Method for producing succinic acid by immobilized cells

Abstract

The invention provides a method for producing succinic acid by immobilized cells, and belongs to the technical field of fermentation. The fermentation culture medium comprises the following components: 50 to 54 g / L of glucose, 8 to 10 g / L of corn steep liquor, 5.7 to 5.9 g / L of monopotassium phosphate, 6.3 to 6.5 g / L of dipotassium phosphate, 0.17 to 0.22 mg / L of calcium chloride, 1.67 to 1.73 mg / L of cobalt chloride hexahydrate, 0.46 to 0.50 mg / L of zinc sulfate heptahydrate, 1.6 to 1.8 mg / L of biotin, 6.0 to 6.5 mg / L of nicotinamide, 8.2 to 8.5 mg / L of vitamin B5 and 0.32 to 0.34 g / L of an active agent. By adopting the method disclosed by the invention, the succinic acid production rate is high, the malic acid content is low, the immobilized microspheres can be repeatedly fermented for multiple times, and the succinic acid content of a single batch is stable and is always kept at a relatively high level.

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 using immobilized cells. Background Art

[0002] Succinic acid, also known as butyric acid, has a molecular formula of C4H6O4 and a molecular weight of 118.09. It is a natural organic acid that is widely found in the tissues of many plants and animals. Succinic acid can be used as a preservative, pH regulator, and cosolvent. It can also be used to synthesize antidotes, diuretics, sedatives, and vitamin A. Succinic acid is a good surfactant and a component of detergents, soaps, and demulsifiers. Succinic acid can be used to produce depilatories, toothpastes, cleaning agents, and high-efficiency wrinkle-removing cosmetic esters. It can also be used in lubricants, additives, and elastomers, and has a wide range of applications.

[0003] At present, the production methods of succinic acid mainly include chemical synthesis and microbial fermentation. However, succinic acid produced by chemical synthesis consumes a large amount of non-renewable fossil raw materials, has a low conversion rate, and is easy to pollute the environment. Microbial fermentation has the advantages of wide raw material sources, low cost, renewable, low energy consumption, and environmental friendliness. However, the microbial fermentation method has problems such as the fermentation products inhibiting the growth of bacteria and the difficulty of subsequent separation process, which limits the acid production level of the microorganisms and results in a high content of by-products such as malic acid in the fermentation broth.

[0004] In order to maintain the activity of the strain, avoid product feedback inhibition, increase the acid production level, and reduce the content of by-products, the prior art usually uses the method of immobilizing the strain, which is as follows: CN103436561B discloses a method for producing succinic acid by fermentation using cotton fiber materials to fix succinic acid actinomycetes. Specifically, succinic acid actinomycetes are cultured and adsorbed and fixed in a fermentation tank equipped with a cotton fiber carrier, and the fermentation cell concentration is increased, the acid production rate and conversion rate are increased, and the yield is increased by repeated batch fermentation, repeated fed-batch fermentation, continuous fermentation or double-tank cycle continuous fermentation process; However, the acid production rate and conversion rate of this patent are still at a low level, the by-product malic acid content is high, and the load between the bacteria and cotton fibers is not firm and easy to fall off, which affects the vitality of the bacteria.

[0005] CN117535280A discloses a method for producing succinic acid by fermentation of immobilized microorganisms using crop straws, which specifically discloses that the microorganisms are firstly adsorbed by straws, then a carrier and an initiator are added for immobilization to obtain immobilized microorganism cell microspheres, then the immobilized microorganism cell microspheres are modified, and finally placed in a fermenter for fermentation and culture; The fermentation method using the above method solves the product inhibition phenomenon and increases the fermentation yield of succinic acid. However, the fermentation cycle is long and the acid production rate is slow. In addition, when the immobilized Escherichia coli is used for continuous multi-batch fermentation of succinic acid, the immobilized cell rate in the first few batches is low and the acid production is low. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a method for producing succinic acid by using immobilized cells, which has high succinic acid yield, short fermentation cycle, low content of byproduct malic acid, and the immobilized bacteria can be fermented continuously in multiple batches, and the succinic acid yield of a single batch is always at a high level.

