Alkali treatment method of straw and method for preparing monosaccharide from straw

Abstract

The invention provides a straw alkali treatment method and a method for preparing monosaccharide from straw. The alkali treatment method comprises the following steps: (1) crushing straws to obtain straw powder; and (2) mixing the straw powder with solid alkali to obtain a reaction system, reacting the reaction system under a steam condition, and then carrying out solid-liquid separation to respectively obtain alkali-treated straw and alkali-treated liquid. The method can solve the problem of more alkali treatment waste liquid generated by alkali treatment of straws in the prior art, and is suitable for the technical field of biology.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for alkali treatment of straw and a method for preparing monosaccharides from straw. Background Technology

[0002] Straw, as one of the most abundant and readily available agricultural residues, is an important raw material for the production of second-generation biofuels and bio-based chemicals.

[0003] The first step in the bioconversion of straw is to pretreat it, with the aim of breaking down the rigid structure of straw and separating cellulose, hemicellulose and lignin, thereby hydrolyzing the cellulose components more efficiently.

[0004] Currently, the main methods for straw pretreatment include physical pretreatment, biological pretreatment, organic solvent pretreatment, and alkaline pretreatment. The first three methods have problems such as high energy consumption, high safety requirements, high cost, and long treatment cycle. However, alkaline pretreatment has attracted much attention because of its characteristics of efficiently removing lignin at moderate temperatures, achieving high cellulose recovery rate, and low inhibitor generation.

[0005] In existing technologies, straw, alkali, steam, and water are generally cooked together under high temperature conditions, as disclosed in Chinese patent application CN101634117A. The above-mentioned straw pretreatment process generates a large amount of black alkali treatment liquid, which not only increases the cost of subsequent treatment but also pollutes the environment.

[0006] Therefore, developing a method to reduce waste liquid from straw alkali treatment is of certain significance for promoting the industrialization of straw bioconversion. Summary of the Invention

[0007] The main objective of this invention is to provide a method for alkali treatment of straw and a method for preparing monosaccharides from straw, so as to solve the problem of excessive alkali treatment waste liquid generated in the prior art.

[0008] To achieve the above objectives, according to a first aspect of the present invention, an alkali treatment method for straw is provided, the alkali treatment method comprising: (1) crushing straw to obtain straw powder; (2) mixing the straw powder with solid alkali to obtain a reaction system, reacting the reaction system under steam conditions and then performing solid-liquid separation to obtain alkali-treated straw and alkali-treated liquid respectively.

[0009] Further, the mass ratio of the straw powder to the solid alkali in the above reaction system is 8-12:1; preferably, the steam conditions include introducing water vapor into the above reaction system to maintain the temperature of the reaction system at 110-130℃, more preferably 120℃; preferably, the mixing speed is 80-120 rpm, more preferably 100 rpm; preferably, the reaction time using the water vapor is 20-40 min, more preferably 25-35 min; preferably, after separating the above alkali-treated straw, the above alkali-treated straw is washed with water and dried to obtain neutral above alkali-treated straw.

[0010] Furthermore, the aforementioned straw is derived from one or more of corn stalks, cotton stalks, wheat stalks, or sweet sorghum stalks, preferably corn stalks.

[0011] Furthermore, the aforementioned solid alkali includes one or more of sodium hydroxide, quicklime, or calcium hydroxide, preferably sodium hydroxide.

[0012] Furthermore, the straw is crushed and then screened through a sieve; preferably, the sieve mesh size is 40-60 mesh, more preferably 45-55 mesh.

[0013] To achieve the above objective, according to a second aspect of the present invention, a method for preparing monosaccharides from straw is provided, the method comprising: (a) treating the straw using the above-described alkali treatment method to obtain alkali-treated straw; (b) mixing the alkali-treated straw with cellulase to obtain a reaction mixture, and enzymatically hydrolyzing the reaction mixture to obtain an enzymatic hydrolysate containing the monosaccharide; wherein the monosaccharide comprises one or more of glucose, galactose, fructose, sorbitol, mannitol, or xylose.

[0014] Further, the monosaccharide includes glucose and / or xylose; preferably, the initial solid content of the straw in the reaction mixture is 5% (w / v); preferably, in step (b), the cellulase is mixed with the alkali-treated straw in batches to obtain an enzymatic hydrolysis system; preferably, step (b) includes: mixing the first alkali-treated straw with the first cellulase and performing the enzymatic hydrolysis to obtain a first enzymatic hydrolysis system; adding the second alkali-treated straw and the second cellulase to the first enzymatic hydrolysis system and performing the enzymatic hydrolysis to obtain a second enzymatic hydrolysis system; adding the third alkali-treated straw and the third cellulase to the second enzymatic hydrolysis system and performing the enzymatic hydrolysis to obtain a third enzymatic hydrolysis system; preferably, the first cellulase, the second cellulase, and the third cellulase each account for 1 / 3 of the total enzyme amount; preferably, the solid content of the alkali-treated straw in the first enzymatic hydrolysis system, the second enzymatic hydrolysis system, and the third enzymatic hydrolysis system is 5% (w / v); preferably, the second alkali-treated straw and the second cellulase are added 12 hours after the reaction of the first enzymatic hydrolysis system. Preferably, the third alkali-treated straw and the third cellulase are added 24 hours after the first enzymatic hydrolysis system has reacted; preferably, the enzymatic hydrolysis system continues to hydrolyze for 72 hours; preferably, the reaction mixture further includes 0.04-0.06M phosphate buffer, more preferably 0.05M phosphate buffer; preferably, the enzyme-to-substrate ratio in the reaction mixture is 15-20 FPU:1g, more preferably 16-19 FPU:1g; preferably, the hydrolysis temperature is 40-60℃, more preferably... Preferably, the temperature is 50°C; preferably, the rotation speed of the enzymatic hydrolysis is 100-250 rpm, more preferably 200 rpm; preferably, the glucose content in the enzymatic hydrolysate is 92-94 g / L, more preferably 92.5-93.5 g / L; preferably, the xylose content in the enzymatic hydrolysate is 36-40 g / L, more preferably 37-39 g / L; the method further includes an adsorption step after the enzymatic hydrolysis step, the adsorption step including: mixing the enzymatic hydrolysate with activated carbon for detoxification treatment to obtain the monosaccharide.

