Production method for improving ethanol yield by treating corn straw with organic acid

Abstract

The invention relates to a production method for improving the yield of ethanol by treating corn straws with organic acid, the corn straws treated with organic acid are subjected to segmented heat treatment by adopting a composite acid solution consisting of citric acid and acetic acid, and the segmented heat treatment comprises the following steps: firstly, carrying out heat treatment at 40-60 DEG C for 0.5-1 hour, and then, heating to 70-90 DEG C, and carrying out heat treatment for 1.5-2.5 hours. According to the method, the corn straw is pretreated by adopting the composite organic acid, so that the degradation efficiency of lignocellulose in the corn straw is effectively improved, more cellulose is reserved, meanwhile, the generation of toxic inhibitors is reduced, the inhibition effect on cellulase and yeast in the subsequent enzymolysis and fermentation process is reduced, and the yield of the corn straw is improved. And the yield of the prepared oxygen-containing organic compound ethanol is improved.

Description

Technical Field

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

[0002] Crop straw is a very valuable biomass energy resource in the agricultural ecosystem, with the characteristic of huge yield. As an important production resource for grain, industrial and agricultural production, corn straw not only contains rich nutrients and utilizable chemical components and can be used as the raw material for livestock feed. Its main component is lignocellulose, and cellulose and hemicellulose are the key carbohydrates that can be fermented to produce ethanol. Lignocellulose is composed of cellulose, hemicellulose and lignin. Cellulose is a macromolecular polysaccharide composed of glucose, insoluble in water and general organic solvents. It is the most widely distributed and most abundant polysaccharide in nature. Usually, it combines with hemicellulose and lignin to form a dense structure. The binding mode and degree cause certain difficulties for the comprehensive utilization of straw. Therefore, it is necessary to pretreat corn straw to open its spatial structure for subsequent utilization.

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

[0004] Organic acid pretreatment has low pollutant emissions and is more environmentally friendly. Due to its special properties, it can selectively dissolve the hemicellulose and lignin components in lignocellulose, thereby exposing more cellulose components, so it has been widely used. However, even if organic acid pretreatment is used, toxic inhibitors that inhibit subsequent enzymatic hydrolysis and fermentation will be produced, resulting in the inhibition of subsequent sugar production efficiency and ethanol production efficiency. Summary of the Invention

[0005] The object of the present invention is to provide a pretreatment method for corn straw, which can reduce the pretreatment cost, while reducing the generation of inhibitors and effectively improving the saccharification rate in the subsequent enzymatic hydrolysis and saccharification process.

[0006] Another object of the present invention is to provide a method for using corn straw as a raw material to improve the yield of ethanol synthesized by straw fermentation through pretreatment of corn straw. Specifically, the corn straw is pretreated by combining a composite acid with segmented heat treatment and then fermented to prepare a saccharified solution, and the yeast is immobilized and then the saccharified solution is fermented to produce ethanol, effectively reducing the generation of inhibitors in the saccharified solution and increasing the yield of ethanol.

[0007] The object of the present invention is achieved by the following technical solutions: A method for pretreating corn straw, characterized in that: a composite acid solution composed of citric acid and acetic acid is used for segmented heat treatment of corn straw, and the segmented heat treatment is to first perform heat treatment at 40-60°C for 0.5-1 h, and then raise the temperature to 70-90°C for heat treatment for 1.5-2.5 h.

[0008] Further, 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.

[0009] Further, the solid-liquid ratio of the composite acid solution to corn straw is 1:8-10.

[0010] In the process of pretreating corn straw with a single citric acid or a single acetic acid, a relatively high treatment temperature is required, and more inhibitors are generated. Without any washing and detoxification treatment, the enzyme activity of the subsequent cellulase is inhibited.

[0011] In the present invention, a composite acid solution formed by citric acid and acetic acid with specific concentrations is used. Through segmented low-temperature heat treatment, the penetration of organic acids and the dissolution of hemicellulose are promoted in the first-stage heat treatment, and the removal of lignin is strengthened in the second-stage heat treatment. The overall heat treatment temperature is relatively low, and the dosage of a single organic acid is reduced, reducing the pretreatment cost while avoiding the rapid degradation of sugars at a higher temperature to generate more inhibitors, improving the enzymatic hydrolysis efficiency of the pretreated straw, and increasing the yield of glucose and xylose.

