powder, nutrient solution, seed solution, and acetate-sodium acetate buffer is 2.9-3.1g:9-9.5mL:3-3.2mL:20-22mL.

[0033] The nutrient solution consists of: 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, and 0.6 g / L magnesium sulfate.

[0034] The concentration of the acetate-sodium acetate buffer solution is 0.05 mol / L, and the pH is 4.8.

[0035] The enzymatic hydrolysis and saccharification process involves mixing pretreated corn stalk powder, corn steep liquor powder, potassium hydroxide, ammonium sulfate, magnesium sulfate, the first portion of water, and crude enzyme solution. The mixture is stirred at 27-29℃ and 180-220 rpm for 30-40 minutes, and the pH is adjusted to 4.7-4.9. The mixture is then sterilized to obtain the fermentation medium. The adjuvant, seed liquid, and the second portion of water are mixed and stirred at 27-29℃ and 180-220 rpm. This mixture is then added to the fermentation medium and cultured with shaking at 27-29℃ and 140-160 rpm for 6-7 days to obtain the in-situ enzymatic hydrolysis and saccharification product.

[0036] In the enzymatic hydrolysis and saccharification process, the ratio of pretreated corn stalk powder, corn steep liquor powder, potassium hydroxide, ammonium sulfate, magnesium sulfate, first portion of water, and crude enzyme solution is 29-31g:4-4.2g:2-2.2g:3-3.2g:0.6-0.65g:500-510mL:5-5.2mL;

[0037] The ratio of pretreated corn stalk powder, adjuvants, seed liquid, and second part water is 29-31g:20-22g:50-52mL:500-520mL;

[0038] When the mixture is shaken and cultured at 27-29℃ and 140-160rpm for 6-7 days, it is fed every 12 hours for the first 48 hours of shaking culture, for a total of 4 feedings. Tween-80 aqueous solution is added each time the mixture is fed. The mass ratio of the pretreated corn straw powder, Tween-80 aqueous solution and the original pretreated corn straw powder in the fermentation medium is 55-60g:28-30mL:29-31g.

[0039] When the culture is shaken at 27-29℃ and 140-160rpm for 6-7 days, the pH value is monitored in real time and always controlled between 4 and 5.

[0040] The second portion of water is sterilized water;

[0041] The Tween-80 aqueous solution has a mass fraction of 10%.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] The in-situ enzymatic hydrolysis and saccharification method of the present invention can achieve high solids enzymatic hydrolysis and high specific sugar yield. When processing corn straw, the specific sugar yield can reach 0.425-0.432 g / g, and the specific sugar yield results show little difference when processing different batches of corn straw. Detailed Implementation

[0044] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0045] The Trichoderma reesei strains used in Examples 1-2 and Comparative Examples 1-5 were donated by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, with accession number CGMCC No. 21470.

[0046] The corn stalk powder used in Examples 1-2 and Comparative Examples 1-5 was prepared as follows: corn stalks were added to a pulverizer and pulverized, passed through a 40-mesh sieve, washed with water, transferred to an oven, and the temperature of the oven was adjusted to 105℃ and kept warm for 5 hours to obtain corn stalk powder.

[0047] The corn stalk powder used in Examples 1-2 and Comparative Examples 1-5 contained 37.24% cellulose, 27.61% hemicellulose, and 15.52% lignin (measured by NREL method).

[0048] Example 1

[0049] A method for in-situ enzymatic hydrolysis and saccharification of straw is as follows:

[0050] Step 1: Pretreatment: Add 200g of corn stalk powder and 2000mL of sodium hydroxide aqueous solution to a wide-mouth bottle, cover the mouth of the wide-mouth bottle with a plate, transfer it to an oven, adjust the temperature of the oven to 55℃, keep it warm and stand for 30h, filter, take the filter residue, wash it with water until the pH value is neutral, transfer it to an oven, adjust the temperature of the oven to 105℃, keep it warm and stand for 4h to obtain the pretreated corn stalk powder;

[0051] The sodium hydroxide aqueous solution has a mass fraction of 1.3%;

[0052] Step 2: Activation and Expansion Culture. Pick one loopful of *Trichoderma reesei* strain and spread it evenly on 60 mL of activation medium. Incubate at 28°C for 7 days. Pick a single colony and inoculate it into 150 mL of expansion medium. Incubate at 28°C and 200 rpm with shaking for 36 hours, adjusting the viable count to 1 × 10⁻⁶. 8 CFU / mL was used to obtain the seed culture;

[0053] The components of the activation culture medium are: 25 g / L glucose, 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, 20 g / L agar powder, and pH 4.8.