[0015] Further, the activated carbon mentioned above includes modified activated carbon; preferably, the preparation method of the modified activated carbon includes: mixing activated carbon with a nitrate metal solution to obtain a modified system, subjecting the modified system to ultrasonic and microwave irradiation treatment to obtain the modified activated carbon; preferably, the solute in the nitrate metal solution includes one or more of ferric nitrate, copper nitrate, silver nitrate, or zinc nitrate; preferably, the solute includes ferric nitrate and zinc nitrate; preferably, the total concentration of ferric nitrate and zinc nitrate is 0.05-0.15M, more preferably 0.1M; preferably, the molar ratio of ferric nitrate to zinc nitrate is 2-4:1, more preferably 3:1; preferably, the ultrasonic treatment time is 20-40 min, more preferably 30 min; preferably, the microwave irradiation power is 700-900W, more preferably 800W; preferably, the microwave irradiation time is 3-6 min, more preferably 5 min.

[0016] Furthermore, the above adsorption step also includes an oscillation treatment; preferably, the concentration of the modified activated carbon added to the enzymatic hydrolysate is 8-12 g / L, more preferably 10 g / L; preferably, the temperature of the above oscillation treatment is 25-40℃, more preferably 30℃; preferably, the time of the above oscillation treatment is 0.5-1.5 h, more preferably 1 h; preferably, the rotation speed of the above oscillation treatment is 150-250 rpm, more preferably 200 rpm.

[0017] According to a third aspect of the present invention, a method for preparing succinic acid is provided, the method comprising: using a monosaccharide obtained by the above-described method of preparing monosaccharides from straw as a carbon source, and subjecting it to fermentation treatment with Escherichia coli to obtain succinic acid; preferably, the Escherichia coli includes an engineered strain capable of synthesizing succinic acid from the monosaccharide as a substrate.

[0018] The present invention involves first crushing straw to obtain straw powder, then mixing the straw powder with solid alkali to obtain a reaction system. After reacting the reaction system under steam conditions, solid-liquid separation is performed to obtain alkali-treated material and alkali-treated liquid, respectively. This treatment method is simple to operate, effectively reduces the generation of alkali-treated liquid, and the obtained alkali-treated material can be used as a raw material for the subsequent preparation of monosaccharide solutions. The present invention, while meeting quality requirements, has better economic value and is environmentally friendly. Detailed Implementation

[0019] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0020] As mentioned in the background section, existing technologies utilize straw, alkali, steam, and water to pretreat straw under high-temperature conditions. However, the addition of water during the preparation process generates a large amount of black alkaline treatment liquid, which not only increases subsequent processing costs but also pollutes the environment. Therefore, in this application, the inventors attempted to pulverize straw to obtain straw powder, and then react the straw powder with solid alkali under steam conditions, thus proposing a series of protection solutions in this application.

[0021] In a first typical embodiment of this application, an alkali treatment method for straw is provided. The alkali treatment method includes: (1) crushing the straw to obtain straw powder; (2) mixing the straw powder with solid alkali to obtain a reaction system, reacting the reaction system under steam conditions, and then performing solid-liquid separation to obtain alkali-treated straw and alkali-treated liquid respectively.

[0022] In this application, the solid alkali refers to alkali in solid form with a water content of less than 1 wt%. In the aforementioned alkali treatment method, by fully reacting straw and solid alkali under steam conditions, straw can be prepared as a high-quality raw material for producing monosaccharide solutions. In this application, the inventors discovered that by adding solid alkali and introducing steam during the alkali treatment of straw, the amount of alkali-treated liquid produced can be reduced, thus achieving the technical effect of reducing the amount of waste liquid generated during the alkali treatment of straw.

[0023] In a preferred embodiment, the mass ratio of the straw powder to the solid alkali in the above reaction system is 8-12:1; preferably, the steam conditions include introducing water vapor into the above reaction system to maintain the temperature of the reaction system at 110-130°C, more preferably 120°C; preferably, the mixing speed is 80-120 rpm, more preferably 100 rpm; preferably, the reaction time using the water vapor is 20-40 min, more preferably 25-35 min; preferably, after separating the above alkali-treated straw, the above alkali-treated straw is washed with water and dried to obtain neutral above alkali-treated straw.

[0024] In this application, the inventors discovered that when the mass ratio of straw powder to solid alkali is, for example, 8-12:1 (including but not limited to 8:1, 9:1, 10:1, 11:1, or 12:1), the solid alkali can achieve a complete reaction with the straw, while avoiding the waste of raw materials caused by excessive alkali. In the aforementioned steam treatment process, the inventors discovered that by introducing steam into the reaction system and maintaining the system temperature at, for example, 110-130℃ (including but not limited to 115℃, 120℃, 125℃, or 130℃), more preferably at, for example, 120℃, a better effect of sufficient reaction between the straw and the solid alkali can be achieved. Furthermore, when the mixing speed is, for example, 80-120 rpm (including but not limited to 85 rpm, 90 rpm, 95 rpm, 100 rpm, 105 rpm, 110 rpm, 115 rpm, or 120 rpm), more preferably at, for example, 100 rpm, and the reaction time using the aforementioned water vapor is, for example, 20-40 min (including but not limited to 25 min, 30 min, or 35 min), more preferably at, for example, 25-35 min, the above-mentioned reaction system can achieve the technical effect of thorough and uniform mixing of straw powder and solid alkali. The inventors also discovered that after obtaining alkali-treated straw by separating the alkali-treated straw from the alkali-treated liquid, the neutral alkali-treated straw obtained by washing and drying the alkali-treated straw can be well used in the subsequent monosaccharide preparation process.