[0012] Although the composite pretreatment of straw with acetic acid and citric acid effectively improves the enzymatic hydrolysis saccharification effect of fiber enzymes, it will cause too high an acetic acid concentration in the saccharified solution. Acetic acid will penetrate the yeast cell membrane and damage the yeast cells, inhibit fermentation, and reduce the yield of ethanol.

[0013] In the present invention, SA-PVA composite immobilized yeast is used to improve the stability and stress resistance of yeast. At the same time, Ca in the immobilization system 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 in contact with yeast cells, reduces the inhibition of acetic acid on yeast through multiple paths, effectively balances the protective effect on yeast and the mass transfer efficiency of the substrate, and improves the yield of sodium ethoxide. In addition, the SA-PVA composite immobilization system also ensures that the immobilized yeast has suitable swelling properties, enabling excellent mass transfer efficiency of the substrate in the immobilized yeast while the immobilized yeast has excellent repeatability.

[0014] A method for improving ethanol production by pretreating corn straw with organic acids, characterized by comprising the following steps: (1) Pretreatment: A composite acid solution composed of citric acid and acetic acid is added to corn straw, and the corn straw is heat-treated in segments. (2) Enzymatic hydrolysis and saccharification: Cellulase is added to the pretreated corn straw for enzymatic hydrolysis to prepare a saccharified solution. (3) Immobilized yeast: Sodium alginate and polyvinyl alcohol are dissolved in water to form a mixed liquid. After sterilization, yeast sludge is added, and the mixture is dropped into a curing agent and cured for 2 - 3 hours. (4) Fermentation to produce ethanol: Immobilized yeast is added to the saccharified solution for fermentation to produce ethanol.

[0015] Further, in the composite acid solution in step (1), the dosage ratio of citric acid, acetic acid, and water is 0.8 - 1.5 g : 1.5 - 2 mL : 100 mL.

[0016] Further, the solid-liquid ratio of the composite acid solution to corn straw is 1:8 - 10.

[0017] Further, the segmented heat treatment is to first heat-treat at 40 - 60 °C for 0.5 - 1 h, and then raise the temperature to 70 - 90 °C for heat treatment for 1.5 - 2.5 h.

[0018] Further, in step (2), for enzymatic hydrolysis and saccharification, the pretreated corn straw is added to a citric acid buffer solution with pH = 4.8, autoclaved at 121 °C for 20 min, cellulase is inoculated, and enzymatic hydrolysis is carried out at 50 °C and 160 rmp for 72 hours. The supernatant is taken by centrifugation and rotary evaporated and concentrated into a saccharified solution with a glucose concentration of 90 - 120 g / L.

[0019] Further, 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, and the dosage ratio of calcium chloride, boric acid, and water is 2 - 2.5 g : 2 - 2.5 g : 100 mL. The yeast sludge is 2.5×10 9 / mL.

[0020] Furthermore, the fermentation temperature in step (4) is 30 - 35°C, and the fermentation time is 36 - 48 hours.

[0021] Most specifically, a method for pretreating corn straw to improve ethanol production, characterized by comprising the following steps: (1) Pretreatment: Cut the corn straw into small sections, crush it, pass through a 40 - mesh sieve, dry it to constant weight at 60°C, and use a composite acid formed by dissolving citric acid and acetic acid in water. Heat - treat the corn straw in segments. The dosage ratio of citric acid, acetic acid, and water in the composite acid solution is 0.8 - 1.5 g: 1.5 - 2 mL: 100 mL. The segmented heat - treatment is to first heat - treat at 40 - 60°C for 0.5 - 1 h, and then raise the temperature to 70 - 90°C for 1.5 - 2.5 h; (2) Enzymatic hydrolysis and saccharification: Add the pretreated corn straw to a citric acid buffer solution with pH = 4.8, sterilize it at 121°C for 20 min, inoculate cellulase, enzymatically hydrolyze at 50°C and 160 rmp for 72 hours, centrifuge to obtain the supernatant, and rotary evaporate and concentrate it into a saccharified solution with a glucose concentration of 90 - 120 g / L; (3) Immobilized yeast: Activate and passage the Saccharomyces cerevisiae in YPD medium, centrifuge to obtain the bacterial sludge. Dissolve sodium alginate and polyvinyl alcohol in water to form a mixed liquid, sterilize it, add the bacterial sludge. The bacterial sludge is 2.5×10 9 / mL in terms of viable yeast cell count, drop it into the curing agent to form a curing system, cure for 2 - 3 hours, then put it into physiological saline, and store it in a 4°C refrigerator for later use. In the curing system, 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, and 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 bacterial sludge is 2.5×10 9 / mL; (4) Fermentation to produce ethanol: Inoculate the immobilized yeast into the saccharified solution prepared in step (2), and ferment at 30 - 35°C for 48 hours.

