A production method for increasing ethanol yield by treating corn straw with organic acid

Abstract

The present invention relates to a production method for increasing ethanol yield by treating corn straw with organic acid. The organic acid treatment of the corn straw comprises performing a staged heat treatment on the corn straw using a composite acid solution composed of citric acid and acetic acid. The staged heat treatment comprises first heat treatment at 40-60°C for 0.5-1 hour, and then heating to 70-90°C for 1.5-2.5 hours. By pretreating the corn straw with the composite organic acid according to the present invention, the degradation efficiency of the lignocellulose in the corn straw is effectively increased, more cellulose is retained, and the generation of toxic inhibitors is reduced, thereby reducing the inhibitory effect on cellulase and yeast in subsequent enzymatic hydrolysis and fermentation processes. Consequently, the yield of the prepared oxygenated organic compound ethanol is increased.

Description

Technical Field

[0001] The invention relates to the technical field of producing oxygen-containing organic compound ethanol by fermenting cellulose, and in particular to a production method for increasing ethanol yield by treating corn straw with organic acid. Background Art

[0002] Crop straw is a valuable biomass energy resource in agricultural ecosystems, boasting enormous yields. Corn straw serves as a crucial resource for food, industrial, and agricultural production. It not only contains a wealth of nutrients and usable chemical compounds, but can also be used as a feedstock for livestock. Its primary component is lignocellulose, of which cellulose and hemicellulose are key carbohydrates for fermentation to produce ethanol. Lignocellulose is composed of cellulose, hemicellulose, and lignin. Cellulose, a macromolecular polysaccharide composed of glucose, is insoluble in water and common organic solvents. It is the most widespread and abundant polysaccharide in nature. It is typically combined with hemicellulose and lignin to form a dense structure. The manner and degree of this combination pose certain challenges to the comprehensive utilization of corn straw, necessitating pretreatment of the corn straw to open up its spatial structure for subsequent utilization.

[0003] Pretreatment is a common means to open the structure of lignocellulose. Common methods include physical, chemical, biological or combined pretreatment. Physical methods include microwaves, steam explosion and other methods, which improve the pretreatment effect by expanding the contact area, but the degree of improvement is limited. Chemical pretreatment includes acid and alkali pretreatment, which can only remove a single component and cause pollution to the environment. Biological pretreatment includes pretreatment using Trichoderma reesei, Aspergillus niger, etc., but the growth cycle is too long. The order of combined pretreatment affects the pretreatment effect, and the process is complicated.

[0004] Organic acid pretreatment has low pollutant emissions and is more environmentally friendly. Its unique properties allow it to selectively dissolve the hemicellulose and lignin components of lignocellulose, exposing more cellulose components, leading to its widespread application. However, even with organic acid pretreatment, toxic inhibitors can be produced that inhibit subsequent enzymatic hydrolysis and fermentation, resulting in reduced sugar and ethanol production efficiency. Summary of the Invention

[0005] The present invention aims to provide a corn straw pretreatment method, which reduces pretreatment costs, reduces the generation of inhibitors, and effectively improves the saccharification rate in the subsequent enzymatic saccharification process.

[0006] Another object of the present invention is to provide a method for increasing the yield of ethanol from corn straw fermentation by pretreating the corn straw. Specifically, the method involves using a composite acid combined with staged heat treatment to produce a saccharified liquid, which is then fermented to produce ethanol using immobilized yeast. This method effectively reduces the formation of inhibitors in the saccharified liquid and increases ethanol yield.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A corn straw pretreatment method is characterized in that: a composite acid solution composed of citric acid and acetic acid is used to perform a segmented heat treatment on the corn straw, wherein the segmented heat treatment is first performed at 40-60° C. for 0.5-1 hour, and then the temperature is raised to 70-90° C. for 1.5-2.5 hours.