[0025] In a preferred embodiment, the straw is derived from one or more of corn stalks, cotton stalks, wheat stalks, or sweet sorghum stalks, preferably corn stalks.

[0026] Corn stalks, cotton stalks, wheat stalks, or sweet sorghum stalks are all common raw materials for the production of monosaccharide solutions in industry. Among them, corn stalks have the characteristics of high cellulose activity, low lignin content, and easy collection compared to other plant stalks. Therefore, in the prior art and the experimental process of this application, the inventors preferably choose corn stalks as the main source for preparing monosaccharides.

[0027] In a preferred embodiment, the solid alkali comprises one or more of sodium hydroxide, quicklime, or calcium hydroxide, preferably sodium hydroxide.

[0028] Alkalis such as sodium hydroxide, quicklime, and calcium hydroxide can effectively remove lignin from straw during the straw treatment process. In this application, the inventors preferably use sodium hydroxide to perform the alkaline treatment of straw.

[0029] In a preferred embodiment, the corn stalks are crushed and then screened through a sieve; preferably, the sieve mesh size is 40-60 mesh, more preferably 45-55 mesh.

[0030] In this application, the inventors discovered that when corn stalks are crushed and then screened through a sieve, and the sieve aperture is, for example, 40-60 mesh (including but not limited to 45 mesh, 50 mesh, 55 mesh or 60 mesh), more preferably, 45-55 mesh, the resulting stalk powder can achieve the technical effect of fully reacting with solid alkali. The alkali-treated stalks obtained after the reaction can be well applied in the subsequent production of monosaccharide solutions.

[0031] In this application, the inventors discovered that by using the above-mentioned alkali treatment method to treat 0.5 kg of straw, the volume of alkali treatment liquid produced is only 300-500 mL. Compared with the traditional straw alkali treatment method, the amount of alkali treatment liquid produced is reduced, and it can even achieve a technical effect of reducing the amount of alkali treatment liquid by 11.25 times, which greatly reduces the subsequent treatment cost and is in line with the development model and requirements of green chemistry.

[0032] In a second typical embodiment of this application, a method for preparing monosaccharides from straw is provided. The method includes: (a) treating the straw using the aforementioned alkali treatment method to obtain alkali-treated straw; (b) mixing the alkali-treated straw with cellulase to obtain a reaction mixture, and enzymatically hydrolyzing the reaction mixture to obtain an enzymatic hydrolysate containing the aforementioned monosaccharide; the monosaccharide includes one or more of glucose, galactose, fructose, sorbitol, mannitol, or xylose.

[0033] In this application, the inventors discovered that by mixing the above-mentioned alkali-treated straw with cellulase and then enzymatically hydrolyzing it, it is possible to obtain a solution containing a variety of monosaccharide components, wherein the monosaccharides include one or more of glucose, galactose, fructose, sorbitol, mannitol or xylose.

[0034] In a preferred embodiment, the monosaccharide comprises glucose and / or xylose; preferably, the initial solid content of the straw in the reaction mixture is 5% (w / v); preferably, in step (b), the cellulase is mixed with the alkali-treated straw in batches to obtain an enzymatic hydrolysis system; preferably, step (b) comprises: mixing the first alkali-treated straw with the first cellulase and performing the enzymatic hydrolysis to obtain a first enzymatic hydrolysis system; adding the second alkali-treated straw and the second cellulase to the first enzymatic hydrolysis system and performing the enzymatic hydrolysis to obtain a second enzymatic hydrolysis system; adding the third alkali-treated straw and the third cellulase to the second enzymatic hydrolysis system and performing the enzymatic hydrolysis to obtain a third enzymatic hydrolysis system; preferably, the first cellulase, the second cellulase, and the third cellulase each account for 1 / 3 of the total enzyme amount; preferably, the solid content of the alkali-treated straw in the first enzymatic hydrolysis system, the second enzymatic hydrolysis system, and the third enzymatic hydrolysis system is 5% (w / v); preferably, the addition time of the second alkali-treated straw and the second cellulase is 1 hour after the reaction of the first enzymatic hydrolysis system. After 2 hours; preferably, the addition of the third alkali-treated straw and the third cellulase is performed 24 hours after the reaction of the first enzymatic hydrolysis system; preferably, the enzymatic hydrolysis system is continuously hydrolyzed for 72 hours; preferably, the reaction mixture further includes 0.04-0.06M phosphate buffer, more preferably 0.05M phosphate buffer; preferably, the enzyme-to-substrate ratio in the reaction mixture is 15-20 FPU:1g, more preferably 16-19 FPU:1g; preferably, the enzymatic hydrolysis temperature is 40-60℃. More preferably, the temperature is 50°C; preferably, the rotation speed of the enzymatic hydrolysis is 100-250 rpm, more preferably 200 rpm; preferably, the glucose content in the enzymatic hydrolysate is 92-94 g / L, more preferably 92.5-93.5 g / L; preferably, the xylose content in the enzymatic hydrolysate is 36-40 g / L, more preferably 37-39 g / L; the method further includes an adsorption step after the enzymatic hydrolysis step, the adsorption step including: mixing the enzymatic hydrolysate with activated carbon for detoxification treatment to obtain the monosaccharide.