[0054] The expanded culture medium consists of: 25 g / L glucose, 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, and a pH of 4.8.

[0055] Step 3: Preparation of Additives. Add 233g sodium hydroxide, 400g urea, and 2700mL water to a reaction vessel. Adjust the stirring speed of the reaction vessel to 100rpm and stir until completely dissolved. Transfer to a freezing device and adjust the temperature of the freezing device to -12℃. Let it stand for 2 hours. Transfer to another reaction vessel and place it in a cold water bath. Adjust the stirring speed of the reaction vessel to 100rpm. Add 100g microcrystalline cellulose and 45g calcium nitrate to the reaction vessel and stir for 2 hours. Carbon dioxide was continuously introduced into the feed liquid at a rate of 60 L / h for 2.5 h. The mixture was then filtered, and the residue was washed twice with water. It was then mixed with 15 g of polyethylene glycol 1000 and 60 mL of water and transferred to a ball mill. Alumina grinding balls with a diameter of 10 mm were added to the ball mill, and the ball-to-material ratio was adjusted to 3:1. The ball milling speed was adjusted to 300 rpm and the mixture was milled for 20 min. The mixture was then transferred to an oven, and the oven temperature was adjusted to 105 °C. The mixture was kept at this temperature for 9 h to obtain the additive.

[0056] Step 4: Preparation of crude enzyme solution. Mix 3g of pretreated corn stalk powder and 9mL of nutrient solution, stir at 28℃ and 200rpm for 30min, adjust the pH to 4.8, sterilize, add 3mL of seed culture, and culture at 28℃ for 7d. Add 20mL of acetate-sodium acetate buffer, shake and culture at 28℃ and 200rpm for 1h, and take the supernatant to obtain crude enzyme solution.

[0057] The nutrient solution consists of: 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, and 0.6 g / L magnesium sulfate.

[0058] The concentration of the acetate-sodium acetate buffer solution is 0.05 mol / L, and the pH is 4.8.

[0059] Step 5: Enzymatic hydrolysis and saccharification. Mix 30g of pretreated corn stalk powder, 4g of corn steep liquor powder, 2g of potassium hydroxide, 3g of ammonium sulfate, 0.6g of magnesium sulfate, 500mL of water, and 5mL of crude enzyme solution. Stir at 28℃ and 200rpm for 30min, adjust the pH to 4.8, and sterilize to obtain the fermentation medium. Mix 20g of adjuvant, 50mL of seed liquid, and 500mL of sterile water. Stir at 28℃ and 200rpm for 5min and add to the above fermentation medium. Shake culture at 28℃ and 150rpm for 6 days. During the first 48h of shaking culture, feed once every 12h, for a total of 4 times. Each time, feed 55g of pretreated corn stalk powder and add 28mL of Tween-80 aqueous solution. At the same time, monitor the pH in real time during shaking culture and keep the pH between 4 and 5. After shaking culture, the in-situ enzymatic hydrolysis and saccharification product is obtained.

[0060] The Tween-80 aqueous solution has a mass fraction of 10%.

[0061] Example 2

[0062] A method for in-situ enzymatic hydrolysis and saccharification of straw is as follows:

[0063] Step 1: Pretreatment: Add 200g of corn stalk powder and 2500mL of sodium hydroxide aqueous solution to a wide-mouth bottle, cover the mouth of the wide-mouth bottle with a plate, transfer it to an oven, adjust the temperature of the oven to 60℃, keep it warm and stand for 35h, filter, take the filter residue, wash it with water until the pH value is neutral, transfer it to an oven, adjust the temperature of the oven to 105℃, keep it warm and stand for 6h to obtain the pretreated corn stalk powder;

[0064] The sodium hydroxide aqueous solution has a mass fraction of 1.5%;

[0065] Step 2: Activation and Expansion Culture. Pick one loopful of *Trichoderma reesei* strain and spread it evenly on 60 mL of activation medium. Incubate at 28°C for 7 days. Pick a single colony and inoculate it into 150 mL of expansion medium. Incubate at 28°C and 200 rpm with shaking for 36 hours, adjusting the viable count to 1 × 10⁻⁶. 8 CFU / mL was used to obtain the seed culture;

[0066] The components of the activation culture medium are: 25 g / L glucose, 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, 20 g / L agar powder, and pH 4.8.