[0022] The present invention has the following technical effects: By using a composite organic acid for pretreating corn straw in the present invention, the degradation efficiency of lignocellulose in corn straw is effectively improved, more cellulose is retained, and at the same time, the generation of toxic inhibitors is reduced, and the inhibitory effect on cellulase and yeast in the subsequent enzymatic hydrolysis and fermentation processes is reduced, and the yield of the oxygen - containing organic compound ethanol is increased. Description of the Drawings

[0023] Figure 1 : Structural characterization of corn straw with different pretreatments.

[0024] Figure 2 : XRD patterns and Fourier transform infrared spectra of pretreated straw.

[0025] Figure 3 : Effects of composite acid cycle pretreatment of straw on inhibitor content.

[0026] Figure 4 : Sugar yield of straw pretreated by composite acid cycle.

[0027] Figure 5 : Effects of different initial sugar concentrations, temperatures, and times on fermentation. Specific implementation manners

[0028] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above content of the present invention.

[0029] The materials used in the present invention are as follows: Corn straw: taken from the experimental field of Jilin Agricultural University in Changchun, Jilin Province, China; citric acid and acetic acid are from Shanghai Macklin Biochemical Co., Ltd.; cellulase is from Novozymes Biotechnology Co., Ltd.

[0030] Sodium alginate is from Tianjin Damao Chemical Reagent Factory; polyvinyl alcohol, calcium chloride, and boric acid are all from Sinopharm Chemical Reagent Co., Ltd.

[0031] The Saccharomyces cerevisiae used for fermentation ( Saccharomyces cerevisiae ) was purchased from the Guangdong Provincial Microbial Culture Collection Center, and the preservation number is GDMCC NO.2.89.

[0032] YPD medium: glucose 20 g / L, peptone 20 g / L, yeast extract powder 10 g / L, deionized water 1 L (autoclaved at 115 °C for 30 min).

[0033] Ethanol fermentation medium: peptone 10 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 2 g / L, saccharified solution 1 L (autoclaved at 115 °C for 30 min).

[0034] Example 1 A method for pretreating corn straw, comprising the following steps: The corn straw is cut into small sections, pulverized, sieved through a 40-mesh sieve, dried to a constant weight at 60 °C, and a composite acid formed by dissolving citric acid and acetic acid in water is used to heat-treat the corn straw in stages. The dosage ratio of citric acid, acetic acid and water in the composite acid solution is 1.0 g: 1.5 mL: 100 mL. The staged heat treatment is to first heat-treat at 50 °C for 0.5 h, and then raise the temperature to 80 °C for 2 h.

[0035] The hemicellulose removal rate of the corn straw pretreated by the method of Example 1 is 66.78%, and the lignin removal rate is 45.86%.

[0036] The pretreated straw is as Figure 1 shown. Scanning electron microscopy shows that (a) and (b) are the original straw observed at magnifications of 500 times and 2000 times respectively, and the surface of the straw is smooth and compact. (c) and (d) are the conditions of citric acid pretreatment at the corresponding magnifications. After treatment, the surface of the corn straw becomes uneven, with irregular fractures and holes, which is more conducive to the subsequent infiltration of cellulase.