[0035] In this application, the inventors discovered that when the initial low solids content of the straw in the above-mentioned reaction mixture is, for example, 5% (w / v), it is possible to achieve the technical effects of improving the efficiency of subsequent feed-in enzymatic hydrolysis and monosaccharide yield while reducing substrate inhibition. The inventors also discovered that by using staged enzyme supplementation (initially adding 1 / 3 of the enzyme, and then adding another 1 / 3 of the enzyme with each subsequent feed), it is possible to fully improve enzyme utilization, increase enzymatic hydrolysis efficiency, increase monosaccharide yield, reduce enzyme usage costs, and avoid enzyme inactivation. Furthermore, the inventors have also discovered that during the above-mentioned enzymatic hydrolysis process, the reaction mixture contains, for example, 0.04-0.06M (including but not limited to 0.04M, 0.05M, 0.06M) phosphate buffer, more preferably, 0.05M phosphate buffer; the enzyme-to-substrate ratio in the reaction mixture is, for example, 15-20 FPU:1g (including but not limited to 16 FPU:1g, 17 FPU:1g, 18 FPU:1g, 19 FPU:1g or 20 FPU:1g), more preferably, 16-19 FPU:1g; the enzymatic hydrolysis temperature is, for example, 40-60℃ (including but not limited to 45℃, 50℃, 55℃ or 60℃), more preferably, 50℃; and the enzymatic hydrolysis rotation speed is, for example, 100-250 rpm (including...). The speeds of the enzymatic hydrolysis solution include, but are not limited to, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, or 200 rpm, and more preferably, at 200 rpm, which can achieve the technical effect of improving the enzymatic hydrolysis efficiency while ensuring that the glucose content in the enzymatic hydrolysate is, for example, 92-94 g / L (including but not limited to 92 g / L, 92.5 g / L, 93 g / L, 93.5 g / L, or 94 g / L), and more preferably, at 92.5-93.5 g / L; preferably, the xylose content in the enzymatic hydrolysate is, for example, 36-40 g / L (including but not limited to 36 g / L, 37 g / L, 38 g / L, 39 g / L, or 40 g / L), and more preferably, at 37-39 g / L.

[0036] In a preferred embodiment, the activated carbon comprises modified activated carbon; preferably, the preparation method of the modified activated carbon comprises: mixing activated carbon with a nitrate metal solution to obtain a modified system, subjecting the modified system to ultrasonic and microwave irradiation treatment to obtain the modified activated carbon; preferably, the solute in the nitrate metal solution comprises one or more of ferric nitrate, copper nitrate, silver nitrate, or zinc nitrate; preferably, the solute comprises ferric nitrate and zinc nitrate; preferably, the total concentration of ferric nitrate and zinc nitrate is 0.05-0.15M, more preferably 0.1M; preferably, the molar ratio of ferric nitrate to zinc nitrate is 2-4:1, more preferably 3:1; preferably, the ultrasonic treatment time is 20-40 min, more preferably 30 min; preferably, the microwave irradiation power is 700-900W, more preferably 800W; preferably, the microwave irradiation time is 3-6 min, more preferably 5 min.

[0037] In this application, the inventors discovered that modifying activated carbon with ferric nitrate and zinc nitrate to form a Fe3O4 and ZnO composite oxide can enhance the selective adsorption of inhibitors such as phenols and furfural, thereby improving detoxification efficiency. Furthermore, microwave irradiation-assisted modification can effectively shorten the preparation time of the modified activated carbon while increasing its specific surface area and adsorption performance. Furthermore, the inventors have discovered that the ultrasonic treatment time is, for example, 20-40 min (including but not limited to 20 min, 25 min, 30 min, 35 min, or 40 min), more preferably 30 min; the total concentration of ferric nitrate and zinc nitrate is, for example, 0.05-0.15 M (including but not limited to 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.11 M, 0.12 M, 0.13 M, 0.14 M, or 0.15 M), more preferably 0.1 M; and preferably, the molar ratio of ferric nitrate to zinc nitrate is, for example, 2-4:1 (including but not limited to 2:1, 2.5:1, 3:1, 3.5:1, or 4:1). The modified activated carbon prepared in the following conditions is more preferably 3:1; the microwave irradiation power is, for example, 700-900W (including but not limited to 700W, 710W, 720W, 730W, 740W, 750W, 760W, 770W, 780W, 790W, 800W, 810W, 820W, 830W, 840W, 850W, 860W, 870W, 880W, 890W or 900W), more preferably 800W; and the microwave irradiation time is, for example, 3-6min (including but not limited to 3min, 4min, 5min or 6min), more preferably 5min.

[0038] In a preferred embodiment, the above adsorption step further includes an oscillation treatment; preferably, the concentration of the modified activated carbon added to the enzymatic hydrolysate is 8-12 g / L, more preferably 10 g / L; preferably, the temperature of the above oscillation treatment is 25-40°C, more preferably 30°C; preferably, the time of the above oscillation treatment is 0.5-1.5 h, more preferably 1 h; preferably, the rotation speed of the above oscillation treatment is 150-250 rpm, more preferably 200 rpm.

[0039] In this application, the inventors discovered that, in the above-mentioned adsorption step, the concentration of the added enzymatic hydrolysate of the modified activated carbon is, for example, 8-12 g / L (including but not limited to 8 g / L, 9 g / L, 10 g / L, 11 g / L or 12 g / L), more preferably 10 g / L; the shaking treatment temperature is, for example, 25-40°C (including but not limited to 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C), more preferably 30°C; and the shaking treatment time is, for example, 0.5-1.5 h (including but not limited to 0.5 h, 0 h, 0 h). The oscillation treatment time is 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h or 1.5h, more preferably 1h; the oscillation speed is, for example, 150-250 rpm (including but not limited to 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm or 250 rpm), more preferably 200 rpm. The modified activated carbon described above can effectively remove toxic substances such as phenols and furfural from monosaccharide solutions, achieving a good detoxification effect.