[0067] The expanded culture medium consists of: 25 g / L glucose, 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, and a pH of 4.8.

[0068] Step 3: Preparation of Additives. Add 233g sodium hydroxide, 400g urea, and 2700mL water to a reaction vessel. Adjust the stirring speed of the reaction vessel to 300rpm and stir until completely dissolved. Transfer to a freezing device and adjust the temperature of the freezing device to -12℃. Let it stand for 2 hours. Transfer to another reaction vessel and place it in a cold water bath. Adjust the stirring speed of the reaction vessel to 300rpm. Add 120g microcrystalline cellulose and 50g calcium nitrate to the reaction vessel and stir for 2.5 hours. Carbon dioxide was continuously introduced into the feed liquid in the reactor at a rate of 70 L / h for 3 hours. The mixture was then filtered, and the residue was washed three times with water. It was then mixed with 18 g of polyethylene glycol 1000 and 70 mL of water and transferred to a ball mill. Alumina grinding balls with a diameter of 10 mm were added to the ball mill, and the ball-to-material ratio was adjusted to 4:1. The ball milling speed was adjusted to 350 rpm and the mixture was milled for 30 minutes. The mixture was then transferred to an oven, and the oven temperature was adjusted to 105℃. The mixture was kept at this temperature and allowed to stand for 10 hours to obtain the additive.

[0069] Step 4: Preparation of crude enzyme solution. Mix 3g of pretreated corn stalk powder and 9mL of nutrient solution, stir at 28℃ and 200rpm for 30min, adjust the pH to 4.8, sterilize, add 3mL of seed culture, and culture at 28℃ for 7d. Add 20mL of acetate-sodium acetate buffer, shake and culture at 28℃ and 200rpm for 1h, and take the supernatant to obtain crude enzyme solution.

[0070] The nutrient solution consists of: 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, and 0.6 g / L magnesium sulfate.

[0071] The concentration of the acetate-sodium acetate buffer solution is 0.05 mol / L, and the pH is 4.8.

[0072] Step 5: Enzymatic hydrolysis and saccharification. Mix 30g of pretreated corn stalk powder, 4g of corn steep liquor powder, 2g of potassium hydroxide, 3g of ammonium sulfate, 0.6g of magnesium sulfate, 500mL of water, and 5mL of crude enzyme solution. Stir at 28℃ and 200rpm for 30min, adjust the pH to 4.8, and sterilize to obtain the fermentation medium. Mix 22g of adjuvant, 50mL of seed liquid, and 500mL of sterile water. Stir at 28℃ and 200rpm for 10min and add to the above fermentation medium. Shake culture at 28℃ and 150rpm for 6 days. During the first 48 hours of shaking culture, feed once every 12 hours for a total of 4 times. Each time, feed 60g of pretreated corn stalk powder and add 30mL of Tween-80 aqueous solution. At the same time, monitor the pH in real time during shaking culture and keep the pH between 4 and 5. After shaking culture, the in-situ enzymatic hydrolysis and saccharification product is obtained.

[0073] The Tween-80 aqueous solution has a mass fraction of 10%.

[0074] Comparative Example 1

[0075] The in-situ enzymatic hydrolysis of straw was used in the same way as in Example 1, except that the polyethylene glycol 1000 in step 3, preparation of the auxiliary agent, was replaced with Tween-80.

[0076] Comparative Example 2

[0077] The in-situ enzymatic hydrolysis and saccharification of straw was carried out using a method that was basically the same as that in Example 1, except that the same volume of Tween-80 aqueous solution in step 5: enzymatic hydrolysis and saccharification was replaced with a 10% (w / w) polyethylene glycol 1000 aqueous solution.