[0007] In view of the above technical problems, the present invention adopts the following technical solutions: A method for producing succinic acid by using immobilized cells, comprising the steps of shake flask culture, seed culture, preparation of immobilized microspheres and fermentation culture; the step of preparing immobilized microspheres comprises the steps of pretreatment, modification, primary coating and secondary coating of polyvinyl alcohol fibers; The specific operations are as follows: 1. Shake flask culture Yeast was taken from the plate and inoculated into a shake flask medium for shake flask culture. The culture time was 11-13 hours, the culture temperature was 33-35° C., and the shaking frequency was 160-164 rpm. After the culture was completed, a shake flask culture solution was obtained. The shaking flask culture medium comprises the following components: 4.5-4.9 g / L tryptone, 4.3-4.7 g / L NaCl, 9.1-9.6 g / L yeast powder, and the rest is water.

[0008] 2. Seed Culture The shake flask culture solution is inoculated into the seed culture medium at an inoculum volume fraction of 1.8-2.2%, the culture time is 16-18 hours, the temperature is 33-35° C., the rotation speed is 173-185 rpm, and the seed culture solution is obtained after the culture is completed; The seed culture medium is composed of the following ingredients: 9-11 g / L glucose, 4.0-4.4 g / L yeast powder, 7.8-8.3 g / L potassium hydrogen phosphate, 0.14-0.16 g / L manganese sulfate, 0.31-0.36 g / L magnesium sulfate heptahydrate, 0.14-0.17 g / L ferrous sulfate, 1.5-1.9 mg / L vitamin B1, 0.7-0.9 mg / L biotin, and the rest is water.

[0009] 3. Preparation of Immobilized Microspheres (1) Polyvinyl alcohol fiber pretreatment The polyvinyl alcohol fiber is sterilized, and then put into a sodium hydroxide solution with a mass of 9 to 12 times and stirred, and chloroacetic acid is added after stirring evenly, and ultrasonic reaction is carried out at room temperature, the ultrasonic time is 4.0 to 4.4 hours, the ultrasonic frequency is 16 to 20 kHz, and the ultrasonic power is 137 to 142 W. After the ultrasonic reaction is completed, the pretreated polyvinyl alcohol fiber is filtered, washed, and dried; The molecular weight of the polyvinyl alcohol fiber is 120,000-140,000 and the diameter is 0.8-1.2 mm; The mass concentration of the sodium hydroxide solution is 14-16%; The mass ratio of the sodium hydroxide solution to chloroacetic acid is 100:12-14; (2) Modification The pretreated polyvinyl alcohol fiber and deionized water are mixed, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is added and stirred to obtain a mixed solution; chitosan is mixed with 1.0-1.2wt% acetic acid solution, and stirred to obtain a chitosan solution; the mixed solution is slowly dripped into the chitosan solution at a dripping rate of 1.0-1.4g / min, and a reaction is carried out after the dripping is completed, the reaction temperature is 8.0-8.5°C, the reaction time is 21-23h, and after the reaction is completed, the modified chitosan is washed and dried to obtain the modified chitosan; The mass ratio of the polyvinyl alcohol fiber, deionized water and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 15.1-15.5:2.0-2.3:127-132; The mass ratio of chitosan to acetic acid solution is 2.6-2.8:85-90; The mass ratio of the mixed solution to the chitosan solution is 2.0-2.2:1.

[0010] (3) Primary coating The modified chitosan is sterilized, and then placed in a seed culture solution, and subjected to ultrasonic oscillation treatment, the ultrasonic time is 4.8-5.2 hours, the ultrasonic power is 117-124W, and the ultrasonic frequency is 11-15kHz. After the ultrasonic treatment, protocatechuic aldehyde is added, the pH is adjusted to 7.1-7.3, the reaction temperature is controlled to 26-28°C, the reaction time is 7.3-7.8 hours, and the primary coating solution is obtained after the reaction is completed; The mass ratio of the modified chitosan to the seed culture solution and protocatechuic aldehyde is 4.0-4.4:117-122:1.8-2.0.

