A method for improving sugar production by high-solid enzymatic hydrolysis of straw and ethanol production by fermentation

Abstract

The present invention relates to a method for improving the production of sugar by high-solid enzymatic hydrolysis of straw and the production of ethanol by fermentation. The method uses water to replace the buffer solution to perform high-solid enzymatic hydrolysis of ball-milled straw to produce reducing sugars and simultaneous saccharification and fermentation to produce cellulosic ethanol. At the same time, ultrafine grinding technology is combined with alkali treatment, and mechanical chemical catalysis is used to improve the enzymatic hydrolysis and fermentation efficiency of wood fiber raw materials such as straw. The present invention improves the enzymatic hydrolysis and fermentation efficiency, and solves the problem of low enzymatic hydrolysis efficiency of straw-based wood fiber. In addition, the use of water to replace the buffer solution reduces the use of chemicals in the enzymatic hydrolysis / fermentation process of straw, is beneficial to the separation of later products, reduces the production cost of cellulosic ethanol, improves the economy of the overall process, and has certain industrial application value.

Description

Technical Field

[0001] The invention relates to the field of biomass energy, and in particular to a method for improving the production of sugar by high-solid enzymatic hydrolysis of straw and ethanol by fermentation. Background Art

[0002] As fossil resources needed for human survival and development are increasingly depleted, it is urgent to find new alternative resources. Biomass is considered to be the most likely new energy source to replace fossil resources because of its wide distribution, large output, no pollution, and it is also the only recyclable carbon resource. Therefore, the use of biomass to produce biochemicals has received more and more attention.

[0003] Bioethanol is the most basic chemical intermediate and can be widely used in many industries such as food, medicine, chemical industry, textile, electronics, transportation, etc. It is both a direct product and a basic raw material. Bioethanol is the world's third largest single chemical, with an annual global output of 94.44 million tons in 2021. The market capacity is conducive to strategic depth. The production of bioethanol from straw requires enzymatic hydrolysis and fermentation processes, and pH is a very important influencing factor for enzymatic hydrolysis and fermentation. Changes in pH during enzymatic hydrolysis will affect the structure and function of the enzyme. The activity of microorganisms during fermentation changes to varying degrees (positive or negative) with changes in pH. Therefore, the pH of the reaction system must be controlled to be stable within a certain range during enzymatic hydrolysis and fermentation. At present, most studies use sodium citrate, sodium acetate or sodium phosphate buffer solutions to maintain pH stability in the enzymatic hydrolysis / fermentation system during the reaction. However, the addition of chemicals not only increases the production cost but also increases the difficulty of product separation in the later stage, limiting industrial applications. In addition, since wood fibers such as straw are a highly dense and stubborn structure, they are the main barrier to their enzymatic hydrolysis and fermentation. Mechanical pulverization such as ball milling can effectively reduce the particle size of straw and improve the efficiency of enzymatic hydrolysis. However, the improvement effect of mechanical treatment is still effective, and the longer ball milling time does not utilize the overall energy. Combining mechanical pulverization with chemical catalysis can reduce the ball milling time and the consumption of chemicals, while effectively improving the enzymatic hydrolysis and fermentation effect of straw. However, the effect of different combinations of mechanical pulverization and chemical catalysis on the enzymatic hydrolysis effect remains to be explored.

[0004] Patent application CN115058467A discloses a composite pretreatment and conversion method for improving the glucose and xylose yields of straw. The method performs enzymatic hydrolysis on straw that has been subjected to ball milling and hydrothermal composite pretreatment to achieve high glucose yield, xylose yield and total sugar yield. However, the method adds sodium citrate buffer during the enzymatic hydrolysis process, which increases the use of chemicals.

[0005] Patent application CN106520845 discloses a surfactant recovery technology for the simultaneous saccharification and fermentation process of lignocellulose, which uses lignocellulose pretreated by acid hydrolysis and steam explosion as raw material, and then pre-enzymatically hydrolyzes it with a buffer solution before ethanol fermentation. However, this method uses chemicals for pH adjustment, specifically, the buffer solution is acetic acid-sodium acetate, citric acid-sodium citrate or sodium phosphate.

[0006] In general, existing processes for enzymatic sugar production and bioethanol production use chemicals as buffer media to adjust pH, which adds unnecessary costs to large-scale production and makes subsequent product separation more difficult. Summary of the invention

[0007] In view of the above, the present invention provides a method for improving the production of sugar by high-solid enzymatic hydrolysis of straw and ethanol by fermentation, which can overcome the defects of the prior art.