[0037] Figure 2 The XRD patterns and Fourier transform infrared spectra of the straw before and after pretreatment are shown. The crystallinity of cellulose is an important index reflecting the structural and compositional changes of corn straw. It can be seen from the XRD pattern that the crystallinity of the pretreated corn straw increases from 43.55% to 49.94%. This change is due to the dissolution of hemicellulose and lignin, resulting in an increase in the relative content of cellulose after pretreatment, thus enhancing the overall crystallinity of corn straw. Fourier transform infrared spectroscopy shows that the absorption peak at 1720 cm -1 disappears because the acetyl group in the hemicellulose structure and the phenyl ester bond between hemicellulose and lignin are broken, indicating that hemicellulose is removed. The change at 1604 cm -1 is due to the vibration of the aromatic skeleton such as lignin and the stretching of the C-O bond; the change at 1515 cm -1 is the stretching of the C-C bond of lignin observed; the absorption at 1253 cm -1 is generated by the bending vibration of CH2; the absorption at 1168 cm -1 belongs to the corresponding peak of the typical structure of xylan; it shows that the lignin in the pretreated corn straw is reduced; the absorption at 1056 cm -1 is the absorption peak of -OH and C-O-C in the sugar unit; the absorption peak at 898 cm -1 indicates the cleavage of the β-glycosidic bond between sugar units, indicating a relative increase in cellulose content. The above results show that citric acid pretreatment of corn straw removes part of the lignin while removing hemicellulose, exposing cellulose, and making it better utilized in the next step.

[0038] Comparative Example 1 Compared with Example 1, single citric acid was used for pretreatment, and pretreatment was carried out according to the parameters that were optimal for the subsequent enzymatic hydrolysis and saccharification effect during the single citric acid pretreatment, as follows: The added concentration of citric acid was 4%, the heat treatment temperature was 125 °C, and the pretreatment time was 3 h.

[0039] Comparative Example 2 Compared with Example 1, single acetic acid was used for pretreatment, and pretreatment was carried out according to the parameters that were optimal for the subsequent enzymatic hydrolysis and saccharification effect during the single acetic acid pretreatment, as follows: The added concentration of acetic acid was 3%, the pretreatment temperature was 140 °C, and the pretreatment time was 60 min.

[0040] Comparative Example 3 Compared with Example 1, one-stage heating was used during the heat treatment process, specifically, it was kept at 80 °C for 3 h.

[0041] Determination of the content of inhibitors in the pretreated straw: High performance liquid chromatography was used to determine the contents of formic acid, acetic acid, furfural and 5-hydroxymethylfurfural (5-HMF) in the inhibitors by using an HPLC (Agilent OpenLAB CDS) equipped with a UV detector and a C18 column.

[0042] For the determination of furfural and 5-HMF, methanol and water at a ratio of 1:9 were used as the mobile phase, isocratic elution was carried out, the detection wavelength was 280 nm, and the flow rate was 0.9 mL / min.

[0043] For the determination of formic acid and acetic acid, 0.02 mol / L potassium dihydrogen phosphate and methanol at a ratio of 9:1 were used as the mobile phase for isocratic elution, the detection wavelength was 210 nm, the flow rate was 0.9 mL / min, and all mobile phases and samples were filtered through a 0.22 μm filter.

[0044] The detection results of the change in the inhibitor content are as Figure 3 shown. (a)-(d) correspond to Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 respectively. It can be seen that the generated amounts of formic acid, acetic acid, furfural and 5-HMF in Example 1 during the initial and cyclic processes are the lowest. During the first pretreatment, under the optimal pretreatment conditions, the contents 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 amount of toxic inhibitors in Comparative Example 3 was also relatively low, its pretreatment effect on corn straw was poor, resulting in an unsatisfactory subsequent enzymatic hydrolysis and saccharification efficiency.

[0045] The pretreated straws of Example 1 and each comparative example were subjected to enzymatic hydrolysis to prepare a fermentation medium (saccharified solution): Add the pretreated corn straw into a citric acid buffer solution with pH = 4.8, sterilize it at 121 °C under high pressure for 20 min, inoculate cellulase, and carry out enzymatic hydrolysis at 50 °C and 160 rmp for 72 hours, then centrifuge to obtain the supernatant. Measure the contents of glucose and xylose in the supernatant by using a glucose kit and a xylose kit.

[0046] Figure 4 It shows the change in sugar production (calculated as glucose) of corn straw after repeated pretreatment with the recycled composite acid solution and further enzymatic hydrolysis of the straw. It can be seen that under the optimal pretreatment conditions, the composite acid was used for six pretreatment processes. The number of cycles is negatively correlated with the effect of citric acid pretreatment. The glucose yield in the first pretreatment was 6.61 g / g of straw, and after six cycles, the obtained glucose yield was 0.37 g / g of straw. It is still higher than the sugar production of untreated corn straw.