[0009] Furthermore, the composite acid solution is prepared by dissolving citric acid and acetic acid in water, and the dosage ratio of citric acid, acetic acid and water is 0.8-1.5 g: 1.5-2 mL: 100 mL.

[0010] Furthermore, the solid-liquid ratio of the composite acid solution to the corn straw is 1:8-10.

[0011] In the process of pretreating corn straw with single citric acid or single acetic acid, a higher treatment temperature is required and more inhibitors are produced, which inhibit the subsequent cellulase activity without any washing and detoxification treatment.

[0012] The present invention adopts citric acid and acetic acid of specific concentrations to form a composite acid solution. Through staged low-temperature heat treatment, the first stage of heat treatment promotes the penetration of organic acid and the dissolution of hemicellulose, and the second stage of heat treatment strengthens the removal of lignin. The overall heat treatment temperature is low, and the amount of single organic acid used is reduced. While reducing the pretreatment cost, it avoids the rapid degradation of sugars caused by high temperature to generate more inhibitors, improves the enzymatic hydrolysis efficiency of the pretreated straw, and increases the yield of glucose and xylose.

[0013] Although the composite pretreatment of straw with acetic acid and citric acid effectively improves the enzymatic saccharification effect of cellulase, it will lead to excessive acetic acid concentration in the saccharification liquid. Acetic acid will penetrate the yeast cell membrane, damage the yeast cells, inhibit fermentation, and reduce ethanol production.

[0014] In the present invention, SA-PVA composite immobilized yeast is used to improve the stability and stress resistance of yeast. 2+It can react with free acetic acid to form acetate, reducing the acetic acid content. In addition, the composite embedded immobilized yeast effectively increases the path for acetic acid to reach the yeast, slows down the diffusion of acetic acid, reduces the acetic acid concentration that yeast cells are exposed to, and reduces the inhibition of acetic acid on yeast under multiple paths, effectively balancing the protective effect on yeast and the mass transfer efficiency of the substrate, thereby increasing the yield of sodium ethanol.

[0015] In addition, the SA-PVA composite immobilization system ensures that the immobilized yeast has suitable swelling properties, which not only ensures excellent mass transfer efficiency of the substrate in the immobilized yeast, but also provides excellent repeatability of the immobilized yeast.

[0016] A method for increasing ethanol production by pretreating corn straw with organic acid, characterized by comprising the following steps:

[0017] (1) Pretreatment: A composite acid solution consisting of citric acid and acetic acid is added to the corn straw and the corn straw is subjected to segmented heat treatment;

[0018] (2) Enzymatic saccharification: Cellulase is added to the pretreated corn straw for enzymatic hydrolysis to prepare saccharification liquid;

[0019] (3) Immobilized yeast: Sodium alginate and polyvinyl alcohol are dissolved in water to form a mixed liquid. After sterilization, yeast sludge is added and a curing agent is added. The mixture is cured for 2 to 3 hours.

[0020] (4) Fermentation to produce ethanol: Immobilized yeast is added to the saccharification liquid for fermentation to produce ethanol.

[0021] Furthermore, the ratio of citric acid, acetic acid and water in the composite acid solution of step (1) is 0.8-1.5 g: 1.5-2 mL: 100 mL.

[0022] Furthermore, the solid-liquid ratio of the composite acid solution to the corn straw is 1:8-10.

[0023] Furthermore, the staged heat treatment is first heat treated at 40-60°C for 0.5-1h, and then heated to 70-90°C for 1.5-2.5h.

[0024] Furthermore, the enzymatic saccharification in step (2) is to add the pretreated corn straw to a citric acid buffer solution with a pH of 4.8, sterilize it under high pressure at 121°C for 20 minutes, add cellulase, perform enzymatic hydrolysis at 50°C and 160 rpm for 72 hours, centrifuge to obtain the supernatant, and condense it by rotary evaporation to obtain a saccharified liquid with a glucose concentration of 90-120 g / L.

