Method for producing succinic acid by fermentation method

Abstract

The invention provides a method for producing succinic acid through a fermentation method, and belongs to the technical field of fermentation. The method comprises the steps of straw pretreatment, fermentation culture and post-treatment. The fermentation step comprises the following steps: inoculating an activated culture solution into a fermentation culture medium, supplementing enzymatic hydrolysate in the fermentation culture medium until the concentration of reducing sugar is 20-25g / L when the content of reducing sugar in a fermentation solution is as low as 4.5-5.2 g / L, and feeding a fermentation promoting solution and carrying out fermentation culture for 57.2-58.5 h when the fermentation is carried out for 20-23 h to obtain a fermentation solution; the fermentation culture medium consists of a primary fermentation culture medium and enzymatic hydrolysate; the fermentation liquor obtained by the method disclosed by the invention is high in succinic acid content, low in byproduct content, short in fermentation period and high in production efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation, and in particular relates to a method for producing succinic acid by fermentation. Background Art

[0002] Succinic acid, also known as succinic acid, is an important dibasic organic acid. It is a colorless crystal at room temperature and pressure, and has good water solubility and stability. The application range of succinic acid is relatively wide. In the food field, succinic acid is mainly used as an acidulant, buffer, preservative, pH adjuster, etc.; in the pharmaceutical field, it can also be used to synthesize antidotes, diuretics, sedatives, hemostatics and vitamins, etc.; in the chemical field, succinic acid is a good surfactant that can be used in lubricants, additives and elastomers, and can also be used as an important raw material for the production of polyester and polyurethane; in the agricultural field, succinic acid can be used to produce plant growth regulators and fertilizers.

[0003] Microbial fermentation has become a research hotspot due to its green and sustainable characteristics. The existing fermentation methods for producing succinic acid mainly include the following two methods: One is to enhance the activity of key enzymes in the metabolic pathway by genetically modifying microorganisms. However, the stability of genetically engineered bacteria is poor, which affects the stability of fermentation during long-term fermentation and ultimately affects the yield of succinic acid. The second method is to use natural microorganisms as fermentation strains and use sugars as substrates for fermentation. Specific inducers are also added to the fermentation system to promote the metabolic activity of microorganisms. However, it has the defects of low fermentation efficiency and low succinic acid production.

[0004] CN117187309A discloses a method for preparing succinic acid by fermenting bagasse, specifically, the bagasse is subjected to dilute acid hydrolysis and detoxification to obtain a detoxified hydrolyzate, the strain is activated and cultured, and then the detoxified hydrolyzate is added for fermentation culture, and succinic acid is obtained by post-treatment; the residual sugar content in the obtained fermented mature mash is 0.85-1.36 g / L, the succinic acid content is 52.03-56.64 g / L, and the succinic acid yield is 62.3-66.8%; The fermentation method of the above patent has a long fermentation cycle, and the content of succinic acid in the obtained fermentation liquid is still low, which is not conducive to large-scale industrial production. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides a method for producing succinic acid by fermentation, which shortens the fermentation cycle, increases the succinic acid content in the fermentation liquid, and improves the production efficiency of succinic acid.

[0006] In view of the above technical problems, the present invention adopts the following technical solutions: A method for producing succinic acid by fermentation, comprising straw pretreatment, fermentation culture and post-treatment steps, specifically as follows: 1. Straw pretreatment (1) Alkaline washing The corn stalks are dried to a moisture content of 0.8-1.4wt%, then crushed to a particle size of 100-130 mesh, put into a closed container, and 6-12 times the mass of sodium hydroxide solution is introduced, the temperature is increased to 104-114°C, and the heat treatment is carried out for 3.6-4.2h. After the heat treatment is completed, the solid matter is filtered out, washed with deionized water to a pH of 7.0-7.5, and dried to obtain alkali-washed stalks; The mass concentration of the sodium hydroxide solution is 12-18%; (2) Secondary processing The alkali-washed straw is put into a supercritical carbon dioxide device, the treatment temperature is controlled to be 44-52°C, the treatment pressure is 12.3-12.8MPa, and the treatment time is 2.0-2.6h. After the treatment, it is added to 8-14 times the mass of the treatment agent, the temperature is increased to 102-108°C, and the temperature is kept at 620-680rpm for 3.2-3.7h. After the treatment, it is centrifuged at 1400-1700rpm for 8-15min, filtered, washed, and then dried to obtain secondary treated straw; The treatment agent is a mixture of choline chloride, glycerol and L-glutamic acid, and the mass ratio of choline chloride, glycerol and L-glutamic acid is 1.8-2.4:2.0-2.7:1.0-1.5; (3) Enzymatic hydrolysis 8-14 times the mass of 1.5-2.0wt% sodium citrate buffer with a pH value of 4.8-5.2 is added to the secondary treated straw, and then cellulase and xylanase are added, with 18-23FPIU cellulase and 26-32FPIU / xylanase per gram of the secondary treated straw, and enzymolysis is carried out at 46-52°C for 40-45h to obtain a primary enzymolysis solution, the temperature of the primary enzymolysis solution is increased to 102-108°C, the heat treatment is carried out for 8-16min, and after cooling, the enzymolysis solution is obtained by filtering.