[0047] Example 2 A method for pretreating corn straw, comprising the following steps: Cut the corn straw into small sections, then crush it, pass through a 40-mesh sieve, dry it at 60 °C to constant weight, and use a composite acid formed by dissolving citric acid and acetic acid in water to heat-treat the corn straw in segments. The dosage ratio of citric acid, acetic acid and water in the composite acid solution is 0.8 g: 2 mL: 100 mL. The segmented heat treatment is to first heat-treat at 60 °C for 0.5 h, and then raise the temperature to 70 °C for heat treatment for 2.5 h.

[0048] The hemicellulose removal rate of the pretreated corn straw is 65.87%, and the lignin removal rate is 44.72%.

[0049] Example 3 A method for pretreating corn straw, comprising the following steps: Cut the corn straw into small sections, then crush it, pass through a 40-mesh sieve, dry it at 60 °C to constant weight, and use a composite acid formed by dissolving citric acid and acetic acid in water to heat-treat the corn straw in segments. The dosage ratio of citric acid, acetic acid and water in the composite acid solution is 1 g: 1.5 mL: 100 mL. The segmented heat treatment is to first heat-treat at 40 °C for 1 h, and then raise the temperature to 90 °C for heat treatment for 1.5 h.

[0050] The hemicellulose removal rate of the pretreated corn straw is 64.54%, and the lignin removal rate is 44.91%.

[0051] Example 4 A method for pretreating corn straw to improve ethanol production, characterized by comprising the following steps: (1) Pretreatment: Cut the corn straw into small sections and then crush it. Sieve it through a 40-mesh sieve and dry it at 60 °C until constant weight. Use a composite acid formed by dissolving citric acid and acetic acid in water to heat-treat the corn straw in stages. The dosage ratio of citric acid, acetic acid, and water in the composite acid solution is 1.0 g: 1.5 mL: 100 mL. The staged heat treatment is to first heat-treat at 50 °C for 0.5 h, and then raise the temperature to 80 °C for 2 h. (2) Enzymatic hydrolysis and saccharification: Add the pretreated corn straw to a citric acid buffer solution with pH = 4.8, sterilize it at 121 °C for 20 min, inoculate cellulase, and carry out enzymatic hydrolysis at 50 °C and 160 rmp for 72 hours. Centrifuge to obtain the supernatant enzymatic hydrolysis solution, and rotary evaporate and concentrate it into a saccharified solution with a glucose concentration of 90 - 120 g / L. (3) Immobilized yeast: Activate and subculture Saccharomyces cerevisiae in YPD medium, centrifuge to obtain the bacterial sludge. Dissolve sodium alginate and polyvinyl alcohol in water to form a mixed liquid, sterilize it, add the bacterial sludge, drop it into the curing agent to form a curing system. After curing for 3 hours, place it in physiological saline and store it in a 4 °C refrigerator for later use. In the curing system, the concentration of sodium alginate is 3%, and the concentration of polyvinyl alcohol is 2%. The curing agent is a mixed solution of calcium chloride and boric acid, and the concentrations of calcium chloride and boric acid are both 2.5%. The bacterial sludge is 2.5×10 9 / mL; (4) Ethanol production by fermentation: Inoculate the immobilized yeast into the saccharified solution prepared in step (2) and ferment at 30 °C for 48 hours.

[0052] Use a universal mechanical testing machine to measure its Young's modulus to represent the mechanical strength of the immobilized beads. Measure the initial diameter of the immobilized particles, liquefy them in the liquefied solution at 30 °C for 1 h, measure the diameter after liquefaction, and calculate its swelling rate; Inoculate the immobilized particles into sterile water at 30 °C and shake at 160 rmp for 24 h. Weigh the mass of the swollen immobilized particles, dry the surface moisture, and weigh the mass of the dried immobilized particles to calculate its swelling degree.

[0053]

[0054]