[0025] Furthermore, in step (3), the concentration of sodium alginate is 3%, the concentration of polyvinyl alcohol is 2%, the curing agent is a mixed solution of calcium chloride and boric acid, wherein the amount ratio of calcium chloride, boric acid and water is 2-2.5 g: 2-2.5 g: 100 mL, and the number of live yeast cells in the bacterial sludge is 2.5×10 9 / mL.

[0026] Furthermore, the fermentation temperature in step (4) is 30-35° C., and the fermentation time is 36-48 hours.

[0027] Most specifically, a method for pretreating corn straw to increase ethanol production is characterized by comprising the following steps:

[0028] (1) Pretreatment: The corn stalks were cut into small pieces and crushed, passed through a 40-mesh sieve, and dried at 60°C to constant weight. The corn stalks were subjected to a staged heat treatment using a composite acid formed by dissolving citric acid and acetic acid in water. The ratio of citric acid, acetic acid, and water in the composite acid solution was 0.8-1.5 g: 1.5-2 mL: 100 mL. The staged heat treatment was first performed at 40-60°C for 0.5-1 h, and then heated to 70-90°C for 1.5-2.5 h.

[0029] (2) Enzymatic saccharification: The pretreated corn straw was added to a citric acid buffer solution with a pH of 4.8, sterilized at 121°C for 20 min, and then incubated with cellulase. The solution was enzymatically hydrolyzed at 50°C and 160 rpm for 72 h. The supernatant was centrifuged and concentrated by rotary evaporation to a saccharification solution with a glucose concentration of 90-120 g / L.

[0030] (3) Immobilized yeast: Saccharomyces cerevisiae was activated and subcultured in YPD medium. The bacterial sludge was obtained by centrifugation. Sodium alginate and polyvinyl alcohol were dissolved in water to form a mixed liquid. The mixture was sterilized and the bacterial sludge was added. The number of live yeast cells in the bacterial sludge was 2.5×10 9 / mL, drop the curing agent to form a curing system, put it into physiological saline after curing for 2-3 hours, and put it into a 4°C refrigerator for standby use. In the curing system, the concentration of sodium alginate is 3%, the concentration of polyvinyl alcohol is 2%, and the curing agent is a mixed solution of calcium chloride and boric acid, wherein the concentrations of calcium chloride and boric acid are both 2.5%. The dosage ratio of the mixed liquid to the curing agent is 1:8-10, and the number of live yeast cells in the bacterial sludge is 2.5×10 9 / mL;

[0031] (4) Fermentation to produce ethanol: The immobilized yeast was inoculated into the saccharification solution prepared in step (2) and fermented at 30-35°C for 48 hours.

[0032] The present invention has the following technical effects:

[0033] The present invention uses composite organic acids to pretreat corn straw, effectively improving the degradation efficiency of lignocellulose in corn straw, retaining more cellulose, while reducing the generation of toxic inhibitors, reducing the inhibitory effects on cellulase and yeast in subsequent enzymatic hydrolysis and fermentation processes, and increasing the yield of the prepared oxygen-containing organic compound ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Structural characterization of corn stover with different pretreatments.

[0035] Figure 2 : XRD patterns and Fourier transform infrared spectra of pretreated straw.

[0036] Figure 3 : Effects of compound acid cycle pretreatment on inhibitor content in straw.

[0037] Figure 4 : Sugar yield of straw pretreated by composite acid cycle.

[0038] Figure 5 : Effects of different initial sugar concentrations, temperatures and times on fermentation. DETAILED DESCRIPTION

[0039] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0040] The materials used in the present invention are as follows:

[0041] Corn straw was obtained from the experimental field of Jilin Agricultural University in Changchun, Jilin Province, China; citric acid and acetic acid were obtained from Shanghai MacLean Biochemical Technology Co., Ltd.; and cellulase was obtained from Novozymes Biotechnology Co., Ltd.