[0078] Comparative Example 3

[0079] The in-situ enzymatic hydrolysis and saccharification of straw is basically the same as in Example 1, except that step 3: preparation of adjuvants is omitted, and the addition of adjuvants is omitted in step 5: enzymatic hydrolysis and saccharification. Specifically, step 5: enzymatic hydrolysis and saccharification is changed to:

[0080] 30g of pretreated corn stalk powder, 4g of corn steep liquor powder, 2g of potassium hydroxide, 3g of ammonium sulfate, 0.6g of magnesium sulfate, 500mL of water, and 5mL of crude enzyme solution were mixed and stirred at 28℃ and 200rpm for 30min. The pH was adjusted to 4.8 and sterilized to obtain the fermentation medium. 50mL of seed liquid and 500mL of sterile water were mixed and stirred at 28℃ and 200rpm for 5min. The mixture was then added to the above fermentation medium and cultured at 28℃ and 150rpm for 6d with shaking. During the first 48h of shaking culture, the mixture was fed every 12h for a total of 4 times. Each feeding consisted of 55g of pretreated corn stalk powder, and 28mL of Tween-80 aqueous solution was added each time. After shaking culture, the in-situ enzymatic hydrolysis and saccharification product was obtained.

[0081] The Tween-80 aqueous solution has a mass fraction of 10%.

[0082] Comparative Example 4

[0083] The in-situ enzymatic hydrolysis of straw was used in essentially the same method as in Example 1, except that step 3: preparation of adjuvants was changed to:

[0084] Mix 100g microcrystalline cellulose, 27g calcium carbonate, 15g polyethylene glycol 1000, and 60g water, and transfer the mixture to a ball mill. Add alumina grinding balls with a diameter of 10mm to the ball mill, adjust the ball-to-material ratio to 3:1, adjust the ball milling speed to 300rpm, and mill for 20 minutes. Transfer the mixture to an oven, adjust the oven temperature to 105℃, and keep it warm for 9 hours to obtain the additive.

[0085] That is, the process of combining microcrystalline cellulose with calcium carbonate is omitted.

[0086] Comparative Example 5

[0087] The in-situ enzymatic hydrolysis of straw was used in essentially the same method as in Example 1, except that step 3: preparation of adjuvants was changed to:

[0088] Mix 127g microcrystalline cellulose, 15g polyethylene glycol 1000, and 60g water, and transfer the mixture to a ball mill. Add alumina grinding balls with a diameter of 10mm to the ball mill, adjust the ball-to-material ratio to 3:1, adjust the ball milling speed to 300rpm, and mill for 20 minutes. Then transfer the mixture to an oven, adjust the oven temperature to 105℃, and keep it at that temperature for 9 hours to obtain the additive.

[0089] That is, the addition of calcium carbonate is omitted.

[0090] Experimental Example 1

[0091] The reducing sugar concentration in the in-situ enzymatic hydrolysis saccharification products obtained in Examples 1-2 and Comparative Examples 1-5 was tested using the DNS method (3,5-dinitrosalicylic acid colorimetric method). Then, the specific sugar yield was calculated according to the method published in Wen Xiaoxia, Bai Guangjian, Li Tao, Ma Yifan, and Zou Wei, "Optimization of In-situ Enzymatic Hydrolysis Saccharification Process of Rice Straw by Liquid Fermentation," *Food and Fermentation Industries*, October 2020. The calculation results are as follows:

[0092]

[0093] The results in the table above show that, compared with Comparative Examples 1-5, the methods of Example 1 and Example 2 can achieve higher specific sugar yields.

[0094] Experimental Example 2

[0095] The corn stalks used in Examples 1-2 and Comparative Examples 1-5 were replaced with another batch, and corn stalk powder was prepared by crushing, sieving, washing, and drying according to the same preparation method. The corn stalk powder contained 36.85% cellulose, 28.72% hemicellulose, and 16.80% lignin (measured by NREL method). Then, in-situ enzymatic hydrolysis and saccharification were carried out according to the methods of Examples 1-2 and Comparative Examples 1-5.

[0096] Following the method in Example 1, the specific sugar yield was calculated, and the results are as follows:

[0097]

[0098] Comparing the results in the table above with those in Experiment 1, it can be seen that when processing different batches of corn stalk powder, the specific sugar yield results of the methods in Examples 1-2 are more stable compared with those in Comparative Examples 1-3.