[0011] (4) Secondary coating Add the wall material solution to the primary coating solution, stir evenly, then add 1.8-2.2wt% calcium chloride solution, stir at 113-126rpm for 1.5-1.7h, filter, wash, and dry at 35-37°C to obtain immobilized microspheres; The mass ratio of the primary coating solution, the wall material solution and the calcium chloride solution is 97-104:12-14:2.5-2.7; The preparation method of the wall material solution is as follows: adding carrageenan to deionized water, then heating it to 80-84° C., stirring it evenly, and sterilizing it at 0.04-0.06 MPa for 22-28 minutes to obtain the wall material solution; The mass ratio of the carrageenan to deionized water is 4-6:81-87.

[0012] 4. Fermentation culture After aseptic treatment of the immobilized microspheres, the immobilized microspheres are put into a fermentation medium for fermentation culture, wherein the volume of the fermentation medium is 18-22 times the volume of the immobilized microspheres, the fermentation temperature is controlled at 36-38°C, and carbon dioxide is continuously introduced at a ventilation flow rate of 0.14-0.16vvm, and the pressure is maintained at 0.04-0.06MPa. When the residual sugar concentration is reduced to 8-12g / L, glucose is added to 17-22g / L. During the fermentation process, sodium carbonate is added to control the pH to 6.5-6.6, and the fermentation time is 45.7-46.0h to obtain a fermentation culture solution; The fermentation medium comprises the following components: 50-54 g / L glucose, 8-10 g / L corn steep liquor, 5.7-5.9 g / L potassium dihydrogen phosphate, 6.3-6.5 g / L potassium hydrogen phosphate, 0.17-0.22 mg / L calcium chloride, 1.67-1.73 mg / L cobalt chloride hexahydrate, 0.46-0.50 mg / L zinc sulfate heptahydrate, 1.6-1.8 mg / L biotin, 6.0-6.5 mg / L nicotinamide, 8.2-8.5 mg / L vitamin B5, 0.32-0.34 g / L active agent, and the pH is 6.7-6.9; The active agent is a mixture of Tween 80 and polyethylene glycol 600, and the mass ratio of Tween 80 to polyethylene glycol 600 is 1:1.1-1.3.

[0013] The yeast is Pichia pastoris, which is classified and named Pichia sp. , deposited in Guangdong Provincial Microbiological Culture Collection Center, the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the deposit number is GDMCC No: 64916, and the deposit date is July 26, 2024.

[0014] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention adopts a specific method to prepare modified polyvinyl alcohol fiber. In the step of pretreating the polyvinyl alcohol fiber, the hydroxyl group of the polyvinyl alcohol fiber is carboxylated so that the surface of the pretreated polyvinyl alcohol fiber is rich in a large number of carboxyl groups. Then, the fiber is modified by grafting the amino group of chitosan with the carboxyl group of the pretreated polyvinyl alcohol fiber so that chitosan and the polyvinyl alcohol fiber can be firmly combined, thereby enhancing the stability of chitosan. Then, the fiber is placed in a seed culture medium for a primary coating so that the outside of the strain is evenly coated with chitosan. Then, a secondary coating is performed to achieve immobilized microspheres with the strain as the microsphere core, chitosan as the inner wall material, and carrageenan as the outer wall material. The immobilized microspheres prepared by the present invention using a specific method have good mass transfer performance, high mechanical properties, and low irritation to the strain. , will not affect the activity of the strain, the strain has high vitality and long-lasting vitality, and has good permeability, which is convenient for the transportation of carbon dioxide gas, solves the product inhibition phenomenon, and is combined with specific fermentation culture and other technical means to increase the yield and production rate of succinic acid, reduce the generation of by-products such as malic acid, and when the immobilized microspheres are used for repeated batch production, the succinic acid yield is stable and can still maintain a high level; the present invention adopts a specific method for fermentation culture, wherein the active agent acts on the protein group on the cell membrane, thereby increasing the permeability of the cell membrane, and vitamin B5 and nicotinamide can provide nutrients to promote the activity of the enzyme, thereby promoting the growth of the bacteria; it is combined with the immobilized microspheres to promote the fermentation culture, increase the yield of the product, and reduce the content of by-products; 2. The fermentation method for producing succinic acid of the present invention has a succinic acid yield of 87.2-87.9 g / L, an acid production rate of 1.90-1.92 g / L / h, a malic acid content of 0.27-0.31 g / L, and an acetic acid content of 0.88-0.90 g / L; 3. The immobilized microspheres prepared by the method of the present invention were used to produce succinic acid. After 25 fermentation batches, the succinic acid content in the first fermentation was 87.2-87.9 g / L, the succinic acid content in the tenth fermentation was 86.1-87.1 g / L, and the succinic acid content in the 25th fermentation was 83.9-85.4 g / L. DETAILED DESCRIPTION

[0015] 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.