[0042] Sodium alginate was obtained from Tianjin Damao Chemical Reagent Factory; polyvinyl alcohol, calcium chloride, and boric acid were all obtained from Sinopharm Chemical Reagent Co., Ltd.

[0043] The fermentation used was the yeast Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) was purchased from Guangdong Provincial Microbiological Culture Collection Center with the collection number GDMCC NO.2.89.

[0044] YPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, and 1 L deionized water (autoclaved at 115°C for 30 min).

[0045] Ethanol fermentation medium: peptone 10 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 2 g / L, saccharification solution 1 L (115 ° C, 30 min high pressure sterilization).

[0046] Example 1

[0047] A corn straw pretreatment method comprises the following steps:

[0048] The corn stalks were cut into small segments and then crushed, passed through a 40-mesh sieve, and dried at 60°C to constant weight. A composite acid formed by dissolving citric acid and acetic acid in water was used to perform a staged heat treatment on the corn stalks. The ratio of citric acid, acetic acid, and water in the composite acid solution was 1.0 g:1.5 mL:100 mL. The staged heat treatment was first performed at 50°C for 0.5 h, and then heated to 80°C for 2 h.

[0049] The hemicellulose removal rate of the corn straw pretreated according to the method of Example 1 was 66.78%, and the lignin removal rate was 45.86%.

[0050] Pretreated straw Figure 1 As shown, scanning electron microscopy shows that (a) and (b) are the original straw surfaces observed to be smooth and compact at magnifications of 500 times and 2000 times, respectively. (c) and (d) are the citric acid pretreatment conditions at the corresponding magnifications. The surface of the treated corn straw becomes uneven, with irregular fractures and holes, which is more conducive to the subsequent penetration of cellulase.

[0051] Figure 2 The XRD patterns and Fourier transform infrared spectra of the corn straw before and after pretreatment. The crystallinity of cellulose is an important indicator of the changes in the structure and composition of corn straw. From the XRD pattern, it can be seen that the crystallinity of the corn straw after pretreatment increased from 43.55% to 49.94%. This change is because the dissolution of hemicellulose and lignin leads to an increase in the relative content of cellulose after pretreatment, thereby improving the overall crystallinity of the corn straw. The Fourier transform infrared spectrum shows that the 1720cm -1 The disappearance of the absorption peak at 1604 cm is due to the cleavage of the acetyl group in the hemicellulose structure and the phenyl ester bond between hemicellulose and lignin, indicating that the hemicellulose has been removed. -1 The change at 1515 cm is due to the vibration of aromatic skeletons such as lignin and the stretching of CO bonds; -1 The C-C bond stretching of lignin was observed at 1253 cm -1 The 1168 cm -1 The peak at 1056 cm corresponds to the typical structure of xylan, indicating that the lignin content of corn straw is reduced after pretreatment. -1 The absorption peaks of -OH and COC in the sugar unit are at 898 cm-1 The absorption peak at 10 indicates that the β-glycosidic bond between sugar units is broken, indicating a relative increase in cellulose content. The above results show that citric acid pretreatment of corn straw not only removes hemicellulose but also removes part of the lignin, exposing the cellulose and making it better for the next step of utilization.

[0052] Comparative Example 1

[0053] Compared with Example 1, a single citric acid pretreatment was used, and the pretreatment was performed according to the parameters that were optimal for subsequent enzymatic saccharification during the single citric acid pretreatment process, as follows:

[0054] The added concentration of citric acid was 4%, the heating treatment temperature was 125°C, and the pretreatment time was 3 h.

[0055] Comparative Example 2

[0056] Compared with Example 1, single acetic acid was used for pretreatment, and pretreatment was performed according to the parameters that were optimal for subsequent enzymatic saccharification during single acetic acid pretreatment, specifically as follows: the added acetic acid concentration was 3%, the pretreatment temperature was 140° C., and the pretreatment time was 60 min.