[0040] In a third typical embodiment of this application, a method for preparing succinic acid is provided. The method includes: using the monosaccharide obtained by the above-mentioned method of preparing monosaccharides from straw as a carbon source, and treating it by Escherichia coli fermentation to obtain succinic acid; preferably, the Escherichia coli includes an engineered strain capable of synthesizing succinic acid using the monosaccharide as a substrate.

[0041] In this application, the inventors discovered that alkali-treated straw, when used as a carbon source with the monosaccharides obtained by the above-mentioned method of preparing monosaccharides from straw, can achieve efficient synthesis of succinic acid and promote the biotransformation of corn straw through fermentation treatment with Escherichia coli.

[0042] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0043] Example 1

[0044] 1. Corn stalks were crushed and passed through a 50-mesh sieve to obtain corn stalk powder. 500g of the corn stalk powder and 50g of sodium hydroxide powder were added to a pretreatment reactor and stirred at 100 rpm for 15 minutes to ensure thorough mixing. Steam was then introduced, and the mixture was maintained at 120℃ for 30 minutes before the reaction was terminated, producing only 400mL of alkaline treatment solution (black liquid). The treated corn stalks were washed with tap water until the pH became neutral (pH=7.0±0.2), and then dried in a 50℃ oven for 48 hours for later use.

[0045] 2. Pretreated corn stalks were added to 0.05M phosphate buffer (pH=4.8) at an initial solids content of 5%, and Novozymes's Novozymes Cellulase III (batch number VDQI0007) was added. The initial enzyme addition was 1 / 3 of the total enzyme amount (17 FPU / g substrate). Enzymatic hydrolysis was performed at 50℃ and 200 rpm. At 12 h and 24 h, 5% substrate and the remaining enzyme were added respectively (1 / 3 of the total enzyme amount was added at the same time as each substrate addition). Enzymatic hydrolysis was continued for 72 h. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 4500 rpm for 15 min, and the supernatant was collected to obtain the enzymatic hydrolysate, which contained 93.2 g / L glucose and 37.5 g / L xylose.

[0046] 3. Immerse the activated carbon in a mixed solution containing ferric nitrate and zinc nitrate, and sonicate for 30 min. The molar ratio of ferric nitrate molecules to zinc nitrate molecules is 3:1, and the total concentration is 0.1 M. Then, under nitrogen protection, irradiate with microwaves at 800 W for 5 min to generate a composite oxide of Fe3O4 and ZnO. Finally, wash with deionized water until neutral, and dry to obtain the modified activated carbon.

[0047] 4. Take the above enzymatic hydrolysate and add 10 g / L modified activated carbon. Shake and adsorb at 30℃ for 1 h (200 rpm). After filtration, the detoxified solution is obtained, which contains 89.7 g / L glucose and 33.1 g / L xylose.

[0048] 5. Prepare fermentation medium II: Detoxification solution, K₂HPO₄ 4.5 g / L, KH₂PO₄ 6 g / L, NaCl 1 g / L, CaCl₂•2H₂O 20 mg / L, MgSO₄•7H₂O 1.5 g / L, trace element solution 0.2%, biotin 4×10⁻⁶ -4 g / L, Vitamin B1 4×10 -4 g / L.

[0049] 6. Seed culture: Select a single colony of Escherichia coli and inoculate it into LBG liquid medium. Incubate at 37°C and 200 rpm for 12 h. Then transfer it to fresh LBG liquid medium for a second activation for 12 h. Use the activated strain for subsequent fermentation to produce succinic acid.

[0050] 7. Load 2L of fermentation medium II into a 5L fermenter. The final inoculation concentration is OD600=1.0, the initial pH is 7.0, the temperature is 37℃, the aeration rate is 4vvm, the stirring speed is 400rpm, and the culture is aerobic for 12h. Then stop the aeration, reduce the stirring speed to 150rpm, and raise the temperature to 38℃ to start the culture. Use 2M sodium carbonate to maintain the fermentation pH at 6.8 for 96h.

[0051] Example 2

[0052] 1. Corn stalks were crushed and passed through a 40-mesh sieve to obtain corn stalk powder. 500g of the corn stalk powder and 50g of sodium hydroxide powder were added to a pretreatment reactor and stirred at 100 rpm for 15 minutes to ensure thorough mixing. Steam was then introduced, and the mixture was maintained at 120℃ for 30 minutes before the reaction was terminated, producing only 400mL of alkaline treatment solution (black liquid). The treated corn stalks were washed with tap water until the pH became neutral (pH=7.0±0.2), and then dried in a 50℃ oven for 48 hours for later use.

[0053] 2. Pretreated corn stalks were added to 0.05M phosphate buffer (pH=4.8) at an initial solids content of 5%, and Novozymes' Novozymes Cellulase III HS was added. The initial enzyme addition was 1 / 3 of the total enzyme amount (15 FPU / g substrate). Enzymatic hydrolysis was performed at 50℃ and 200 rpm. At 12h and 24h, 5% substrate and the remaining enzyme were added respectively (1 / 3 of the total enzyme amount was added at the same time as each substrate addition). Enzymatic hydrolysis was continued for 72h. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 4500 rpm for 15min, and the supernatant was collected to obtain the enzymatic hydrolysate, which contained 92.8 g / L glucose and 36.7 g / L xylose.