[0007] 2. Fermentation culture (1) Bacteria activation The Escherichia coli is inoculated into a liquid culture medium, and activated and cultured at 33-36° C. and 130-160 rpm for 11.0-15.0 hours, and then inoculated into an activation culture medium at an inoculum volume fraction of 1.6-2.2%, and cultured at 32-37° C. and 106-128 rpm for 18.0-24.0 hours to obtain an activated culture solution; The Escherichia coli is purchased from the market; The liquid culture medium is composed of 8-12 g / L tryptone, 4.6-5.4 g / L yeast extract powder, 5.7-6.4 g / L ammonium chloride, and 9.5-10.2 g / L sodium chloride; The activation culture medium is composed of 9-13 g / L glucose, 8-13 g / L yeast extract powder, 4.6-5.3 g / L corn steep liquor, 6.2-6.8 g / L sodium bicarbonate, and 8-13 g / L dipotassium hydrogen phosphate; (2) Fermentation Inoculate the activated culture solution into the fermentation medium at an inoculum amount of 4.6-5.3% by volume, adjust the pH to 6.5-7.0, and culture at 405-430 rpm in a mixed atmosphere at 35-38° C. When the reducing sugar content in the fermentation solution decreases to 4.5-5.2 g / L, add the enzymatic hydrolyzate in the fermentation medium until the reducing sugar concentration is 20-25 g / L, and when the fermentation culture reaches 20-23 hours, feed 75-82 g / L of fermentation promoting solution, control the feeding time to 9.5-10.2 hours, and ferment for 57.2-58.5 hours to obtain the fermentation solution; The mixed atmosphere is carbon dioxide and hydrogen, and the volume ratio of carbon dioxide to hydrogen is 90-94:6-10; The fermentation medium is composed of a primary fermentation medium and an enzymatic hydrolysate, and the volume ratio of the primary fermentation medium to the enzymatic hydrolysate is 1:0.8-1.3; The fermentation medium is composed of 12.0-12.5 g / L of glucose, 9.4-10.2 g / L of yeast extract powder, 3.2-3.8 g / L of corn steep liquor powder, 7.2-7.7 g / L of dipotassium hydrogen phosphate, 2.2-2.8 g / L of potassium dihydrogen phosphate, 3.7-4.3 / L of ammonium chloride, 0.3-0.6 g / L of carrageenan, 0.40-0.45 mg / L of sodium molybdate, 2.0-2.5 mg / L of manganese sulfate, and 1.0-1.5 g / L of magnesium sulfate; The fermentation promoting liquid is a mixture of deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid, and the mass ratio of the deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid is 1000g: 0.23-0.28g: 0.04-0.08g: 0.10-0.14mg.

[0008] 3. Post-processing The fermentation liquid is filtered, the filtrate is centrifuged, the supernatant is collected, and then it is concentrated under reduced pressure to 32-40% of the original volume to obtain a concentrated liquid; the concentrated liquid is cooled to 3.8-4.4°C, kept warm for 47-54 hours, filtered and dried to obtain a succinic acid product.

[0009] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention adopts a specific method to ferment and produce succinic acid, adopts Escherichia coli as a fermentation agent, and adopts plant straw as a fermentation substrate. First, the plant straw is alkali-washed to destroy the wood cellulose structure in the straw, dissolve part of the lignin, and fully expose cellulose and hemicellulose. Then, supercritical carbon dioxide is treated, which can penetrate into the cell wall of the straw, so that the cell wall expands, destroys the connection between cellulose and hemicellulose, and increases the porosity between the straws. Then, a treatment agent is used for treatment. Choline chloride in the treatment agent serves as a hydrogen bond acceptor, and glycerol and L-glutamic acid serve as hydrogen bond donors. The treatment agent composed of the choline chloride and L-glutamic acid has good solubility and biocompatibility, and can penetrate into the crystallization area of ​​cellulose and hemicellulose to destroy their hydrogen bonds. The bond network structure makes cellulose and hemicellulose loose, increasing their contact area with the enzyme, thereby better achieving enzymatic hydrolysis, converting the macromolecular sugars in the straw into fermentable sugars, and providing a nutrient source for the fermentation production of succinic acid; in the fermentation step, carrageenan can not only serve as a carbon source, but also regulate the growth environment of microorganisms. The gel network formed by it helps to distribute the nutrient solution and metabolites more evenly; the γ-aminobutyric acid in the fermentation promoting liquid can better regulate cell metabolism and conduction, enhance the cell's stress resistance, and combine with vitamin B2 and indoleacetic acid to provide sufficient energy and reducing power for the enzymes in the succinic acid synthesis pathway, promote the synthesis reaction of succinic acid, thereby increasing the yield and production efficiency of succinic acid and reducing the generation of by-products; 2. The fermentation broth obtained by the present invention was tested, and the succinic acid content was measured to be 68.5-72.3 g / L, the acetic acid content was 0.941.18 g / L, and the lactic acid content was 1.22-1.36 g / L. DETAILED DESCRIPTION