[0057] Comparative Example 3

[0058] Compared with Example 1, the heating process adopts one-stage heating, specifically keeping warm at 80° C. for 3 hours.

[0059] Determination of inhibitor content in pretreated straw:

[0060] The contents of formic acid, acetic acid, furfural and pentahydroxymethylfurfural (5-HMF) in the inhibitors were determined by high performance liquid chromatography (HPLC) (Agilent OpenLAB CDS) equipped with a UV detector and a C18 column.

[0061] The determination of furfural and 5-HMF used methanol and water in a ratio of 1:9 as the mobile phase with isocratic elution, the detection wavelength was 280 nm, and the flow rate was 0.9 mL / min.

[0062] Formic acid and acetic acid were determined by isocratic elution using a mobile phase of 0.02 mol / L potassium dihydrogen phosphate and methanol in a ratio of 9:1. The detection wavelength was 210 nm and the flow rate was 0.9 mL / min. All mobile phases and samples were filtered through a 0.22 μm filter.

[0063] The results of inhibitor content change detection are as follows Figure 3As shown. (a)-(d) correspond to Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively. It can be seen that in Example 1, the production of formic acid, acetic acid, furfural, and 5-HMF was the lowest during the initial and cyclic processes. During the first pretreatment, under optimal pretreatment conditions, the levels of formic acid, acetic acid, furfural, and 5-hydroxymethylfurfural were 26.67 g / L, 55.14 g / L, 0.27 g / L, and 0.18 g / L, respectively. Although the production of toxic inhibitors in Comparative Example 3 was also low, its pretreatment effect on corn straw was poor, resulting in suboptimal enzymatic saccharification efficiency.

[0064] The straw pretreated in Example 1 and the comparative examples was enzymatically hydrolyzed to prepare fermentation medium (saccharification liquid):

[0065] The pretreated corn straw was added to a pH 4.8 citric acid buffer solution and autoclaved at 121°C for 20 minutes. Cellulase was then added and enzymatically hydrolyzed at 50°C and 160 rpm for 72 hours. The supernatant was then centrifuged and the glucose and xylose contents in the supernatant were measured using a glucose and xylose assay kits.

[0066] Figure 4 Figure 3 shows the changes in sugar yield (measured in glucose) after repeated pretreatment of corn straw with the recycled composite acid solution and subsequent enzymatic hydrolysis. Under optimal pretreatment conditions, six pretreatment cycles with the composite acid solution were performed. The number of cycles was negatively correlated with the effectiveness of citric acid pretreatment. The glucose yield in the first pretreatment was 6.61 g / g straw, and after six cycles, the glucose yield was 0.37 g / g straw. This is still higher than the sugar yield of untreated corn straw.

[0067] Example 2

[0068] A corn straw pretreatment method comprises the following steps:

[0069] The corn stalks were cut into small segments and then crushed, passed through a 40-mesh sieve, and dried at 60°C to constant weight. A composite acid formed by dissolving citric acid and acetic acid in water was used to perform a staged heat treatment on the corn stalks. The ratio of citric acid, acetic acid, and water in the composite acid solution was 0.8 g:2 mL:100 mL. The staged heat treatment was first performed at 60°C for 0.5 h, and then heated to 70°C for 2.5 h.

[0070] The hemicellulose removal rate of the pretreated corn straw was 65.87%, and the lignin removal rate was 44.72%.

[0071] Example 3

[0072] A corn straw pretreatment method comprises the following steps:

[0073] The corn stalks were cut into small segments and then crushed, passed through a 40-mesh sieve, and dried at 60°C to constant weight. A composite acid formed by dissolving citric acid and acetic acid in water was used to perform a staged heat treatment on the corn stalks. The ratio of citric acid, acetic acid, and water in the composite acid solution was 1 g:1.5 mL:100 mL. The staged heat treatment was first performed at 40°C for 1 hour, and then heated to 90°C for 1.5 hours.