[0008] A method for improving the production of sugar by high-solid enzymatic hydrolysis of straw and ethanol by fermentation of straw comprises the following steps:

[0009] (1) Pretreatment of straw, prepared by one of the following methods:

[0010] Single ball mill pretreatment: ball mill the straw raw material to obtain pretreated raw material;

[0011] Alkali water boiling and composite ball milling pretreatment: boil the straw raw material in alkali water, dry the straw sample after the reaction is completed; ball mill the sample to obtain the pretreated raw material;

[0012] Ball milling composite alkali boiling pretreatment: ball milling the straw raw material to obtain the pretreated raw material; mixing the pretreated corn straw with sodium hydroxide solution and placing it in a reaction container for boiling, and drying the straw sample for later use after the reaction is completed;

[0013] Alkali-assisted ball milling pretreatment: Sodium hydroxide is mixed with the straw raw material for ball milling pretreatment, and the pretreated straw raw material is washed and dried for later use;

[0014] Alkaline hydrogen peroxide-assisted ball milling pretreatment: prepare a mixture of sodium hydroxide and hydrogen peroxide, mix it with the straw raw material for ball milling pretreatment, wash the pretreated straw raw material with water and then dry it for use;

[0015] (2) adding the pretreated straw material, cellulase and buffer medium of step (1) into a reaction container for enzymatic hydrolysis reaction, and controlling the solid content to be 20% to 30% (w / w); or adding the pretreated straw material, cellulase, brewer's yeast cells and buffer medium of step (1) into a reaction container for enzymatic hydrolysis and simultaneous fermentation, and controlling the solid content to be 20% to 30% (w / w).

[0016] In a specific case, the straw raw material in the above step (1) is at least one of corn straw, cotton straw, wheat straw or rice straw;

[0017] Preferably, the alkaline water boiling composite ball milling pretreatment is specifically performed as follows: alkaline water boiling treatment: coarse corn stalks are mixed with sodium hydroxide solution and placed in a reaction container for boiling, the sodium hydroxide concentration is 5%, the solid-liquid ratio is 1:10, the reaction temperature is 80°C, the reaction time is 2h, and after the reaction, the stalk sample is washed with water to a neutral pH and placed in a 60°C oven for drying; ultrafine grinding treatment: the above sample is ball milled for 60 minutes to obtain a pretreated raw material.

[0018] Preferably, the ball milling composite alkali water boiling pretreatment is specifically operated as follows: ultrafine grinding treatment: ball milling pretreatment is performed on the straw raw material (preferably 45-75 minutes) to obtain a pretreated raw material; alkali water boiling treatment: the pretreated corn straw is mixed with a sodium hydroxide solution and placed in a reaction container for boiling, the sodium hydroxide concentration is 5%, the solid-liquid ratio is 1:10, the reaction temperature is 80°C, the reaction time is 2h, and after the reaction is completed, the straw sample is washed with water until the pH is neutral and placed in a 60°C oven for drying.

[0019] Preferably, the alkaline hydrogen peroxide-assisted ball milling pretreatment is specifically performed as follows: a mixture of sodium hydroxide (0.15 g / g solid) and hydrogen peroxide (0.05 g / g solid) is prepared, the mixture is mixed with corn straw and ball milled for 60 minutes for pretreatment, and the ultrafine powder straw raw material is washed with water (washed to pH neutrality) and dried for later use.

[0020] Preferably, the buffer medium in the above steps is water.

[0021] Preferably, in the above step (2), the enzymatic hydrolysis reaction temperature is 50° C., the stirring speed is 100-200 rpm, and the reaction time is 10-72 h.

[0022] Preferably, in the above step (2), the enzymatic hydrolysis and simultaneous fermentation are carried out at a reaction temperature of 30 to 40° C., a stirring speed of 100 to 200 rpm, and a reaction time of 10 to 72 h.

[0023] In one embodiment, in step (2), the enzymatic hydrolysis and fermentation method is a step-by-step saccharification and co-fermentation, and the specific steps are to add 15-25% solid content of the pretreated straw raw material in step (1) and 10-20 FPU / g straw weight of cellulase into a conical flask, set the reaction temperature to 45-55°C, the rotation speed to 150-250rpm, react for 64-96h, take a sample and centrifuge to obtain a sugar solution, add the sterilized filter membrane (preferably 0.22μm) to the conical flask, add 8-12g / L peptone and 3-8g / L yeast extract, and inoculate yeast cells for fermentation.