[0010] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0011] Example 1 1. Straw pretreatment (1) Alkaline washing The corn stalks were dried to a moisture content of 1.0 wt%, then crushed to a particle size of 120 mesh, put into a closed container, and 8 times the mass of 16 wt% sodium hydroxide solution was introduced, the temperature was raised to 110° C., and the heat treatment was carried out for 4.0 h. After the heat treatment was completed, the solid matter was filtered out, washed with deionized water to a pH of 7.3, and dried to obtain alkali-washed stalks; (2) Secondary processing The alkali-washed straw was put into a supercritical carbon dioxide device, and the treatment temperature was controlled to be 48°C, the treatment pressure was 12.5MPa, and the treatment time was 2.2h. After the treatment, it was added to a treatment agent of 10 times the mass, and the temperature was raised to 106°C. At 650rpm, the heat preservation and sealing treatment was carried out for 3.5h. After the treatment, it was centrifuged at 1600rpm for 10min, filtered, washed, and dried to obtain secondary treated straw; The treatment agent is a mixture of choline chloride, glycerol and L-glutamic acid, and the mass ratio of choline chloride, glycerol and L-glutamic acid is 2:2.4:1.3; (3) Enzymatic hydrolysis Add 10 times the mass of 2.0wt% sodium citrate buffer with a pH value of 5.0 to the secondary treated straw, and then add cellulase and xylanase, 20FPIU cellulase and 30FPIU / xylanase per gram of secondary treated straw, enzymolysis at 50°C for 42h to obtain a primary enzymolysis solution, raise the temperature of the primary enzymolysis solution to 100°C, keep it warm for 10min, cool it, and filter it to obtain an enzymolysis solution.

[0012] 2. Fermentation culture (1) Bacteria activation The Escherichia coli was inoculated into a liquid culture medium, and activated and cultured at 34°C and 140 rpm for 12.0 hours, and then inoculated into an activation culture medium at an inoculum volume fraction of 2%, and cultured at 36°C and 120 rpm for 20.0 hours to obtain an activated culture solution; The Escherichia coli is purchased from the market; The liquid culture medium is composed of 10 g / L tryptone, 5.0 g / L yeast extract powder, 6.0 g / L ammonium chloride, and 10.0 g / L sodium chloride; The activation culture medium is composed of 10 g / L glucose, 12 g / L yeast extract powder, 5.0 g / L corn steep liquor, 6.5 g / L sodium bicarbonate, and 10 g / L dipotassium hydrogen phosphate; (2) Fermentation The activated culture solution was inoculated into the fermentation medium at an inoculum volume fraction of 5.0%, the pH was adjusted to 6.7, and the culture was shaken at 420 rpm in a mixed atmosphere at 37°C. When the reducing sugar content in the fermentation solution decreased to 5.0 g / L, the enzymatic hydrolyzate in the fermentation medium was added to the reducing sugar concentration of 23 g / L. When the fermentation culture reached the 22nd hour, 80 g / L of fermentation promoting solution was fed, the feeding time was controlled to be 10.0 hours, and the fermentation culture was carried out for 57.8 hours to obtain the fermentation solution. The mixed atmosphere is carbon dioxide and hydrogen, and the volume ratio of carbon dioxide to hydrogen is 92:8; The fermentation medium is composed of a primary fermentation medium and an enzymatic hydrolysate, and the volume ratio of the primary fermentation medium to the enzymatic hydrolysate is 1:1; The fermentation medium is composed of 12.3 g / L glucose, 10 g / L yeast extract powder, 3.5 g / L corn steep liquor powder, 7.5 g / L dipotassium hydrogen phosphate, 2.4 g / L potassium dihydrogen phosphate, 4.0 / L ammonium chloride, 0.5 g / L carrageenan, 0.42 mg / L sodium molybdate, 2.2 mg / L manganese sulfate, and 1.2 g / L magnesium sulfate; The fermentation promoting liquid is a mixture of deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid, and the mass ratio of the deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid is 1000g: 0.26g: 0.06g: 0.11mg.

[0013] 3. Post-processing The fermentation liquid was filtered, the filtrate was centrifuged, the supernatant was collected, and then it was concentrated under reduced pressure to 35% of the original volume to obtain a concentrated liquid; the concentrated liquid was cooled to 4.2°C, kept warm for 50 hours, filtered and dried to obtain a succinic acid product.

[0014] The fermentation broth obtained in Example 1 was tested, and the succinic acid content was measured to be 72.3 g / L, the acetic acid content was 0.94 g / L, and the lactic acid content was 1.22 g / L.