[0054] 3. Immerse the activated carbon in a mixed solution containing ferric nitrate and zinc nitrate, and sonicate for 30 min. The molar ratio of ferric nitrate molecules to zinc nitrate molecules is 3:1, and the total concentration is 0.1 M. Then, under nitrogen protection, irradiate with microwaves at 800 W for 5 min to generate a composite oxide of Fe3O4 and ZnO. Finally, wash with deionized water until neutral, and dry to obtain the modified activated carbon.

[0055] 4. Take the above enzymatic hydrolysate and add 10 g / L modified activated carbon. Shake and adsorb at 30℃ for 1 h (200 rpm). After filtration, the detoxified solution was obtained, which contained 87.6 g / L glucose and 32.4 g / L xylose.

[0056] 5. Prepare fermentation medium II: Detoxification solution, K₂HPO₄ 4.5 g / L, KH₂PO₄ 6 g / L, NaCl 1 g / L, CaCl₂•2H₂O 20 mg / L, MgSO₄•7H₂O 1.5 g / L, trace element solution 0.2%, biotin 4×10⁻⁶ -4 g / L, Vitamin B1 4×10 -4 g / L.

[0057] 6. Seed culture: Select a single colony of Escherichia coli and inoculate it into LBG liquid medium. Incubate at 37°C and 200 rpm for 12 h. Then transfer it to fresh LBG liquid medium for a second activation for 12 h. Use the activated strain for subsequent fermentation to produce succinic acid.

[0058] 7. Load 2L of fermentation medium II into a 5L fermenter. The final inoculation concentration is OD600=1.0, the initial pH is 7.0, the temperature is 37℃, the aeration rate is 4 vvm, the stirring speed is 300 rpm, and the culture is aerobic for 12h. Then stop the aeration, reduce the stirring speed to 100 rpm, and raise the temperature to 38℃ to start the culture. Use 2M sodium carbonate to maintain the fermentation pH at 6.8 for 96h.

[0059] Example 3

[0060] 1. Corn stalks were crushed and passed through a 60-mesh sieve to obtain corn stalk powder. 500g of the corn stalk powder and 50g of sodium hydroxide powder were added to a pretreatment reactor and stirred at 100 rpm for 15 minutes to ensure thorough mixing. Steam was then introduced, and the mixture was maintained at 120℃ for 30 minutes before the reaction was terminated, producing only 400mL of alkaline treatment solution (black liquid). The treated corn stalks were washed with tap water until the pH became neutral (pH=7.0±0.2), and then dried in a 50℃ oven for 48 hours for later use.

[0061] 2. Pretreated corn stalks were added to 0.05M phosphate buffer (pH=4.8) at an initial solids content of 5%, and Novozymes' Novozymes Cellulase III HS was added. The initial enzyme addition was 1 / 3 of the total enzyme amount (20 FPU / g substrate). Enzymatic hydrolysis was performed at 50℃ and 200 rpm. At 12h and 24h, 5% substrate and the remaining enzyme were added respectively (1 / 3 of the total enzyme amount was added at the same time as each substrate addition). Enzymatic hydrolysis was continued for 72h. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 4500 rpm for 15min, and the supernatant was collected to obtain the enzymatic hydrolysate, which contained 94.1 g / L glucose and 38.6 g / L xylose.

[0062] 3. Immerse the activated carbon in a mixed solution containing ferric nitrate and zinc nitrate, and sonicate for 30 min. The molar ratio of ferric nitrate molecules to zinc nitrate molecules is 3:1, and the total concentration is 0.1 M. Then, under nitrogen protection, irradiate with microwaves at 800 W for 5 min to generate a composite oxide of Fe3O4 and ZnO. Finally, wash with deionized water until neutral, and dry to obtain the modified activated carbon.

[0063] 4. Take the above enzymatic hydrolysate and add 10 g / L modified activated carbon. Shake and adsorb at 30℃ for 1 h (200 rpm). After filtration, the detoxified solution is obtained, which contains 90.5 g / L glucose and 34.6 g / L xylose.

[0064] 5. Prepare fermentation medium II: Detoxification solution, K₂HPO₄ 4.5 g / L, KH₂PO₄ 6 g / L, NaCl 1 g / L, CaCl₂•2H₂O 20 mg / L, MgSO₄•7H₂O 1.5 g / L, trace element solution 0.2%, biotin 4×10⁻⁶ -4 g / L, Vitamin B1 4×10 -4 g / L.

[0065] 6. Seed culture: Select a single colony of Escherichia coli and inoculate it into LBG liquid medium. Incubate at 37°C and 200 rpm for 12 hours. Then transfer it to fresh LBG liquid medium for a second activation of 12 hours. Use the activated strain for subsequent fermentation to produce succinic acid.

[0066] 7. Load 2L of fermentation medium II into a 5L fermenter. The final inoculation concentration is OD600=1.0, the initial pH is 7.0, the temperature is 37℃, the aeration rate is 4 vvm, the stirring speed is 500 rpm, and the culture is aerobic for 12h. Then stop the aeration, reduce the stirring speed to 200 rpm, and raise the temperature to 38℃ to start the culture. Use 2M sodium carbonate to maintain the fermentation pH at 6.8 for 96h.

[0067] Example 4

[0068] The ratio of corn stalk powder to sodium hydroxide powder in step 1 of Example 1 was replaced with 8:1 (500g corn stalk powder: 62.5g sodium hydroxide powder). Other operations were the same as in Example 1. The glucose content in the enzymatic hydrolysate obtained in step 2 was measured to be 88.1g / L and the xylose content to be 33.54g / L.