[0024] In another embodiment, in step (2), the enzymatic hydrolysis and fermentation method is synchronous saccharification and co-fermentation, and the specific steps are to add 15-25% solid content of the pretreated straw raw material in step (1), 10-20 FPU / g straw weight cellulase, 8-12 g / L peptone, 3-8 g / L yeast extract into a conical flask, and inoculate yeast cells for fermentation.

[0025] In another embodiment, in step (2), the enzymatic hydrolysis and fermentation method is pre-saccharification co-fermentation, and the specific steps are to add 15-25% solid content of the pretreated straw raw material in step (1), 10-20 FPU / g straw weight of cellulase into a conical flask, 150-250 rpm, 45-55°C enzymatic hydrolysis for 10-18h, add 8-12g / L peptone and 3-8g / L yeast extract after cooling, and inoculate yeast cells for fermentation.

[0026] The advantages of the present invention are: (1) water is used instead of buffer as a buffer medium to carry out high-solid enzymatic hydrolysis of straw to produce sugar and simultaneous saccharification and fermentation to produce bioethanol (it can also achieve similar effects as using buffer), thereby reducing the use of chemicals in the enzymatic hydrolysis / fermentation reaction process, avoiding the introduction of exogenous components, and reducing the difficulty of separating fermentation products. According to cost accounting, processing 1 ton of straw material (solid concentration of 20% to 30%) can save at least about 100-200 yuan, effectively reducing the production cost of enzymatic hydrolysis of sugar and bioethanol. (2) The combination of ultrafine grinding and alkali treatment is adopted, through mechanical force and chemical catalysis, while reducing the particle size, lignin is removed, cellulose reaction sites are enhanced, and ineffective adsorption is reduced, thereby effectively improving the product concentration and yield of high-solid enzymatic hydrolysis of straw to produce sugar and simultaneous saccharification and fermentation to produce ethanol, and solving the problem of low efficiency of existing straw enzymatic hydrolysis processes. Among them, the glucose yield of alkaline hydrogen peroxide-assisted ball milling pretreatment can reach 89.88%, and the xylose yield can reach 90.78%, which are significantly better than single ball milling treatment; the ethanol yield of alkali-assisted ball milling pretreatment can reach 72.3%, which is significantly better than single ball milling treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The features and advantages of the present invention will become more apparent with reference to the detailed description of the following drawings:

[0028] Figure 1 The results of enzymatic hydrolysis and sugar production of corn stalks pretreated with ball milling in Example 1 are shown at 20% solid content and 15 FPU / g stalk enzyme addition. Figure 1 It can be seen that the concentrations of glucose, xylose and arabinose in the water and buffer systems are similar, and there is no significant difference in the saccharification results.

[0029] Figure 2The pH change during the enzymatic hydrolysis process was monitored for Example 1. By monitoring the pH change during the enzymatic hydrolysis process, it was found that the pH of the two systems gradually decreased with the extension of the enzymatic hydrolysis time, and changed slowly after 36 hours, and the change trend was almost synchronous, indicating that corn straw can play a buffering role in the enzymatic hydrolysis system.

[0030] Figure 3 The results of simultaneous saccharification and fermentation of ethanol from corn straw pretreated by ball milling in Example 2 at a solid content of 20% and an enzyme addition amount of 15 FPU / g straw. Figure 3 It can be seen that there is no significant difference in ethanol concentration and ethanol yield between the water and buffer systems.

[0031] Figure 4 The pH changes during the simultaneous saccharification and fermentation were monitored for Example 2. The pH of the two systems gradually decreased with the extension of the enzymatic hydrolysis time, and changed slowly after 36 hours, and the change trend was almost synchronous.

[0032] Figure 1-Figure 4 The changes in sugar concentration, ethanol concentration and pH in the results indicate that corn stover contains inorganic ions that can play a buffering role. Therefore, water can completely replace the buffer for enzymatic fermentation, and there is no need to add additional buffer substances to adjust the pH.