[0099] Further analysis using the methods in Examples 1 and 2 demonstrates that the methods of Examples 1-2 are superior to those of Comparative Examples 1-5. Analysis suggests this may be because, in the preparation of the additives in Examples 1-2, microcrystalline cellulose and calcium carbonate are first combined through intermolecular forces, ensuring that calcium ions can rapidly bind with cellulase after the enzymatic hydrolysis of microcrystalline cellulose. Then, polyethylene glycol 1000 is used for treatment. Polyethylene glycol 1000 connects to microcrystalline cellulose and calcium carbonate through its oxygen-containing groups. Furthermore, as a nonionic surfactant, polyethylene glycol 1000 can bind to *Trichoderma reesei* through its hydrophobic groups, thus binding the microcrystalline cellulose... Vitamin C and calcium carbonate bind to the surface of *Trichoderma reesei*. According to Li Xiaojuan's "Study on the Inhibitory Effect and Mechanism of CaCO3 on Butanol Fermentation by Reducing Toxic Substances in Lignocellulose Hydrolysate," published by Henan Agricultural University in June 2023, calcium carbonate has a certain detoxification and pH buffering effect, which can protect *Trichoderma reesei* and avoid the difficulty in pH adjustment due to high viscosity during high-solids fermentation. Microcrystalline cellulose can reduce the influence of other substances in corn straw on enzyme production by coating *Trichoderma reesei*, ensuring rapid enzyme production. With the decomposition of microcrystalline cellulose in the adjuvant, according to Li Feiyang's "Based on CaCO3...", calcium carbonate has a certain detoxification and pH buffering effect, protecting *Trichoderma reesei* and avoiding the difficulty in pH adjustment due to high viscosity during high-solids fermentation. 2+ Preparation and Properties of Stable Peptide-Microcrystalline Cellulose Pickering Emulsion. Beijing Forestry University. June 2022. Calcium ions can act as a bridge, promoting the binding between cellulase and microcrystalline cellulose, thereby promoting the decomposition of straw. Then, by adding Tween-80 later, according to Yan Rixiang and Zhao Xiaoli. Cellulase Secretion under Certain Chemical Factors—The Effects of Suitable Concentrations of Several Surfactants and Organic Acids on Cellulase Secretion and Time Process. Journal of Northwest University (Natural Science Edition). June 1992, utilizing the different effects of different surfactants on cellulase activity, polyethylene glycol 1000 and Tween-80 were used synergistically to further improve cellulase activity.

Claims

1. A method for in-situ enzymatic hydrolysis and saccharification of straw, characterized in that, It consists of the following steps: pretreatment of corn straw powder, activation and expansion culture of Trichoderma reesei to prepare seed liquid, preparation of adjuvants, preparation of crude enzyme solution, and enzymatic hydrolysis and saccharification; The preparation of the auxiliary agent involves mixing sodium hydroxide, urea, and the first portion of water, stirring until completely dissolved, keeping it at -12°C for 2 hours, transferring it to a cold water bath, mixing it with microcrystalline cellulose and calcium nitrate, stirring, continuously bubbling carbon dioxide into it, filtering, taking the filter residue, washing it with water, mixing it with polyethylene glycol 1000 and the second portion of water, ball milling it, and drying it to obtain the auxiliary agent. To prepare the crude enzyme solution, pretreated corn stalk powder and nutrient solution are mixed and stirred at 27-29℃ and 180-220 rpm for 30-40 min. The pH value is adjusted to 4.7-4.9, sterilized, and seed solution is added. The mixture is then cultured at 27-29℃ for 7-7.5 days. Acetate-sodium acetate buffer is added, and the mixture is shaken and cultured at 27-29℃ and 180-220 rpm for 1-1.5 h. The supernatant is then collected to obtain the crude enzyme solution. In the preparation of the crude enzyme solution, the nutrient solution consists of: 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, and 0.6 g / L magnesium sulfate. The enzymatic hydrolysis and saccharification process involves mixing pretreated corn stalk powder, corn steep liquor powder, potassium hydroxide, ammonium sulfate, magnesium sulfate, the first portion of water, and crude enzyme solution. The mixture is stirred at 27-29℃ and 180-220 rpm for 30-40 minutes, and the pH is adjusted to 4.7-4.