[0016] The yeast involved in the embodiment is Pichia pastoris ( Pichia sp. ), with the deposit number GDMCC NO:64916, collected and isolated and purified in Nanning, Guangxi Zhuang Autonomous Region, China in April 2024; Example 1 1. Shake flask culture Yeast was taken from the plate and inoculated into a shake flask medium for shake flask culture. The culture time was 12 hours, the culture temperature was 34°C, and the shaking frequency was 162 rpm. After the culture was completed, a shake flask culture solution was obtained. The shake flask culture medium comprises the following components: 4.7 g / L tryptone, 4.5 g / L NaCl, 9.4 g / L yeast powder, and the rest is water.

[0017] 2. Seed Culture The shake flask culture solution was inoculated into the seed culture medium at an inoculum volume fraction of 2%, the culture time was 17 hours, the temperature was 34° C., and the rotation speed was 180 rpm. After the culture was completed, the seed culture solution was obtained; The seed culture medium is composed of the following ingredients: 10 g / L glucose, 4.2 g / L yeast powder, 8 g / L dipotassium hydrogen phosphate, 0.15 g / L manganese sulfate, 0.34 g / L magnesium sulfate heptahydrate, 0.16 g / L ferrous sulfate, 1.7 mg / L vitamin B1, 0.8 mg / L biotin, and the rest is water.

[0018] 3. Preparation of Immobilized Microspheres (1) Polyvinyl alcohol fiber pretreatment The polyvinyl alcohol fiber was sterilized, and then put into a sodium hydroxide solution with a mass of 10 times and stirred, and chloroacetic acid was added after stirring evenly, and ultrasonic reaction was carried out at room temperature, the ultrasonic time was 4.2 hours, the ultrasonic frequency was 18kHz, and the ultrasonic power was 140W. After the ultrasonic reaction was completed, the pretreated polyvinyl alcohol fiber was filtered, washed, and dried. The molecular weight of the polyvinyl alcohol fiber is 130,000 and the diameter is 1.0 mm; The mass concentration of the sodium hydroxide solution is 15%; The mass ratio of the sodium hydroxide solution to chloroacetic acid is 100:13; (2) Modification The pretreated polyvinyl alcohol fiber and deionized water are mixed, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is added and stirred to obtain a mixed solution; chitosan is mixed with a 1.1wt% acetic acid solution, and stirred to obtain a chitosan solution; the mixed solution is slowly added dropwise to the chitosan solution at a dropping rate of 1.2g / min, and a reaction is carried out after the addition is completed. The reaction temperature is 8.2°C and the reaction time is 22h. After the reaction is completed, the modified chitosan is washed and dried to obtain the modified chitosan; The mass ratio of the polyvinyl alcohol fiber, deionized water and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 15.3:2.1:130; The mass ratio of chitosan to acetic acid solution is 2.7:88; The mass ratio of the mixed solution to the chitosan solution is 2.1:1.

[0019] (3) Primary coating The modified chitosan was sterilized, and then placed in a seed culture solution, and subjected to ultrasonic oscillation treatment. The ultrasonic time was 5 hours, the ultrasonic power was 121 W, and the ultrasonic frequency was 13 kHz. After the ultrasonic treatment, protocatechuic aldehyde was added, the pH was adjusted to 7.2, the reaction temperature was controlled to 27° C., the reaction time was 7.5 hours, and the primary coating solution was obtained after the reaction was completed. The mass ratio of the modified chitosan to the seed culture solution and protocatechuic aldehyde is 4.2:120:1.9.

[0020] (4) Secondary coating Add the wall material solution to the primary coating solution, stir evenly, then add 2 wt % calcium chloride solution, stir at 120 rpm for 1.6 h, filter, wash, and dry at 36° C. to obtain immobilized microspheres; The mass ratio of the primary coating solution, the wall material solution and the calcium chloride solution is 100:13:2.6; The wall material solution is prepared by adding carrageenan to deionized water, heating the water to 82° C., stirring the water evenly, and sterilizing the water at 0.05 MPa for 25 min to obtain the wall material solution. The mass ratio of the carrageenan to deionized water is 5:83.