[0015] Example 2 1. Straw pretreatment (1) Alkaline washing The corn stalks were dried to a moisture content of 0.8 wt %, then crushed to a particle size of 110 mesh, put into a closed container, and 6 times the mass of 12 wt % sodium hydroxide solution was introduced, the temperature was raised to 104° C., and the heat treatment was carried out for 3.6 h. After the heat treatment was completed, the solid matter was filtered out, washed with deionized water to a pH of 7.0, and dried to obtain alkali-washed stalks; (2) Secondary processing The alkali-washed straw was put into a supercritical carbon dioxide device, and the treatment temperature was controlled to be 44°C, the treatment pressure was 12.3MPa, and the treatment time was 2.0h. After the treatment, it was added to a treatment agent of 8 times the mass, and the temperature was raised to 102°C. At 620rpm, the heat preservation and sealing treatment was carried out for 3.2h. After the treatment, it was centrifuged at 1400rpm for 15min, filtered, washed, and dried to obtain secondary treated straw; The treatment agent is a mixture of choline chloride, glycerol and L-glutamic acid, and the mass ratio of choline chloride, glycerol and L-glutamic acid is 1.8:2.0:1.0; (3) Enzymatic hydrolysis 1.8 wt% sodium citrate buffer with a pH value of 4.8 was added to 8 times the mass of the secondary treated straw, and then cellulase and xylanase were added, with 18 FPIU cellulase and 26 FPIU / xylanase per gram of the secondary treated straw. The enzymatic hydrolysis was carried out at 46°C for 45 hours to obtain a primary enzymatic hydrolyzate. The temperature of the primary enzymatic hydrolyzate was raised to 102°C, kept warm for 16 minutes, cooled, and filtered to obtain an enzymatic hydrolyzate.

[0016] 2. Fermentation culture (1) Bacteria activation The Escherichia coli was inoculated into a liquid culture medium, and activated and cultured at 33°C and 130 rpm for 11.0 hours, and then inoculated into an activated culture medium at an inoculum volume fraction of 1.6%, and cultured at 32°C and 106 rpm for 18.0 hours to obtain an activated culture solution; The Escherichia coli is purchased from the market; The liquid culture medium is composed of 8 g / L tryptone, 4.6 g / L yeast extract powder, 5.7 g / L ammonium chloride, and 9.5 g / L sodium chloride; The activation culture medium is composed of 9 g / L glucose, 8 g / L yeast extract powder, 4.6 g / L corn steep liquor, 6.2 g / L sodium bicarbonate, and 8 g / L dipotassium hydrogen phosphate; (2) Fermentation The activated culture solution was inoculated into the fermentation medium at an inoculum volume fraction of 4.6%, the pH was adjusted to 6.5, and the culture was shaken at 405 rpm in a mixed atmosphere at 35°C. When the reducing sugar content in the fermentation solution decreased to 4.5 g / L, the enzymatic hydrolyzate in the fermentation medium was added to the reducing sugar concentration of 20 g / L. When the fermentation culture reached the 20th hour, 75 g / L of fermentation promoting solution was fed, the feeding time was controlled to be 9.5 hours, and the fermentation culture was carried out for 57.2 hours to obtain the fermentation solution. The mixed atmosphere is carbon dioxide and hydrogen, and the volume ratio of carbon dioxide to hydrogen is 90:10; The fermentation medium is composed of a primary fermentation medium and an enzymatic hydrolysate, and the volume ratio of the primary fermentation medium to the enzymatic hydrolysate is 1:0.8; The fermentation medium is composed of 12.0 g / L glucose, 9.4 g / L yeast extract powder, 3.2 g / L corn steep liquor powder, 7.2 g / L dipotassium hydrogen phosphate, 2.2 g / L potassium dihydrogen phosphate, 3.7 g / L ammonium chloride, 0.3 g / L carrageenan, 0.40 mg / L sodium molybdate, 2.0 mg / L manganese sulfate, and 1.0 g / L magnesium sulfate; The fermentation promoting liquid is a mixture of deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid, and the mass ratio of the deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid is 1000g: 0.23g: 0.04g: 0.10mg.

[0017] 3. Post-processing The fermentation liquid was filtered, the filtrate was centrifuged, the supernatant was collected, and then it was concentrated under reduced pressure to 32% of the original volume to obtain a concentrated liquid; the concentrated liquid was cooled to 4.0°C, kept warm for 52 hours, filtered and dried to obtain a succinic acid product.

[0018] The fermentation broth obtained in Example 2 was tested, and the succinic acid content was measured to be 68.5 g / L, the acetic acid content was 1.18 g / L, and the lactic acid content was 1.36 g / L.

[0019] Example 3 1. Straw pretreatment (1) Alkaline washing The corn stalks were dried to a moisture content of 1.2 wt%, then crushed to a particle size of 100 mesh, put into a closed container, and 12 times the mass of a 14 wt% sodium hydroxide solution was introduced, the temperature was raised to 108°C, and the heat treatment was carried out for 3.8 hours. After the heat treatment, the solids were filtered out, washed with deionized water to a pH of 7.2, and dried to obtain alkali-washed stalks; (2) Secondary processing The alkali-washed straw was put into a supercritical carbon dioxide device, and the treatment temperature was controlled to be 46°C, the treatment pressure was 12.6MPa, and the treatment time was 2.4h. After the treatment, it was added to a treatment agent of 12 times the mass, and the temperature was raised to 105°C. At 640rpm, the heat preservation and sealing treatment were carried out for 3.4h. After the treatment, it was centrifuged at 1500rpm for 12min, filtered, washed, and dried to obtain secondary treated straw; The treatment agent is a mixture of choline chloride, glycerol and L-glutamic acid, and the mass ratio of choline chloride, glycerol and L-glutamic acid is 2.2:2.3:1.2; (3) Enzymatic hydrolysis Twelve times the mass of 2.0 wt % sodium citrate buffer with a pH of 5.0 was added to the secondary treated straw, and then cellulase and xylanase were added, with 22 FPIU cellulase and 28 FPIU / xylanase per gram of secondary treated straw. The enzymatic hydrolysis was carried out at 48°C for 43 hours to obtain a primary enzymatic hydrolysate. The temperature of the primary enzymatic hydrolysate was raised to 106°C, kept warm for 12 minutes, cooled, and filtered to obtain an enzymatic hydrolysate.