[0033] Figure 5 The results of enzymatic sugar production in different pretreatment methods in Examples 4-6 (20% solid content, enzyme addition amount of 20 FPU / g straw). Figure 5 It can be seen that compared with single ball milling pretreatment, the composite pretreatment method of alkali-assisted ball milling has a significant effect on improving the enzymatic hydrolysis and sugar production of straw. The total monosaccharide concentrations of alkaline water boiling followed by ball milling pretreatment, ball milling followed by alkaline water boiling pretreatment and alkaline hydrogen peroxide-assisted ball milling pretreatment can reach 186g / L, 178g / L and 169g / L, respectively. Compared with single ball milling pretreatment, the total monosaccharide concentration is increased by 59% to 75%. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments.

[0035] The straw raw material used in the embodiment of the present invention is corn straw, wherein the cellulose content is 32.6%, the hemicellulose content is 16.5%, and the lignin content is 16.6%, and the straw is crushed to a particle size of 1 mm by a crusher. The coarse straw is subjected to ultrafine powder processing by a nano impact mill, the ball-to-material volume ratio is 2:1, and the ball milling time is 60 minutes.

[0036] Cellulase (Novozymes Cellic CTec3), wherein the enzyme activity of cellulase CTec3 was determined according to the determination method of NREL, and the enzyme activity was measured to be 240 FPU / mL.

[0037] The strain used is Saccharomyces cerevisiae WXY74, which realizes the utilization of xylose by Saccharomyces cerevisiae by introducing genes related to xylose metabolism in Pichia pastoris. After four years of laboratory adaptive evolution, it can simultaneously utilize glucose and xylose to co-ferment ethanol. The seed culture medium is Yeast extract-Peptone-Dextrose medium (YPD liquid culture medium), including 20g / L glucose, 20g / L peptone and 10g / L yeast extract. First, pick a single colony from the plate and put it in a 150mL conical flask, in which the conical flask contains 100mL seed culture medium after high pressure sterilization, and then place the conical flask containing bacteria and seed culture medium in a constant temperature air bath shaker at 200rpm and 30℃ for 24h, then centrifuge at 6000r / min for 10min, remove the supernatant, and retain the yeast cells for subsequent fermentation inoculation.

[0038] Embodiment 1:

[0039] 4.5 g of corn straw pretreated by ball milling for 60 min, 16 g of water (pH = 6.2) were added to a conical flask, the amount of cellulase added was 15 FPU / g of straw, the reaction temperature was set to 50°C, the rotation speed was 200 rpm, and the sample was taken after 72 hours of reaction. After analysis and calculation after high performance liquid chromatography, the glucose concentration was 73.33 g / L, the xylose concentration was 28.39 g / L, and the arabinose concentration was 1.49 g / L.

[0040] Comparative Example 1 was designed accordingly: 4.5 g of corn stover pretreated by ball milling for 60 min, 16 g of sodium citrate buffer (initial pH = 4.8) were added to a conical flask, the amount of cellulase added was 15 FPU / g, the reaction temperature was set to 50°C, the rotation speed was 200 rpm, and the sample was taken after 72 hours of reaction. After analysis and calculation after high performance liquid chromatography, the glucose concentration was 72.49 g / L, the xylose concentration was 27.48 g / L, and the arabinose concentration was 1.05 g / L.

[0041] in, Figure 1 The results are shown in Example 1, where the corn straw was pretreated by ball milling and the solid content was 20% and the enzyme addition amount was 15 FPU / g straw to produce sugar. Figure 1 It can be seen that the concentrations of glucose, xylose and arabinose in the water and buffer systems are similar, and there is no significant difference in the saccharification results.

[0042] Embodiment 2:

[0043] 4.5 g of corn straw pretreated by ball milling for 60 min, 10 g / L peptone, 5 g / L yeast extract, 16 g of water (initial pH = 6.2), 15 FPU / g of cellulase were added to a conical flask, and yeast cells (initial OD 600 =2), the reaction temperature was set at 35°C, the rotation speed was 200 rpm, and samples were taken after 72 hours of reaction. After analysis and calculation by high performance liquid chromatography, the ethanol concentration was 35.38 g / L and the ethanol yield was 52.27%.

[0044] Comparative Example 2 was designed accordingly: 4.5 g of corn stalks pretreated by ball milling for 60 min, 10 g / L peptone, 5 g / L yeast extract, 16 g of sodium citrate buffer (initial pH = 5.5), 15 FPU / g of cellulase were added to a conical flask, and yeast cells (initial OD 600 =2), the reaction temperature was set at 35°C, the rotation speed was set at 200 rpm, and samples were taken after the reaction for 72 hours. After analysis and calculation by high performance liquid chromatography, the ethanol concentration was 34.71 g / L and the ethanol yield was 51.28%.