[0021] 4. Fermentation culture After the immobilized microspheres were aseptically treated, they were put into a fermentation medium for fermentation culture. The volume of the fermentation medium was 20 times the volume of the immobilized microspheres. The fermentation temperature was controlled at 37°C. Carbon dioxide was continuously introduced at a ventilation flow rate of 0.15 vvm. The pressure was maintained at 0.05 MPa. When the residual sugar concentration decreased to 10 g / L, glucose was added to 20 g / L. Sodium carbonate was added during the fermentation process to control the pH to 6.6. The fermentation time was 45.7 h to obtain a fermentation culture solution. The fermentation medium comprises the following ingredients: 52 g / L glucose, 9 g / L corn steep liquor, 5.8 g / L potassium dihydrogen phosphate, 6.4 g / L potassium hydrogen phosphate, 0.2 mg / L calcium chloride, 1.70 mg / L cobalt chloride hexahydrate, 0.48 mg / L zinc sulfate heptahydrate, 1.7 mg / L biotin, 6.3 mg / L nicotinamide, 8.4 mg / L vitamin B5, 0.33 g / L active agent, and the pH is 6.8; The active agent is a mixture of Tween 80 and polyethylene glycol 600, and the mass ratio of Tween 80 to polyethylene glycol 600 is 1:1.2.

[0022] The fermentation broth prepared by the method of Example 1 had a succinic acid yield of 87.9 g / L, an acid production rate of 1.92 g / L / h, a malic acid content of 0.27 g / L, and an acetic acid content of 0.88 g / L.

[0023] Example 2 1. Shake flask culture Yeast was taken from the plate and inoculated into a shake flask medium for shake flask culture. The culture time was 11 hours, the culture temperature was 33°C, and the shaking frequency was 160 rpm. After the culture was completed, a shake flask culture solution was obtained. The shaking flask culture medium comprises the following components: 4.5 g / L tryptone, 4.3 g / L NaCl, 9.1 g / L yeast powder, and the rest is water.

[0024] 2. Seed Culture The shake flask culture solution was inoculated into the seed culture medium at an inoculum volume fraction of 1.8%, the culture time was 16 hours, the temperature was 33° C., and the rotation speed was 173 rpm. After the culture was completed, the seed culture solution was obtained; The seed culture medium is composed of the following ingredients: 9 g / L glucose, 4.0 g / L yeast powder, 7.8 g / L dipotassium hydrogen phosphate, 0.14 g / L manganese sulfate, 0.31 g / L magnesium sulfate heptahydrate, 0.14 g / L ferrous sulfate, 1.5 mg / L vitamin B1, 0.7 mg / L biotin, and the rest is water.

[0025] 3. Preparation of Immobilized Microspheres (1) Polyvinyl alcohol fiber pretreatment The polyvinyl alcohol fiber was sterilized, and then put into a sodium hydroxide solution of 9 times the mass and stirred, and chloroacetic acid was added after stirring evenly, and ultrasonic reaction was carried out at room temperature, the ultrasonic time was 4.0 hours, the ultrasonic frequency was 16kHz, and the ultrasonic power was 137W. After the ultrasonic reaction was completed, the pretreated polyvinyl alcohol fiber was filtered, washed, and dried. The molecular weight of the polyvinyl alcohol fiber is 120,000 and the diameter is 0.8 mm; The mass concentration of the sodium hydroxide solution is 14%; The mass ratio of the sodium hydroxide solution to chloroacetic acid is 100:12; (2) Modification The pretreated polyvinyl alcohol fiber and deionized water are mixed, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is added and stirred to obtain a mixed solution; chitosan is mixed with a 1.0wt% acetic acid solution, and stirred to obtain a chitosan solution; the mixed solution is slowly added dropwise to the chitosan solution at a dropping rate of 1.0g / min, and a reaction is carried out after the addition is completed. The reaction temperature is 8.0°C and the reaction time is 21h. After the reaction is completed, the modified chitosan is washed and dried to obtain the modified chitosan; The mass ratio of the polyvinyl alcohol fiber, deionized water and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 15.1:2.0:127; The mass ratio of chitosan to acetic acid solution is 2.6:85; The mass ratio of the mixed solution to the chitosan solution is 2.0:1.