[0074] The hemicellulose removal rate of the pretreated corn straw was 64.54%, and the lignin removal rate was 44.91%.

[0075] Example 4

[0076] A method for pretreating corn straw to increase ethanol production, characterized by comprising the following steps:

[0077] (1) Pretreatment: The corn stalks were cut into small pieces and crushed, passed through a 40-mesh sieve, and dried at 60°C to constant weight. The corn stalks were subjected to a staged heat treatment using a composite acid formed by dissolving citric acid and acetic acid in water. The ratio of citric acid, acetic acid, and water in the composite acid solution was 1.0 g:1.5 mL:100 mL. The staged heat treatment was first performed at 50°C for 0.5 h, and then heated to 80°C for 2 h.

[0078] (2) Enzymatic saccharification: The pretreated corn straw was added to a citric acid buffer solution with a pH of 4.8, sterilized at 121°C for 20 min, and then incubated with cellulase. The solution was enzymatically hydrolyzed at 50°C and 160 rpm for 72 h. The supernatant was centrifuged and concentrated by rotary evaporation to a saccharification solution with a glucose concentration of 90-120 g / L.

[0079] (3) Immobilized yeast: Saccharomyces cerevisiae was activated and subcultured in YPD medium, and the bacterial sludge was obtained by centrifugation. Sodium alginate and polyvinyl alcohol were dissolved in water to form a mixed liquid, which was sterilized. The bacterial sludge was added and a curing agent was added dropwise to form a curing system. After curing for 3 hours, the mixture was placed in physiological saline and placed in a refrigerator at 4°C for standby use. In the curing system, the concentration of sodium alginate was 3%, the concentration of polyvinyl alcohol was 2%, and the curing agent was a mixed solution of calcium chloride and boric acid, wherein the concentrations of calcium chloride and boric acid were both 2.5%. The number of live yeast cells in the bacterial sludge was 2.5×10 9 / mL;

[0080] (4) Fermentation to produce ethanol: The immobilized yeast was inoculated into the saccharification solution prepared in step (2) and fermented at 30°C for 48 hours.

[0081] The Young's modulus, representing the mechanical strength of the immobilized spheres, was measured using a universal mechanical testing machine. The initial diameter of the immobilized spheres was determined, and the spheres were liquefied in a liquefaction solution at 30°C for 1 hour. The diameter after liquefaction was measured, and the swelling rate was calculated. The immobilized spheres were then placed in sterile water and shaken at 160 rpm at 30°C for 24 hours. The mass of the swollen immobilized spheres was measured, and the surface moisture was removed. The mass of the dried immobilized spheres was then weighed, and the swelling degree was calculated.

[0082]

[0083]

[0084] Conditions were optimized by measuring the mechanical strength of the immobilized particles, residual sugar in the culture medium after 6 hours of fermentation, and the swelling rate and degree of solubility. Excessively low sodium alginate concentrations resulted in low mechanical strength and potentially caused bacterial leakage. Excessively high sodium alginate concentrations led to the formation of calcium bridges between the alginate molecules in the system, resulting in a denser structure. This resulted in a dense exterior structure, while the interior was not fully reacted, leading to lower mechanical strength. Excessively high polyvinyl alcohol concentrations may have lowered mechanical strength, possibly due to increased rigidity and poorer toughness. The curing agent in the SA-PVA dual-crosslink system is a mixture of calcium chloride and boric acid. At low concentrations, the immobilized particles did not fully react, resulting in poor performance. Excessive concentrations resulted in a dense outer shell and the formation of a nutrient gradient within. Short curing times can also lead to incomplete reaction. After refining the sodium alginate concentration and repeating the experiment, the optimal conditions were determined to be 3% SA, 2% PVA, 2.5% curing agent, and 3 hours of curing time. At this time, the residual sugar content after 6 hours of fermentation was 16.2 g / L; the expansion rate was 55%; the swelling degree was 0.37 g / g; and the mechanical strength of the immobilized brewer's yeast under the optimal conditions, characterized by Young's modulus, was 1085.14 MPa.