9. The mixture is then sterilized to obtain the fermentation medium. The auxiliary agent, seed liquid, and the second portion of water are mixed and stirred at 27-29℃ and 180-220 rpm. This mixture is then added to the fermentation medium and cultured with shaking at 27-29℃ and 140-160 rpm for 6-7 days to obtain the in-situ enzymatic hydrolysis and saccharification product. During the enzymatic hydrolysis and saccharification, when the corn straw powder was pre-treated and cultured at 27-29℃ and 140-160rpm for 6-7 days, it was fed with the pre-treated corn straw powder every 12 hours for the first 48 hours of the culture, for a total of 4 feedings. Tween-80 aqueous solution was added each time the feed was added. The preservation number of the Trichoderma reesei is CGMCC No. 21470.

2. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 1, characterized in that, The pretreatment of the corn stalk powder involves adding corn stalk powder and sodium hydroxide aqueous solution into a container, covering the mouth of the container, keeping it at 55-60℃ for 30-35 hours, filtering, taking the filter residue, washing it with water until the pH value is neutral, and drying it to obtain the pretreated corn stalk powder.

3. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 2, characterized in that, In the pretreatment of corn stalk powder, the ratio of corn stalk powder to sodium hydroxide aqueous solution is 200g:2000-2500mL; The sodium hydroxide aqueous solution has a mass fraction of 1.3-1.5%.

4. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 1, characterized in that, The seed culture of Trichoderma reesei was prepared by activation and expansion culture. One loopful of Trichoderma reesei strain was picked and evenly spread on 60 mL of activation medium and cultured at 28 °C for 7 days. A single colony was picked and inoculated into 150 mL of expansion medium and cultured at 28 °C and 200 rpm for 36 h with shaking. The viable number was adjusted to obtain the seed culture.

5. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 4, characterized in that, In the preparation of seed culture for the activation and expansion culture of Trichoderma reesei, the components of the activation culture medium are: 25 g / L glucose, 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, 20 g / L agar powder, and pH 4.

8. The expanded culture medium consists of: 25 g / L glucose, 4 g / L corn steep liquor powder, 1.66 g / L potassium hydroxide, 2.8 g / L ammonium sulfate, 0.6 g / L magnesium sulfate, and a pH of 4.

8. The viable bacteria count in the seed liquid was 1×10⁻⁶. 8 CFU / mL.

6. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 1, characterized in that, In the preparation aids, the ratio of sodium hydroxide, urea, first part water, microcrystalline cellulose, calcium nitrate, polyethylene glycol 1000, and second part water is 233g:400g:2700mL:100-120g:45-50g:15-18g:60-70mL. When continuously introducing carbon dioxide, the introduction rate is 60-70 L / h, and the continuous introduction time is 2.5-3 h. In the ball milling process, alumina grinding balls with a diameter of 10 mm are used. In the ball milling process, the ball-to-material ratio is 3-4:1, the ball milling speed is 300-350 rpm, and the ball milling time is 20-30 min.

7. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 1, characterized in that, In the preparation of the crude enzyme solution, the ratio of pretreated corn stalk powder, nutrient solution, seed solution, and acetate-sodium acetate buffer is 2.9-3.1g:9-9.5mL:3-3.2mL:20-22mL.

8. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 1, characterized in that, In the enzymatic hydrolysis and saccharification process, the ratio of pretreated corn stalk powder, corn steep liquor powder, potassium hydroxide, ammonium sulfate, magnesium sulfate, first portion of water, and crude enzyme solution is 29-31g:4-4.2g:2-2.2g:3-3.2g:0.6-0.65g:500-510mL:5-5.2mL; The ratio of pretreated corn stalk powder, additives, seed liquid, and second part water is 29-31g:20-22g:50-52mL:500-520mL.

9. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 1, characterized in that, In the enzymatic hydrolysis and saccharification process, when the culture is shaken at 27-29℃ and 140-160rpm for 6-7 days, the mass ratio of the pretreated corn straw powder and Tween-80 aqueous solution added each time to the original pretreated corn straw powder in the fermentation medium is 55-60g:28-30mL:29-31g. During the shaking culture at 27-29℃ and 140-160rpm for 6-7 days, the pH value was monitored in real time and kept between 4 and 5.

10. The method for in-situ enzymatic hydrolysis and saccharification of straw according to claim 1, characterized in that, In the enzymatic hydrolysis and saccharification process, the second portion of water is sterile water; The Tween-80 aqueous solution has a mass fraction of 10%.

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