[0069] Example 5

[0070] The ratio of corn stalk powder to sodium hydroxide powder in step 1 of Example 1 was replaced with 12:1 (500g corn stalk powder: 41.7g sodium hydroxide powder). Other operations were the same as in Example 1. The glucose content in the enzymatic hydrolysate obtained in step 2 was measured to be 90.3g / L and the xylose content to be 34.4g / L.

[0071] Example 6

[0072] The steam treatment time in step 1 of Example 1 was changed to 20 min, and other operations were the same as in Example 1. The glucose content in the enzymatic hydrolysate obtained in step 2 was measured to be 81.4 g / L and the xylose content to be 27.7 g / L.

[0073] Example 7

[0074] The steam treatment time in step 1 of Example 1 was changed to 40 min, and other operations were the same as in Example 1. The glucose content in the enzymatic hydrolysate obtained in step 2 was measured to be 93 g / L and the xylose content to be 36.9 g / L.

[0075] Comparative Example 1

[0076] The only difference between this comparative example and Example 1 is that the corn stalks were crushed and passed through a sieve with a mesh size of 40-60 to obtain corn stalk powder. 500g of corn stalk powder and 50g of sodium hydroxide powder were added to 5L of water, and the reaction was stopped after maintaining at 120°C for 60min, producing about 4.5L of alkaline treatment solution (black liquid).

[0077] In summary, the high-temperature steam solid alkali treatment method of the present invention reduces the amount of alkali treatment liquid produced by 11.25 times.

[0078] Comparative Example 2

[0079] Replace the modified activated carbon with commercially available ordinary activated carbon (200 mesh, specific surface area 800 m²). 2 / g), other operations are the same as in Example 1, after filtration, the detoxified solution is obtained, which is found to contain 87.3 g / L glucose and 32.4 g / L xylose.

[0080] Comparative Example 3

[0081] Replace the modified activated carbon with commercially available ordinary activated carbon (200 mesh, specific surface area 800 m²). 2 / g), other operations are the same as in Example 2, after filtration, the detoxified solution is obtained, which is found to contain 85.2 g / L glucose and 30.9 g / L xylose.

[0082] Comparative Example 4

[0083] Replace the modified activated carbon with commercially available ordinary activated carbon (200 mesh, specific surface area 800 m²). 2 / g), other operations are the same as in Example 3, after filtration, the detoxified solution is obtained, and it is found to contain 88.1 g / L glucose and 32.8 g / L xylose.

[0084] Experimental Example 1

[0085] The enzymatic hydrolysate prepared by steps 1 and 2 in Example 1 of this application was processed with different activated carbons.

[0086] (1) Experimental group: Take the enzymatic hydrolysate and add 10 g / L of the modified activated carbon prepared in Example 1. Shake and adsorb at 30°C for 1 h (200 rpm). After filtration, the detoxification treatment solution is obtained.

[0087] (2) Control group: Commercially available ordinary activated carbon (200 mesh, specific surface area 800 m²) was used. 2 / g) to treat the enzyme hydrolysate, add 10g / L ordinary activated carbon to the enzyme hydrolysate, shake and adsorb at 30℃ for 1h (200rpm), and filter to obtain the detoxified treatment solution.

[0088] (3) Blank group: The enzyme hydrolysate was not treated.

[0089] The detoxification effect of each treatment group was tested, and the test results are shown in Table 1 below.

[0090] Table 1

[0091]

[0092] As shown in Table 1 above, the modified activated carbon of this invention has a significantly better adsorption capacity for phenols and furfural than ordinary activated carbon.

[0093] Experimental Example 2

[0094] The experimental groups and design are shown in Table 2 below, comparing the differences between different enzymatic hydrolysis methods, namely, batch feeding and one-time addition of substrate and enzyme.

[0095] Table 2

[0096]

[0097] The sugar yield results are shown in Table 3 below:

[0098] Table 3

[0099]

[0100] As shown in Table 3 above, batch feeding avoids enzyme inhibition caused by the initial high solids content, and dynamic enzyme supplementation maintains cellulase activity, ultimately improving sugar yield.

[0101] Experimental Example 3

[0102] The monosaccharides from Examples 1-3 and Comparative Examples 2-4 were used as carbon sources for fermentation by engineered bacteria capable of producing succinic acid (corresponding to step 7 in Examples 1-3 and the corresponding step in Comparative Examples 2-4). The succinic acid content in the fermentation broth was detected using an UltiMate 3000 high-performance liquid chromatograph (HPLC, Thermo Fisher Scientific), with a Bio-Rad Aminex HPX-87H organic acid column. The results of succinic acid detection are shown in Table 4.

[0103] Table 4

[0104]

[0105] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The present invention provides a method for alkali treatment of straw and a method for preparing monosaccharides from straw. By optimizing the pretreatment, enzymatic hydrolysis, detoxification, and fermentation processes, a corn straw hydrolysate with high monosaccharide content and low production of alkali treatment solution and toxic inhibitors is obtained, which is then used for the efficient fermentation production of succinic acid. The present invention achieves efficient utilization of corn straw resources, has advantages such as simple process and low production of alkali treatment solution and toxic inhibitors, and provides a technical reference for the production of succinic acid from corn straw hydrolysate.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for alkali treatment of straw, characterized in that, The alkaline treatment method includes: (1) Crush the straw to obtain straw powder; (2) The straw powder is mixed with solid alkali to obtain a reaction system. The reaction system is reacted under steam conditions and then separated into solid and liquid to obtain alkali-treated straw and alkali-treated liquid respectively.