[0045] in, Figure 3 This is the result of simultaneous saccharification and fermentation of ethanol from corn straw pretreated by ball milling at 20% solid content and 15 FPU / g of straw. Figure 3 It can be seen that there is no significant difference in ethanol concentration and ethanol yield between the water and buffer systems.

[0046] Embodiment 3:

[0047] 4.5 g of alkali-assisted ball-milled pretreated corn straw and 13.5 g of water (initial pH = 6.2) were added to a conical flask and enzymatic hydrolysis was carried out in batches. The initial solid loading was 20%, which was replenished to 25% in 24 hours. The amount of cellulase added was 30 FPU / g straw. The reaction temperature was set to 50°C and the rotation speed was 200 rpm. Samples were taken after 72 hours of reaction. After analysis and calculation after high performance liquid chromatography, the glucose concentration was 148.29 g / L, the xylose concentration was 33.97 g / L, and the arabinose concentration was 2.29 g / L.

[0048] Comparative Example 3 was designed accordingly: 4.5 g of alkali-assisted ball-milling pretreated corn straw and 13.5 g of sodium citrate buffer (initial pH = 4.8) were added to a conical flask, and enzymatic hydrolysis was carried out in batches. The initial solid loading was 20%, which was replenished to 25% in 24 hours. The amount of cellulase added was 30 FPU / g straw, the reaction temperature was set to 50°C, the rotation speed was 200 rpm, and the samples were taken after 72 hours of reaction. After analysis and calculation after high performance liquid chromatography, the glucose concentration was 148.24 g / L, the xylose concentration was 29.73 g / L, and the arabinose concentration was 2.30 g / L.

[0049] The alkali-assisted ball milling pre-treated corn stalk raw material is prepared by the following method:

[0050] (1) Straw pretreatment: ball milling the straw raw material to obtain pretreated raw material;

[0051] (2) placing a sodium hydroxide solution and a straw sample in a reaction container and mixing them with water to boil, washing the alkali-treated straw sample with water, and adjusting the pH to neutral, placing the sample in an oven at 60° C. to dry to constant weight, and finally pre-treating the alkali-treated raw material by ball milling to obtain a pre-treated raw material;

[0052] Embodiment 4:

[0053] Add 4.5g of alkaline boiled composite ball mill pretreated corn straw and 18g of water into a conical flask, add 20FPU / g of cellulase, set the reaction temperature to 50℃, the rotation speed to 200rpm, and take samples after 72h of reaction. After high performance liquid chromatography detection, analysis and calculation showed that the glucose concentration was 152.90g / L, the xylose concentration was 29.96g / L, and the arabinose concentration was 3.01g / L.

[0054] Comparative Example 4 was designed accordingly: 4.5 g of corn straw pretreated by ball milling for 60 min and 18 g of water were added to a conical flask, the amount of cellulase added was 20 FPU / g of straw, the reaction temperature was set to 50°C, the rotation speed was 200 rpm, the reaction was performed for 72 hours, and the samples were taken. After high performance liquid chromatography detection, the glucose concentration was 74.55 g / L, the xylose concentration was 30.50 g / L, and the arabinose concentration was 1.77 g / L.

[0055] The corn stalk raw material pretreated by alkali water boiling and composite ball milling (alkaline water boiling and then ball milling) is prepared by the following method:

[0056] (1) Alkaline boiling treatment: Coarse corn stalks were mixed with sodium hydroxide solution and placed in a reaction vessel for boiling. The sodium hydroxide concentration was 5%, the solid-liquid ratio was 1:10, the reaction temperature was 80°C, and the reaction time was 2h. After the reaction, the stalk samples were washed with water until the pH was neutral and dried in an oven at 60°C.

[0057] (2) Ultrafine grinding treatment: The above sample was ball-milled for 60 min to obtain a pre-treated raw material;

[0058] Embodiment 5:

[0059] Add 4.5g ball-milled composite alkali-boiled pretreated corn straw and 18g water into a conical flask, add 20FPU / g of cellulase, set the reaction temperature to 50°C and the rotation speed to 200rpm, take samples after 72h of reaction, and analyze and calculate after high performance liquid chromatography detection. The glucose concentration is 161.38g / L, the xylose concentration is 14.95g / L, and the arabinose concentration is 1.23g / L.