[0085] Effects of different initial sugar concentrations, temperatures, and times on fermentation Figure 5As shown, fermentation experiments were conducted at initial sugar concentrations of 30 g / L, 60 g / L, 90 g / L, 120 g / L, and 150 g / L, and residual sugar levels were measured after 48 hours of fermentation. Ethanol yields were low at lower initial sugar concentrations, while higher initial sugar concentrations inhibited bacterial growth. At an initial sugar concentration of 90 g / L, ethanol yield reached 39.75 g / L, indicating complete glucose consumption with a small amount of xylose remaining. Fermentation at different temperatures (20°C, 25°C, 30°C, 35°C, and 40°C) showed that yeast growth was extremely slow at lower or higher temperatures, resulting in low ethanol yields. Yields were higher at temperatures between 25 and 35°C, with the highest yield reaching 39.75 g / L at 30°C, indicating complete glucose consumption. Fermentation was carried out under the conditions of 12h, 24h, 36h, 48h, 60h and 72h. The ethanol yield was low when the time was shorter. As time increased, the ethanol yield increased significantly, reaching the highest at 48h. Finally, the ethanol yield did not increase significantly when the fermentation time was further extended. Therefore, the single-factor experiment finally determined that the optimal fermentation conditions were: initial sugar concentration 90g / L, fermentation temperature 30℃, fermentation time 48h, at which time the ethanol yield was 39.75g / L, the residual xylose sugar content was 5.04g / L, and the glucose was completely consumed.

[0086] Comparative Example 4

[0087] The difference from Example 4 is that the concentration of calcium chloride in the solidifying agent used in the yeast immobilization process is 1.5%. The remaining steps are the same as in Example 4.

[0088] The enzymatic hydrolyzate prepared by enzymatic hydrolysis in Comparative Examples 1-3 was also concentrated into a saccharified liquid with a glucose concentration of 90 g / L. Yeast was immobilized according to Example 4, and the same fermentation was carried out at 30° C. for 48 h to produce ethanol.

[0089] The results of fermentation to produce ethanol in Example 2 and Comparative Example 1 are shown in Table 1.

[0090] Table 1:

[0091]

[0092] As can be seen, the same concentration of saccharified liquid prepared from straw pretreated with either citric acid or acetic acid alone resulted in lower ethanol production during fermentation than in Example 4. The ethanol yield was lowest in the composite acid one-step heating pretreatment group, with significantly higher residual glucose and xylose levels in the saccharified liquid. Comparative Example 4 also had higher residual sugar levels, and ethanol production was significantly lower than in Example 4.

[0093] It should be noted that the yeast used in the present invention is conventional fermentation ethanol-producing yeast. If it is replaced with yeast with better ethanol production efficiency, the same pretreatment of corn straw in the present invention and enzymatic saccharification can also achieve better ethanol production efficiency.

Claims

1. A corn straw pretreatment method, characterized in that: The method uses a composite acid solution composed of citric acid and acetic acid to perform a segmented heat treatment on corn stalks. The segmented heat treatment comprises first performing a heat treatment at 40-60°C for 0.5-1 hour and then heating the corn stalks to 70-90°C for 1.5-2.5 hours. The composite acid solution is prepared by dissolving citric acid and acetic acid in water. The dosage ratio of citric acid, acetic acid and water is 0.8-1.5 g:1.5-2 mL:100 mL. The solid-liquid ratio of the composite acid solution to the corn stalks is 1:8-10.