[0020] 2. Fermentation culture (1) Bacteria activation The Escherichia coli was inoculated into a liquid culture medium, and activated and cultured at 35°C and 150 rpm for 13.0 hours, and then inoculated into an activated culture medium at an inoculum volume fraction of 1.8%, and cultured at 34°C and 114 rpm for 22.0 hours to obtain an activated culture solution; The Escherichia coli is purchased from the market; The liquid culture medium is composed of 11 g / L tryptone, 5.2 g / L yeast extract powder, 6.1 g / L ammonium chloride, and 9.7 g / L sodium chloride; The activation culture medium is composed of 12 g / L glucose, 10 g / L yeast extract powder, 4.8 g / L corn steep liquor, 6.4 g / L sodium bicarbonate, and 12 g / L dipotassium hydrogen phosphate; (2) Fermentation The activated culture solution was inoculated into the fermentation medium at an inoculum volume fraction of 4.8%, the pH was adjusted to 6.8, and the culture was shaken at 415 rpm in a mixed atmosphere at 36°C. When the reducing sugar content in the fermentation solution decreased to 4.8 g / L, the enzymatic hydrolyzate in the fermentation medium was added to the reducing sugar concentration of 22 g / L. When the fermentation culture reached the 21st hour, 77 g / L of fermentation promoting solution was fed, the feeding time was controlled to be 9.8 hours, and the fermentation culture was carried out for 58.2 hours to obtain the fermentation solution. The mixed atmosphere is carbon dioxide and hydrogen, and the volume ratio of carbon dioxide to hydrogen is 93:7; The fermentation medium is composed of a primary fermentation medium and an enzymatic hydrolysate, and the volume ratio of the primary fermentation medium to the enzymatic hydrolysate is 1:1.2; The fermentation medium is composed of 12.2 g / L glucose, 9.6 g / L yeast extract powder, 3.6 g / L corn steep liquor powder, 7.4 g / L dipotassium hydrogen phosphate, 2.6 g / L potassium dihydrogen phosphate, 4.2 / L ammonium chloride, 0.4 g / L carrageenan, 0.44 mg / L sodium molybdate, 2.3 mg / L manganese sulfate, and 1.3 g / L magnesium sulfate; The fermentation promoting liquid is a mixture of deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid, and the mass ratio of the deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid is 1000g:0.25g:0.07g:0.12mg.

[0021] 3. Post-processing The fermentation liquid was filtered, the filtrate was centrifuged, the supernatant was collected, and then it was concentrated under reduced pressure to 36% of the original volume to obtain a concentrated liquid; the concentrated liquid was cooled to 3.8°C, kept warm for 54 hours, filtered and dried to obtain a succinic acid product.

[0022] The fermentation broth obtained in Example 3 was tested, and the succinic acid content was measured to be 70.6 g / L, the acetic acid content was 1.14 g / L, and the lactic acid content was 1.30 g / L.

[0023] Example 4 1. Straw pretreatment (1) Alkaline washing The corn stalks were dried to a moisture content of 1.4 wt%, then crushed to a particle size of 130 mesh, put into a closed container, and 10 times the mass of 18 wt% sodium hydroxide solution was introduced, the temperature was raised to 114°C, and the heat treatment was carried out for 4.2 hours. After the heat treatment, the solid matter was filtered out, washed with deionized water to a pH of 7.5, and dried to obtain alkali-washed stalks; (2) Secondary processing The alkali-washed straw was put into a supercritical carbon dioxide device, and the treatment temperature was controlled to be 52°C, the treatment pressure was 12.8MPa, and the treatment time was 2.6h. After the treatment, it was added to a treatment agent of 14 times the mass, and the temperature was raised to 108°C. At 680rpm, the heat preservation and sealing treatment was carried out for 3.7h. After the treatment, it was centrifuged at 1700rpm for 8min, filtered, washed, and dried to obtain secondary treated straw; The treatment agent is a mixture of choline chloride, glycerol and L-glutamic acid, and the mass ratio of choline chloride, glycerol and L-glutamic acid is 2.4:2.7:1.5; (3) Enzymatic hydrolysis 14 times the mass of 1.5wt% sodium citrate buffer with a pH value of 5.2 was added to the secondary treated straw, and then cellulase and xylanase were added, with 23FPIU cellulase and 32FPIU / xylanase per gram of secondary treated straw. The enzymatic hydrolysis was carried out at 52°C for 40 hours to obtain a primary enzymatic hydrolyzate. The temperature of the primary enzymatic hydrolyzate was raised to 108°C, and the temperature was kept for 8 minutes. After cooling, it was filtered to obtain an enzymatic hydrolyzate.