[0060] Comparative Example 5 was designed accordingly: 4.5 g of corn straw pretreated by ball milling for 60 min and 18 g of water were added to a conical flask, the amount of cellulase added was 20 FPU / g of straw, the reaction temperature was set to 50°C, the rotation speed was 200 rpm, and the samples were taken after 72 hours of reaction. After analysis and calculation after high performance liquid chromatography, the glucose concentration was 74.55 g / L, the xylose concentration was 30.50 g / L, and the arabinose concentration was 1.77 g / L.

[0061] The corn stalks pretreated by ball milling and alkaline boiling (ball milling followed by alkaline boiling) are prepared by the following method:

[0062] (1) Ultrafine grinding treatment: The straw raw material is pretreated by ball milling (60 min) to obtain pretreated raw material;

[0063] (2) Alkaline boiling treatment: The pretreated corn stalks were mixed with sodium hydroxide solution and placed in a reaction vessel for boiling. The sodium hydroxide concentration was 5%, the solid-liquid ratio was 1:10, the reaction temperature was 80°C, and the reaction time was 2h. After the reaction, the stalk samples were washed with water until the pH was neutral and dried in an oven at 60°C.

[0064] Embodiment 6:

[0065] 4.5 g of alkaline hydrogen peroxide-assisted ball milling pretreated corn straw and 18 g of water were added to a conical flask. The amount of cellulase added was 20 FPU / g of straw. The reaction temperature was set to 50°C and the rotation speed was 200 rpm. Samples were taken after 72 hours of reaction. After analysis and calculation after high performance liquid chromatography, the glucose concentration was 138.38 g / L, the xylose concentration was 27.96 g / L, and the arabinose concentration was 2.73 g / L.

[0066] Comparative Example 6 was designed accordingly: 4.5 g of alkali-assisted ball-milled pretreated corn straw and 18 g of water were added to a conical flask, the amount of cellulase added was 20 FPU / g of straw, the reaction temperature was set to 50°C, the rotation speed was 200 rpm, the reaction was performed for 72 hours, and the samples were taken. After high performance liquid chromatography detection, the glucose concentration was 93.25 g / L, the xylose concentration was 26.99 g / L, and the arabinose concentration was 1.78 g / L.

[0067] The alkaline hydrogen peroxide-assisted ball milling pre-treated corn stalk raw material is prepared by the following method:

[0068] A mixture of sodium hydroxide (0.15 g / g solid) and hydrogen peroxide (0.05 g / g solid) of a certain concentration was prepared, and the mixture was mixed with corn straw for ball milling for 60 min pretreatment, and the ultrafine powder straw raw material was washed with water (washed with water to neutral pH) and dried for use;

[0069] Embodiment 7:

[0070] 4.5 g of alkali-assisted ball-milled corn straw, 18 g of water, and 20 FPU / g of cellulase were added to a conical flask. The reaction temperature was set to 50 °C and the speed was 200 rpm. The reaction was continued for 72 h. The sugar solution was obtained by centrifugation. The filter membrane (0.22 μm) was sterilized and added to the conical flask. 10 g / L peptone and 5 g / L yeast extract were added. Saccharomyces cerevisiae cells (initial OD 600 =2), the reaction temperature was set at 35°C, the rotation speed was 200 rpm, and samples were taken after 72 hours of reaction. After analysis and calculation by high performance liquid chromatography, the ethanol concentration was 53.80 g / L and the ethanol yield was 72.50%.

[0071] Comparative Example 7 was designed accordingly: 4.5 g of corn straw pretreated by ball milling for 60 min, 18 g of water, 20 FPU / g of cellulase were added to a conical flask, the reaction temperature was set to 50 ° C, the speed was set to 200 rpm, the reaction was performed for 72 h, and the sugar solution was obtained by centrifugation. The filter membrane (0.22 μm) was sterilized and added to the conical flask, 10 g / L of peptone and 5 g / L of yeast extract were added, and Saccharomyces cerevisiae cells (initial OD 600 =2), the reaction temperature was set at 35°C, the rotation speed was 200 rpm, and samples were taken after 72 hours of reaction. After analysis and calculation by high performance liquid chromatography, the ethanol concentration was 53.80 g / L and the ethanol yield was 72.50%.

[0072] The alkali-assisted ball milling pre-treated corn stalk raw material is prepared by the following method:

[0073] Sodium hydroxide (0.12 g / g solid) was mixed with corn straw and ball-milled for 60 min for pretreatment. The ultrafine straw powder raw material was washed with water (washed to pH neutrality) and dried for later use.