2. The alkali treatment method according to claim 1, characterized in that, The mass ratio of the straw powder to the solid alkali in the reaction system is 8-12:1; Preferably, the steam conditions include introducing water vapor into the reaction system to maintain the temperature of the reaction system at 110-130°C, more preferably 120°C; Preferably, the mixing speed is 80-120 rpm, more preferably 100 rpm; Preferably, the reaction time using the water vapor is 20-40 min, more preferably 25-35 min; Preferably, after separating and obtaining the alkali-treated straw, the alkali-treated straw is washed with water and dried to obtain neutral alkali-treated straw.

3. The alkali treatment method according to claim 1, characterized in that, The straw is derived from one or more of corn stalks, cotton stalks, wheat stalks, or sweet sorghum stalks, preferably corn stalks.

4. The alkali treatment method according to claim 1, characterized in that, The solid alkali includes one or more of sodium hydroxide, quicklime, or calcium hydroxide, preferably sodium hydroxide.

5. The alkali treatment method according to claim 1, characterized in that, The straw is crushed and then screened through a sieve. Preferably, the mesh size of the sieve is 40-60 mesh, more preferably 45-55 mesh.

6. A method for preparing monosaccharides using straw, characterized in that, The method includes: (a) The straw is treated using the alkali treatment method for straw according to any one of claims 1-5 to obtain the alkali-treated straw; (b) The alkali-treated straw is mixed with cellulase to obtain a reaction mixture, and the reaction mixture is enzymatically hydrolyzed to obtain an enzymatic hydrolysate containing the monosaccharide; The monosaccharides include one or more of glucose, galactose, fructose, sorbitol, mannitol, or xylose.

7. The method according to claim 6, characterized in that, The monosaccharides include glucose and / or xylose; Preferably, the initial solid content of the straw in the reaction mixture is 5% (W / V). Preferably, in step (b), the cellulase is mixed with the alkali-treated straw in batches to obtain an enzymatic hydrolysis system; Preferably, (b) includes: The straw treated with the first alkali was mixed with the first cellulase and subjected to enzymatic hydrolysis to obtain the first enzymatic hydrolysis system. Add the second alkali-treated straw and the second cellulase to the first enzymatic hydrolysis system to carry out the enzymatic hydrolysis and obtain the second enzymatic hydrolysis system; Add a third alkali-treated straw and a third cellulase to the second enzymatic hydrolysis system to carry out the enzymatic hydrolysis and obtain the third enzymatic hydrolysis system; Preferably, the first cellulase, the second cellulase, and the third cellulase each account for 1 / 3 of the total enzyme amount; Preferably, the solid content of the alkali-treated straw in the first enzymatic hydrolysis system, the second enzymatic hydrolysis system, and the third enzymatic hydrolysis system is 5% (W / V). Preferably, the second alkali-treated straw and the second cellulase are added after the first enzymatic hydrolysis system has reacted for 12 hours; Preferably, the third alkali-treated straw and the third cellulase are added after the first enzymatic hydrolysis system has reacted for 24 hours; Preferably, the enzymatic hydrolysis system continues enzymatic hydrolysis for 72 hours; Preferably, the reaction mixture further includes 0.04-0.06M phosphate buffer, more preferably 0.05M phosphate buffer; Preferably, the ratio of enzyme to substrate in the reaction mixture is 15-20 FPU:1g, more preferably 16-19 FPU:1g; Preferably, the enzymatic hydrolysis temperature is 40-60℃, more preferably 50℃; Preferably, the rotation speed of the enzymatic hydrolysis is 100-250 rpm, more preferably 200 rpm; Preferably, the glucose content in the enzymatic hydrolysate is 92-94 g / L, more preferably 92.5-93.5 g / L; Preferably, the xylose content in the enzymatic hydrolysate is 36-40 g / L, more preferably 37-39 g / L; The method further includes an adsorption step following the enzymatic hydrolysis step, the adsorption step comprising: mixing the enzymatic hydrolysate with activated carbon for detoxification treatment to obtain the monosaccharide.

8. The method according to claim 7, characterized in that, The activated carbon includes modified activated carbon; Preferably, the method for preparing the modified activated carbon includes: mixing activated carbon with a nitrate metal solution to obtain a modified system, and subjecting the modified system to ultrasonic and microwave irradiation treatment to obtain the modified activated carbon; Preferably, the solute in the nitrate metal solution includes one or more of ferric nitrate, copper nitrate, silver nitrate, or zinc nitrate; Preferably, the solute includes ferric nitrate and zinc nitrate; Preferably, the total concentration of ferric nitrate and zinc nitrate is 0.05-0.15M, more preferably 0.1M; Preferably, the molar ratio of ferric nitrate to zinc nitrate is 2-4:1, more preferably 3:1; Preferably, the ultrasonic treatment time is 20-40 minutes, more preferably 30 minutes; Preferably, the power of the microwave irradiation is 700-900W, more preferably 800W; Preferably, the microwave irradiation time is 3-6 minutes, more preferably 5 minutes.

9. The method according to claim 8, characterized in that, The adsorption step further includes an oscillation process; Preferably, the concentration of the modified activated carbon added to the enzymatic hydrolysate is 8-12 g / L, more preferably 10 g / L; Preferably, the temperature of the oscillation treatment is 25-40°C, more preferably 30°C; Preferably, the oscillation treatment time is 0.5-1.5 hours, more preferably 1 hour; Preferably, the rotational speed of the oscillation process is 150-250 rpm, more preferably 200 rpm.

10. A method for preparing succinic acid, characterized in that, The preparation method includes: using the monosaccharide obtained by the method of preparing monosaccharides from straw according to any one of claims 6-9 as a carbon source, and treating it by Escherichia coli fermentation to obtain succinic acid; Preferably, the *Escherichia coli* includes an engineered strain capable of synthesizing succinic acid using the monosaccharide as a substrate.

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