[0026] (3) Primary coating The modified chitosan was sterilized, and then placed in a seed culture solution, and subjected to ultrasonic oscillation treatment. The ultrasonic time was 4.8 h, the ultrasonic power was 117 W, and the ultrasonic frequency was 11 kHz. After the ultrasonic treatment, protocatechuic aldehyde was added, the pH was adjusted to 7.1, the reaction temperature was controlled to 26° C., the reaction time was 7.3 h, and the primary coating solution was obtained after the reaction was completed. The mass ratio of the modified chitosan to the seed culture solution and protocatechuic aldehyde is 4.0:117:1.8.

[0027] (4) Secondary coating Add the wall material solution to the primary coating solution, stir evenly, then add 1.8 wt % calcium chloride solution, stir at 113 rpm for 1.5 h, filter, wash, and dry at 35 ° C to obtain immobilized microspheres; The mass ratio of the primary coating solution, the wall material solution and the calcium chloride solution is 97:12:2.5; The preparation method of the wall material solution is as follows: adding carrageenan to deionized water, then heating to 80° C., stirring evenly, and sterilizing at 0.04 MPa for 22 minutes to obtain the wall material solution; The mass ratio of the carrageenan to deionized water is 4:81.

[0028] 4. Fermentation culture After aseptic treatment, the immobilized microspheres were put into a fermentation medium for fermentation culture. The volume of the fermentation medium was 18 times the volume of the immobilized microspheres. The fermentation temperature was controlled at 36°C. Carbon dioxide was continuously introduced at a ventilation flow rate of 0.14 vvm. The pressure was maintained at 0.04 MPa. When the residual sugar concentration decreased to 8 g / L, glucose was added to 17 g / L. Sodium carbonate was added during the fermentation process to control the pH to 6.5. The fermentation time was 46.0 h to obtain a fermentation culture solution. The fermentation medium comprises the following ingredients: 50 g / L glucose, 8 g / L corn steep liquor, 5.7 g / L potassium dihydrogen phosphate, 6.3 g / L potassium hydrogen phosphate, 0.17 mg / L calcium chloride, 1.67 mg / L cobalt chloride hexahydrate, 0.46 mg / L zinc sulfate heptahydrate, 1.6 mg / L biotin, 6.0 mg / L nicotinamide, 8.2 mg / L vitamin B5, 0.32 g / L active agent, and the pH is 6.7; The active agent is a mixture of Tween 80 and polyethylene glycol 600600, and the mass ratio of Tween 80 to polyethylene glycol 600600 is 1:1.1.

[0029] The fermentation broth prepared by the method of Example 2 had a succinic acid yield of 87.2 g / L, an acid production rate of 1.90 g / L / h, a malic acid content of 0.31 g / L, and an acetic acid content of 0.90 g / L.

[0030] Example 3 1. Shake flask culture Yeast was taken from the plate and inoculated into a shake flask medium for shake flask culture. The culture time was 13 hours, the culture temperature was 35°C, and the shaking frequency was 164 rpm. After the culture was completed, a shake flask culture solution was obtained. The shake flask culture medium comprises the following components: 4.9 g / L tryptone, 4.7 g / L NaCl, 9.6 g / L yeast powder, and the rest is water.

[0031] 2. Seed Culture The shake flask culture solution was inoculated into the seed culture medium at an inoculum volume fraction of 2.2%, the culture time was 18 hours, the temperature was 35° C., and the rotation speed was 185 rpm. After the culture was completed, the seed culture solution was obtained; The seed culture medium is composed of the following ingredients: 11 g / L glucose, 4.4 g / L yeast powder, 8.3 g / L dipotassium hydrogen phosphate, 0.16 g / L manganese sulfate, 0.36 g / L magnesium sulfate heptahydrate, 0.17 g / L ferrous sulfate, 1.9 mg / L vitamin B1, 0.9 mg / L biotin, and the rest is water.