2. A method for pretreating corn straw to increase ethanol production, characterized in that: The steps include: (1) Pretreatment: A composite acid solution consisting of citric acid, acetic acid, and water in a ratio of 0.8-1.5 g:1.5-2 mL:100 mL was added to corn straw, and the solid-liquid ratio of the composite acid solution to the corn straw was 1:8-10. The corn straw was subjected to a staged heat treatment, wherein the staged heat treatment was first performed at 40-60°C for 0.5-1 h, and then heated to 70-90°C for 1.5-2.5 h. (2) Enzymatic saccharification: Cellulase is added to the pretreated corn straw for enzymatic hydrolysis to prepare saccharification liquid; (3) Immobilized yeast: Sodium alginate and polyvinyl alcohol are dissolved in water to form a mixed liquid. After sterilization, yeast sludge is added and a curing agent is added. The mixture is cured for 2 to 3 hours. (4) Fermentation to produce ethanol: Immobilized yeast is added to the saccharification liquid for fermentation to produce ethanol.

3. The method for pretreating corn straw to increase ethanol production according to claim 2, wherein: The enzymatic saccharification in step (2) is to add the pretreated corn straw to a citric acid buffer solution with a pH of 4.8, sterilize it under high pressure at 121°C for 20 minutes, add cellulase, perform enzymatic hydrolysis at 50°C and 160 rpm for 72 hours, centrifuge to obtain the supernatant, and condense it by rotary evaporation to obtain a saccharified liquid with a glucose concentration of 90-120 g / L.

4. The method for increasing ethanol production by pretreating corn straw according to claim 3, wherein: In step (3), the concentration of sodium alginate is 3%, the concentration of polyvinyl alcohol is 2%, the curing agent is a mixed solution of calcium chloride and boric acid, wherein the amount ratio of calcium chloride, boric acid and water is 2-2.5 g: 2-2.5 g: 100 mL, and the number of live yeast cells in the bacterial sludge is 2.5×10 9 / mL.

5. The method for increasing ethanol production by pretreating corn straw according to claim 4, wherein: The fermentation temperature in step (4) is 30-35° C., and the fermentation time is 36-48 hours.

6. A method for pretreating corn straw to increase ethanol production, characterized in that: The steps include: (1) Pretreatment: The corn stalks were cut into small pieces and crushed, passed through a 40-mesh sieve, and dried at 60°C to constant weight. The corn stalks were subjected to a staged heat treatment using a composite acid formed by dissolving citric acid and acetic acid in water. The ratio of citric acid, acetic acid, and water in the composite acid solution was 0.8-1.5 g: 1.5-2 mL: 100 mL. The staged heat treatment was first performed at 40-60°C for 0.5-1 h, and then heated to 70-90°C for 1.5-2.5 h. (2) Enzymatic saccharification: The pretreated corn straw was added to a citric acid buffer solution with a pH of 4.8, sterilized at 121°C for 20 min, and then incubated with cellulase. The solution was enzymatically hydrolyzed at 50°C and 160 rpm for 72 h. The supernatant was centrifuged and concentrated by rotary evaporation to a saccharification solution with a glucose concentration of 90-120 g / L. (3) Immobilized yeast: Saccharomyces cerevisiae was activated and subcultured in YPD medium. The bacterial sludge was obtained by centrifugation. Sodium alginate and polyvinyl alcohol were dissolved in water to form a mixed liquid. The mixture was sterilized and the bacterial sludge was added. The number of live yeast cells in the bacterial sludge was 2.5×10 9 / mL, dropwise add curing agent to form a curing system, and after curing for 2-3 hours, place in physiological saline and put in a 4°C refrigerator for standby use. In the curing system, the concentration of sodium alginate is 3%, the concentration of polyvinyl alcohol is 2%, and the curing agent is a mixed solution of calcium chloride and boric acid, wherein the concentrations of calcium chloride and boric acid are both 2.5%, and the amount ratio of the mixed liquid to the curing agent is 1:8-10; (4) Fermentation to produce ethanol: The immobilized yeast was inoculated into the saccharification solution prepared in step (2) and fermented at 30-35°C for 48 hours.

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