[0055] Through the determination of the mechanical strength of the immobilized particles, the residual sugar in the culture medium after 6 h of fermentation, the swelling ratio and the degree of swelling, the conditions were optimized. When the sodium alginate concentration was too low, the mechanical strength was low, resulting in the leakage of the bacterial cells. When the sodium alginate concentration was too high, calcium bridges were formed among the sodium alginate molecules in the system, resulting in a dense structure, a dense external structure, and incomplete internal reaction, leading to low mechanical strength. When the polyvinyl alcohol concentration was too high, the mechanical strength was even lower, probably because the rigidity was increased, resulting in poor toughness. The curing agent for the SA-PVA double-crosslinked system was a mixed liquid of calcium chloride and boric acid. At low concentrations, the immobilized particles were not fully reacted, resulting in poor performance. At high concentrations, the outer shell was dense, and a nutrient gradient was formed inside. A short curing time would lead to incomplete reaction. After repeating the experiments with refined sodium alginate concentrations, it was finally determined that the optimal conditions were: SA concentration of 3%, PVA concentration of 2%, curing agent concentration of 2.5%, and curing time of 3 h. At this time, the residual sugar content after 6 h of fermentation was 16.2 g / L; the swelling ratio was 55%; the degree of swelling was 0.37 g / g; the mechanical strength of the immobilized Saccharomyces cerevisiae under the optimal conditions characterized by Young's modulus was 1085.14 MPa.

[0056] The effects of different initial sugar concentrations, temperatures, and times on fermentation are as Figure 5 shown. Fermentation experiments were carried out at initial sugar concentrations of 30 g / L, 60 g / L, 90 g / L, 120 g / L, and 150 g / L, and the residual sugar content after 48 h of fermentation was measured. At lower initial sugar concentrations, the ethanol yield was low. Higher initial sugar concentrations would inhibit the growth of the bacterial cells. At an initial sugar concentration of 90 g / L, the ethanol yield was 39.75 g / L. At this time, glucose was completely consumed, and there was a small amount of xylose remaining. Fermentation was carried out under different conditions of 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C. The growth of yeast was extremely slow at lower or higher temperatures, resulting in a low ethanol yield. The yield was higher at 25-35 °C, and the highest yield was 39.75 g / L at 30 °C. At this time, glucose was completely consumed. Fermentation was carried out under the conditions of 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. The ethanol yield was low at shorter times. As the time increased, the ethanol yield increased significantly and reached the highest at 48 h. Finally, continuing to extend the fermentation time did not result in a significant increase in the ethanol yield. Therefore, the optimal fermentation conditions determined by the single-factor experiment were: initial sugar concentration of 90 g / L, fermentation temperature of 30 °C, and fermentation time of 48 h. At this time, the ethanol yield was 39.75 g / L, the residual xylose content was 5.04 g / L, and glucose was completely consumed.

[0057] Comparative Example 4 Different from Example 4, the calcium chloride concentration in the curing agent used in the immobilization of yeast was 1.5%. The remaining steps were the same as those in Example 4.

[0058] The enzymolysis solutions prepared by enzymolysis in Comparative Examples 1-3 were also concentrated into saccharified solutions with a glucose concentration of 90 g / L, the yeast was immobilized according to Example 4, and ethanol was produced by the same fermentation at 30°C for 48 h.

[0059] The results of ethanol production by fermentation in Example 2 and each of Comparative Examples 1 are shown in Table 1.

[0060] Table 1:

[0061] It can be seen that for the straws pretreated with single citric acid and single acetic acid to prepare saccharified solutions with the same concentration, the amount of ethanol produced by the same fermentation decreased compared with that in Example 4, while the ethanol yield in the composite acid one-step heating pretreatment group was the lowest, and the residual amounts of glucose and xylose in the saccharified solution were significantly higher. In Comparative Example 4, the sugar residue solution was also relatively high, and the ethanol yield decreased significantly compared with that in Example 4.

[0062] It should be noted that the yeast used in the present invention is a conventional ethanol-producing yeast by fermentation. If it is replaced with a yeast with more excellent ethanol production efficiency, for the same pretreated corn straw in the present invention and subjected to enzymatic hydrolysis and saccharification, a more excellent ethanol production efficiency can also be achieved.

Claims

1. A method for pretreating corn straw, characterized in that: The method uses a composite acid solution composed of citric acid and acetic acid to perform segmented heat treatment on corn straw. The segmented heat treatment is to first perform heat treatment at 40 - 60 °C for 0.5 - 1 h, and then raise the temperature to 70 - 90 °C for heat treatment for 1.5 - 2.5 h.

2. The method for pretreating corn straw according to claim 1, characterized in that: 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 and corn straw is 1:8 - 10.