[0024] 2. Fermentation culture (1) Bacteria activation The Escherichia coli was inoculated into a liquid culture medium, and activated and cultured at 36°C and 160 rpm for 15.0 hours, and then inoculated into an activated culture medium at an inoculum volume fraction of 2.2%, and cultured at 37°C and 128 rpm for 24.0 hours to obtain an activated culture solution; The Escherichia coli is purchased from the market; The liquid culture medium is composed of 12 g / L tryptone, 5.4 g / L yeast extract powder, 6.4 g / L ammonium chloride, and 10.2 g / L sodium chloride; The activation culture medium is composed of 13 g / L glucose, 13 g / L yeast extract powder, 5.3 / L corn steep liquor, 6.8 g / L sodium bicarbonate, and 13 g / L dipotassium hydrogen phosphate; (2) Fermentation The activated culture solution was inoculated into the fermentation medium at an inoculum volume fraction of 5.3%, the pH was adjusted to 7.0, and the culture was shaken at 430 rpm in a mixed atmosphere at 38°C. When the reducing sugar content in the fermentation solution decreased to 5.2 g / L, the enzymatic hydrolyzate in the fermentation medium was added to the reducing sugar concentration of 25 g / L. When the fermentation culture reached the 23rd hour, 82 g / L of fermentation promoting solution was fed, the feeding time was controlled to be 10.2 hours, and the fermentation culture was carried out for 58.5 hours to obtain the fermentation solution. The mixed atmosphere is carbon dioxide and hydrogen, and the volume ratio of carbon dioxide to hydrogen is 94:6; The fermentation medium is composed of a primary fermentation medium and an enzymatic hydrolysate, and the volume ratio of the primary fermentation medium to the enzymatic hydrolysate is 1:1.3; The fermentation medium is composed of 12.5 g / L glucose, 10.2 g / L yeast extract powder, 3.8 g / L corn steep liquor powder, 7.7 g / L dipotassium hydrogen phosphate, 2.8 g / L potassium dihydrogen phosphate, 4.3 g / L ammonium chloride, 0.6 g / L carrageenan, 0.45 mg / L sodium molybdate, 2.5 mg / L manganese sulfate, and 1.5 g / L magnesium sulfate; The fermentation promoting liquid is a mixture of deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid, and the mass ratio of the deionized water, γ-aminobutyric acid, vitamin B2, and indoleacetic acid is 1000g: 0.28g: 0.08g: 0.14mg.

[0025] 3. Post-processing The fermentation liquid was filtered, the filtrate was centrifuged, the supernatant was collected, and then it was concentrated under reduced pressure to 40% of the original volume to obtain a concentrated liquid; the concentrated liquid was cooled to 4.4°C, kept warm for 47 hours, filtered and dried to obtain a succinic acid product.

[0026] The fermentation broth obtained in Example 4 was tested, and the succinic acid content was measured to be 71.3 g / L, the acetic acid content was 1.02 g / L, and the lactic acid content was 1.27 g / L.

[0027] The invention adopts a specific method to ferment and produce succinic acid, adopts Escherichia coli as a fermentation agent, adopts plant straw as a fermentation substrate, firstly performs alkali washing on the plant straw to destroy the wood cellulose structure in the straw, dissolves part of the lignin, and fully exposes the cellulose and hemicellulose, and then performs supercritical carbon dioxide treatment, which can penetrate into the cell wall of the straw to make the cell wall swell, destroy the connection between cellulose and hemicellulose, and increase the porosity between the straws, and then uses a treatment agent for treatment, wherein choline chloride in the treatment agent serves as a hydrogen bond acceptor, and glycerol and L-glutamic acid serve as hydrogen bond donors, and the treatment agent composed of the choline chloride and L-glutamic acid has good solubility and biocompatibility, and can penetrate into the crystallization area of ​​cellulose and hemicellulose to destroy their hydrogen bonds. The network structure makes cellulose and hemicellulose loose, increasing their contact area with the enzyme, thereby better realizing enzymatic hydrolysis, converting the macromolecular sugar in the straw into fermentable sugar, and providing a nutrient source for the fermentation production of succinic acid; in the fermentation step, carrageenan can not only serve as a carbon source, but also regulate the growth environment of microorganisms. The gel network formed by it helps to distribute the nutrient solution and metabolites more evenly; the γ-aminobutyric acid in the fermentation promoting liquid can better regulate cell metabolism and conduction, enhance the cell's stress resistance, and combine with vitamin B2 and indoleacetic acid to provide sufficient energy and reducing power for the enzymes in the succinic acid synthesis pathway, promote the synthesis reaction of succinic acid, thereby increasing the yield and production efficiency of succinic acid and reducing the formation of by-products.