[0074] Embodiment 8:

[0075] 4.5 g of corn straw pretreated by alkali-assisted ball milling for 60 min, 10 g / L peptone, 5 g / L yeast extract, 16 g of water (initial pH = 6.2), and 15 FPU / g of cellulase were added to a conical flask. Saccharomyces cerevisiae cells (initial OD 600=2), the reaction temperature was set at 35°C, the rotation speed was set at 200 rpm, and samples were taken after the reaction for 72 hours. After analysis and calculation by high performance liquid chromatography, the ethanol concentration was 53.60 g / L and the ethanol yield was 72.30%.

[0076] Comparative Example 8 was designed accordingly: 4.5 g of corn straw pretreated by ball milling for 60 min, 10 g / L peptone, 5 g / L yeast extract, 16 g of water (initial pH = 6.2), 15 FPU / g of cellulase were added to a conical flask, and Saccharomyces cerevisiae cells (initial OD 600 =2), the reaction temperature was set at 35°C, the rotation speed was set at 200 rpm, and samples were taken after the reaction for 72 hours. After analysis and calculation by high performance liquid chromatography, the ethanol concentration was 35.38 g / L and the ethanol yield was 52.27%.

[0077] The alkali-assisted ball milling pre-treated corn stalk raw material is prepared by the following method:

[0078] Sodium hydroxide (0.12 g / g solid) was mixed with corn straw and ball-milled for 60 min for pretreatment. The ultrafine straw powder raw material was washed with water (washed to pH neutrality) and dried for later use.

[0079] Embodiment 9:

[0080] 4.5 g of corn straw pretreated by alkali-assisted ball milling for 60 min, 16 g of water (initial pH = 6.2), 15 FPU / g of cellulase were added to a conical flask, and the enzyme was hydrolyzed at 200 rpm and 50 °C for 14 h. After cooling, 10 g / L of peptone and 5 g / L of yeast extract were added, and Saccharomyces cerevisiae cells (initial OD 600 =2), the reaction temperature was set at 35°C, the rotation speed was set at 200 rpm, and samples were taken after the reaction for 72 hours. After analysis and calculation by high performance liquid chromatography, the ethanol concentration was 61.10 g / L and the ethanol yield was 82.30%.

[0081] Comparative Example 9 was designed accordingly: 4.5 g of corn straw pretreated by ball milling for 60 min, 16 g of water (initial pH = 6.2) were added to a conical flask, the amount of cellulase added was 15 FPU / g straw, 200 rpm, 50 ° C enzymolysis for 14 h, 10 g / L peptone and 5 g / L yeast extract were added after cooling, and Saccharomyces cerevisiae cells (initial OD 600 =2), the reaction temperature was set at 35°C, the rotation speed was 200 rpm, and samples were taken after reaction for 72 hours. After analysis and calculation by high performance liquid chromatography, the ethanol concentration was 43.80 g / L and the ethanol yield was 59.00%.

[0082] The alkali-assisted ball milling pre-treated corn stalk raw material is prepared by the following method:

[0083] Sodium hydroxide (0.12 g / g solid) was mixed with corn straw and ball-milled for 60 min for pretreatment. The ultrafine straw powder raw material was washed with water (washed to pH neutrality) and dried for later use.

Claims

1. A method for improving the production of sugar by high-solid enzymatic hydrolysis of straw and ethanol by fermentation, characterized in that The following steps are involved: (1) Straw pretreatment: Mix the corn straw raw material with sodium hydroxide in a weight ratio of 0.12:1 and perform ball milling pretreatment, and wash the ultrafine powdered straw raw material with water until it is neutral and dry it for later use; (2) performing enzymatic fermentation in the form of pre-saccharification and co-fermentation: adding 15-25% solid content of the pre-treated straw raw material in step (1), water with an initial pH of 6.2, and 10-20 FPU / g of straw weight of cellulase into a conical flask, performing enzymatic hydrolysis at 150-250 rpm and 45-55°C for 10-18 h, adding 8-12 g / L peptone and 3-8 g / L yeast extract after cooling, and inoculating an initial OD 600 =2 yeast cell fermentation; In step (2), the fermentation temperature is 30-40°C, the stirring speed is 100-200 rpm, and the reaction time is 12-72 h.

2. The method according to claim 1, characterized in that The ball milling pretreatment time is 60 min.

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