3. A method for preprocessing corn straw to increase ethanol production, characterized in that, It includes the following steps: (1) Pretreatment: Use a composite acid solution composed of citric acid and acetic acid, add it to corn straw, and perform segmented heat treatment on the corn straw; (2) Enzymatic hydrolysis and saccharification: Add cellulase to the pretreated corn straw for enzymatic hydrolysis to prepare a saccharified solution; (3) Immobilized yeast: Dissolve sodium alginate and polyvinyl alcohol in water to form a mixed liquid. After sterilization, add the yeast sludge, drop it into the curing agent, and cure for 2 - 3 hours; (4) Fermentation to produce ethanol: Add the immobilized yeast to the saccharified solution for fermentation to produce ethanol.

4. The method for pretreating corn straw to increase ethanol production according to claim 3, characterized in that: In the composite acid solution in step (1), the dosage ratio of citric acid, acetic acid and water is 0.8 - 1.5 g : 1.5 - 2 mL : 100 mL.

5. A method for pretreating corn straw to increase ethanol production according to claim 3 or 4, characterized in that: The solid - liquid ratio of the composite acid solution and corn straw is 1:8 - 10.

6. A method for pretreating corn straw to increase ethanol production according to any one of claims 3-5, characterized in that: The segmented heat treatment is to first perform heat treatment at 40 - 60 °C for 0.5 - 1 h, and then raise the temperature to 70 - 90 °C for heat treatment for 1.5 - 2.5 h.

7. The method for preprocessing corn straw to increase ethanol production according to claim 6, characterized in that: In step (2), for enzymatic hydrolysis and saccharification, add the pretreated corn straw to a citric acid buffer solution with pH = 4.8, sterilize at 121 °C under high pressure for 20 min, add cellulase, perform enzymatic hydrolysis at 50 °C and 160 rmp for 72 hours, centrifuge to obtain the supernatant, and rotary evaporate and concentrate it into a saccharified solution with a glucose concentration of 90 - 120 g / L.

8. A method for pre-treating corn straw to increase ethanol production according to claim 7, characterized in that: In step (3), the concentration of sodium alginate is 3% and the concentration of polyvinyl alcohol is 2%. The curing agent is a mixed solution of calcium chloride and boric acid, where the dosage ratio of calcium chloride, boric acid and water is 2 - 2.5 g: 2 - 2.5 g: 100 mL. The bacterial sludge is 2.5×10 9 / mL in terms of the viable yeast count.

9. A method for pre-treating corn straw to increase ethanol production according to claim 8, characterized in that: In step (4), the fermentation temperature is 30 - 35 °C, and the fermentation time is 36 - 48 hours.

10. A method for pretreating corn straw to increase ethanol production, characterized in that, It includes the following steps: (1) Pretreatment: Cut the corn straw into small sections, crush it, pass through a 40 - mesh sieve, dry it to constant weight at 60 °C, and use a composite acid formed by dissolving citric acid and acetic acid in water to perform segmented heat treatment on the corn straw. In the composite acid solution, the dosage ratio of citric acid, acetic acid and water is 0.8 - 1.5 g : 1.5 - 2 mL : 100 mL. The segmented heat treatment is to first perform heat treatment at 40 - 60 °C for 0.5 - 1 h, and then raise the temperature to 70 - 90 °C for heat treatment for 1.5 - 2.5 h; (2) Enzymatic hydrolysis and saccharification: Add the pretreated corn straw to a citric acid buffer solution with pH = 4.8, sterilize at 121 °C under high pressure for 20 min, add cellulase, perform enzymatic hydrolysis at 50 °C and 160 rmp for 72 hours, centrifuge to obtain the supernatant, and rotary evaporate and concentrate it into a saccharified solution with a glucose concentration of 90 - 120 g / L; (3) Immobilized yeast: Activate and subculture Saccharomyces cerevisiae in YPD medium, centrifuge to obtain the bacterial sludge. Dissolve sodium alginate and polyvinyl alcohol in water to form a mixed liquid, sterilize it, add the bacterial sludge. The bacterial sludge is 2.5×10 9 / mL in terms of the viable yeast cell count. Drop it into the curing agent to form a curing system. After curing for 2 - 3 hours, place it in physiological saline and store it in a 4°C refrigerator for standby. In the curing system, the concentration of sodium alginate is 3% and the concentration of polyvinyl alcohol is 2%. The curing agent is a mixed solution of calcium chloride and boric acid, where 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. The bacterial sludge is 2.5×10 9 / mL; (4) Fermentation to produce ethanol: Add the immobilized yeast to the saccharified solution prepared in step (2), and ferment at 30 - 35 °C for 48 hours.

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