[0028] Example 1-1 1. Straw pretreatment (1) Alkaline washing The corn stalks were dried to a moisture content of 1.0 wt%, then crushed to a particle size of 120 mesh, put into a closed container, and 8 times the mass of 16 wt% sodium hydroxide solution was introduced, the temperature was raised to 110° C., and the heat treatment was carried out for 4.0 h. After the heat treatment was completed, the solid matter was filtered out, washed with deionized water to a pH of 7.3, and dried to obtain alkali-washed stalks; (2) Enzymatic hydrolysis Add 10 times the mass of 2.0wt% sodium citrate buffer with a pH value of 5.0 to the alkali-washed straw, and then add cellulase and xylanase, 20FPIU cellulase and 30FPIU / xylanase per gram of alkali-washed straw, and enzymolyze at 50°C for 42 hours to obtain a primary enzymatic hydrolyzate. Raise the temperature of the primary enzymatic hydrolyzate to 100°C, keep it warm for 10 minutes, cool it, and filter it to obtain an enzymatic hydrolyzate.

[0029] 2. Fermentation culture (1) Bacteria activation The Escherichia coli was inoculated into a liquid culture medium, and activated and cultured at 34°C and 140 rpm for 12.0 hours, and then inoculated into an activation culture medium at an inoculum volume fraction of 2%, and cultured at 36°C and 120 rpm for 20.0 hours to obtain an activated culture solution; The Escherichia coli is purchased from the market; The liquid culture medium is composed of 10 g / L tryptone, 5.0 g / L yeast extract powder, 6.0 g / L ammonium chloride, and 10.0 g / L sodium chloride; The activation culture medium is composed of 10 g / L glucose, 12 g / L yeast extract powder, 5.0 g / L corn steep liquor, 6.5 g / L sodium bicarbonate, and 10 g / L dipotassium hydrogen phosphate; (2) Fermentation The activated culture solution was inoculated into the fermentation medium at an inoculum volume fraction of 5.0%, the pH was adjusted to 6.7, and the culture was shaken at 420 rpm in a mixed atmosphere at 37°C. When the reducing sugar content in the fermentation solution decreased to 5.0 g / L, the mixed solution in the fermentation medium was supplemented to a reducing sugar concentration of 23 g / L, and the fermentation was cultured for 57.8 hours to obtain a fermentation solution. The mixed atmosphere is carbon dioxide and hydrogen, and the volume ratio of carbon dioxide to hydrogen is 92:8; The fermentation medium is composed of a primary fermentation medium and an enzymatic hydrolysate, and the volume ratio of the primary fermentation medium to the enzymatic hydrolysate is 1:1; The fermentation medium is composed of 12.3 g / L glucose, 10 g / L yeast extract powder, 3.5 g / L corn steep liquor dry powder, 7.5 g / L dipotassium hydrogen phosphate, 2.4 g / L potassium dihydrogen phosphate, 4.0 / L ammonium chloride, 0.5 g / L carrageenan, 0.42 mg / L sodium molybdate, 2.2 mg / L manganese sulfate and 1.2 g / L magnesium sulfate.

[0030] 3. Post-processing The fermentation liquid was filtered, the filtrate was centrifuged, the supernatant was collected, and then it was concentrated under reduced pressure to 35% of the original volume to obtain a concentrated liquid; the concentrated liquid was cooled to 4.2°C, kept warm for 50 hours, filtered and dried to obtain a succinic acid product.

[0031] The fermentation broth obtained in Example 1-1 was tested, and the succinic acid content was measured to be 60.5 g / L, the acetic acid content was 1.7 g / L, and the lactic acid content was 2.6 g / L.

[0032] It can be seen that in Example 1-1, the alkali-washed straw is enzymatically hydrolyzed, the contact area between the enzyme and the alkali-washed straw is small, and the structure between the hemicellulose and the cellulose is still relatively tight, which makes the enzymatic hydrolysis process incomplete and affects the conversion to fermentable sugars. During the fermentation process, the fermentation promoting liquid is not added, which affects the synthesis of succinic acid and increases the formation of by-products, ultimately resulting in low succinic acid production efficiency and low yield.

[0033] Unless otherwise specified, all percentages used in the present invention are by mass.

[0034] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for producing succinic acid by fermentation, characterized in that: It includes straw pretreatment, fermentation culture and post-treatment steps; The straw pretreatment step includes alkali washing, secondary treatment and enzymatic hydrolysis steps; The secondary treatment step is to treat the alkali-washed straw with supercritical carbon dioxide, then add it to the treatment agent, and treat it in a closed environment at 102-108° C. for 3.2-3.7 hours to obtain the secondary treated straw; The fermentation and culturing step includes bacterial activation and fermentation steps; The fermentation step comprises inoculating the activated culture solution into the fermentation medium, and when the reducing sugar content in the fermentation medium is as low as 4.5-5.2 g / L, adding the enzymatic hydrolyzate in the fermentation medium until the reducing sugar concentration is 20-25 g / L, and when the fermentation reaches 20-23 hours, feeding the fermentation promoting solution, and fermenting and culturing for 57.2-58.5 hours to obtain the fermentation solution; The fermentation medium is composed of primary fermentation medium and enzymatic hydrolysate; The fermentation medium is composed of glucose, yeast extract powder, corn steep liquor powder, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium chloride, carrageenan, sodium molybdate, manganese sulfate and magnesium sulfate; The fermentation promoting liquid is a mixture of deionized water, gamma-aminobutyric acid, vitamin B2 and indoleacetic acid.

2. The method for producing succinic acid by fermentation according to claim 1, characterized in that: In the secondary treatment step, the supercritical carbon dioxide treatment has a treatment temperature of 44-52° C., a treatment pressure of 12.3-12.8 MPa, and a treatment time of 2.0-2.6 h; The treatment agent is a mixture of choline chloride, glycerol and L-glutamic acid, and the mass ratio of choline chloride, glycerol and L-glutamic acid is 1.8-2.4:2.0-2.7:1.0-1.5; The mass ratio of the treatment agent to the alkali-washed straw is 8-14:

1.

3. The method for producing succinic acid by fermentation according to claim 1, characterized in that: The alkali washing step comprises drying the corn stalks to a moisture content of 0.8-1.4wt%, then crushing them to a particle size of 100-130 meshes, putting them into a closed container, introducing a sodium hydroxide solution of 6-12 times the mass, raising the temperature to 104-114°C, and heat-treating them for 3.6-4.2h. After the heat-treating is completed, filtering out the solid matter, washing them with deionized water to a pH of 7.0-7.5, and drying them to obtain alkali-washed stalks; The mass concentration of the sodium hydroxide solution is 12-18%.

4. The method for producing succinic acid by fermentation according to claim 1, characterized in that: The enzymatic hydrolysis step comprises: adding 8-14 times the mass of 1.5-2.0wt% sodium citrate buffer having a pH value of 4.8-5.2 to the secondary treated straw, then adding cellulase and xylanase, with 18-23FPIU cellulase and 26-32FPIU / xylanase per gram of the secondary treated straw, performing enzymatic hydrolysis at 46-52°C for 40-45h to obtain a primary enzymatic hydrolyzate, raising the temperature of the primary enzymatic hydrolyzate to 102-108°C, heat-insulating for 8-16min, cooling, and filtering to obtain an enzymatic hydrolyzate.

5. The method for producing succinic acid by fermentation according to claim 1, characterized in that: The bacterial strain activation step is to inoculate Escherichia coli into a liquid culture medium, activate and culture it at 33-36° C. and 130-160 rpm for 11.0-15.0 hours, then inoculate it into an activation culture medium at an inoculum volume fraction of 1.6-2.2%, and culture it at 32-37° C. and 106-128 rpm for 18.0-24.0 hours to obtain an activated culture solution.

6. The method for producing succinic acid by fermentation according to claim 1, characterized in that: In the bacterial activation step, the liquid culture medium is composed of 8-12 g / L tryptone, 4.6-5.4 g / L yeast extract powder, 5.7-6.4 g / L ammonium chloride, and 9.5-10.2 g / L sodium chloride; The activation culture medium is composed of 9-13 g / L glucose, 8-13 g / L yeast extract powder, 4.6-5.3 g / L corn steep liquor, 6.2-6.8 g / L sodium bicarbonate and 8-13 g / L dipotassium hydrogen phosphate.

7. The method for producing succinic acid by fermentation according to claim 1, characterized in that: The fermentation step comprises the following steps: inoculating the activated culture solution into the fermentation medium at an inoculation amount of 4.6-5.3% by volume, adjusting the pH to 6.5-7.0, oscillating and culturing at 405-430 rpm in a mixed atmosphere at 35-38° C., adding the enzymatic hydrolyzate in the fermentation medium until the reducing sugar content in the fermentation solution decreases to 4.5-5.2 g / L, adding 75-82 g / L of fermentation promoting solution at 20-23 hours of fermentation, controlling the addition time to 9.5-10.2 hours, and fermenting and culturing for 57.2-58.5 hours to obtain the fermentation solution.

8. The method for producing succinic acid by fermentation according to claim 1, characterized in that: In the fermentation step, the mixed atmosphere is carbon dioxide and hydrogen, and the volume ratio of carbon dioxide to hydrogen is 90-94:6-10; The volume ratio of the primary fermentation medium to the enzymatic hydrolysate is 1:0.8-1.3; The fermentation medium is composed of 12.0-12.5 g / L glucose, 9.4-10.2 g / L yeast extract powder, 3.2-3.8 g / L corn steep liquor powder, 7.2-7.7 g / L dipotassium hydrogen phosphate, 2.2-2.8 g / L potassium dihydrogen phosphate, 3.7-4.3 / L ammonium chloride, 0.3-0.6 g / L carrageenan, 0.40-0.45 mg / L sodium molybdate, 2.0-2.5 mg / L manganese sulfate, and 1.0-1.5 g / L magnesium sulfate; In the fermentation promoting liquid, the mass ratio of the deionized water, γ-aminobutyric acid, vitamin B2 and indoleacetic acid is 1000g: 0.23-0.28g: 0.04-0.08g: 0.10-0.14mg.

9. The method for producing succinic acid by fermentation according to claim 1, characterized in that: The post-treatment step is to filter the fermentation liquid, collect the supernatant after centrifugation of the filtrate, and then concentrate it under reduced pressure to 32-40% of the original volume to obtain a concentrated solution; reduce the temperature of the concentrated solution to 3.8-4.4° C., keep it warm and stand it for 47-54 hours, filter it and dry it to obtain a succinic acid product.