[0032] 3. Preparation of Immobilized Microspheres (1) Polyvinyl alcohol fiber pretreatment The polyvinyl alcohol fiber was sterilized, and then put into a sodium hydroxide solution with a mass of 12 times and stirred, and chloroacetic acid was added after stirring evenly, and ultrasonic reaction was carried out at room temperature, the ultrasonic time was 4.4 hours, the ultrasonic frequency was 20kHz, and the ultrasonic power was 142W. After the ultrasonic reaction was completed, the pretreated polyvinyl alcohol fiber was filtered, washed, and dried; The molecular weight of the polyvinyl alcohol fiber is 140,000 and the diameter is 1.2 mm; The mass concentration of the sodium hydroxide solution is 16%; The mass ratio of the sodium hydroxide solution to chloroacetic acid is 100:14; (2) Modification The pretreated polyvinyl alcohol fiber and deionized water are mixed, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is added and stirred to obtain a mixed solution; chitosan is mixed with a 1.2wt% acetic acid solution, and stirred to obtain a chitosan solution; the mixed solution is slowly added dropwise to the chitosan solution at a dropping rate of 1.4g / min, and a reaction is carried out after the addition is completed. The reaction temperature is 8.5°C and the reaction time is 23h. After the reaction is completed, the modified chitosan is washed and dried to obtain the modified chitosan; The mass ratio of the polyvinyl alcohol fiber, deionized water and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 15.5:2.3:132; The mass ratio of chitosan to acetic acid solution is 2.8:90; The mass ratio of the mixed solution to the chitosan solution is 2.2:1.

[0033] (3) Primary coating The modified chitosan was sterilized, and then placed in a seed culture solution, and subjected to ultrasonic oscillation treatment. The ultrasonic time was 5.2 h, the ultrasonic power was 124 W, and the ultrasonic frequency was 15 kHz. After the ultrasonic treatment, protocatechuic aldehyde was added, the pH was adjusted to 7.3, the reaction temperature was controlled to 28° C., the reaction time was 7.8 h, and the primary coating solution was obtained after the reaction was completed. The mass ratio of the modified chitosan to the seed culture solution and protocatechuic aldehyde is 4.4:122:2.0.

[0034] (4) Secondary coating Add the wall material solution to the primary coating solution, stir evenly, then add 2.2 wt % calcium chloride solution, stir at 126 rpm for 1.7 h, filter, wash, and dry at 37 ° C to obtain immobilized microspheres; The mass ratio of the primary coating solution, the wall material solution and the calcium chloride solution is 104:14:2.7; The wall material solution is prepared by adding carrageenan to deionized water, heating the water to 84° C., stirring the water evenly, and sterilizing the water at 0.06 MPa for 28 minutes to obtain the wall material solution. The mass ratio of the carrageenan to deionized water is 6:87.

[0035] 4. Fermentation culture After aseptic treatment, the immobilized microspheres were put into a fermentation medium for fermentation culture. The volume of the fermentation medium was 22 times the volume of the immobilized microspheres. The fermentation temperature was controlled at 38°C. Carbon dioxide was continuously introduced at a ventilation flow rate of 0.16 vvm. The pressure was maintained at 0.06 MPa. When the residual sugar concentration decreased to 12 g / L, glucose was added to 22 g / L. Sodium carbonate was added during the fermentation process to control the pH to 6.6. The fermentation time was 45.9 h to obtain a fermentation culture solution. The fermentation medium comprises the following ingredients: 54 g / L glucose, 10 g / L corn steep liquor, 5.9 g / L potassium dihydrogen phosphate, 6.5 g / L potassium hydrogen phosphate, 0.22 mg / L calcium chloride, 1.73 mg / L cobalt chloride hexahydrate, 0.50 mg / L zinc sulfate heptahydrate, 1.8 mg / L biotin, 6.5 mg / L nicotinamide, 8.5 mg / L vitamin B5, 0.34 g / L active agent, and the pH is 6.9; The active agent is a mixture of Tween 80 and polyethylene glycol 600600, and the mass ratio of Tween 80 to polyethylene glycol 600600 is 1:1.3.

[0036] The fermentation broth prepared by the method of Example 3 had a succinic acid yield of 87.5 g / L, an acid production rate of 1.91 g / L / h, a malic acid content of 0.30 g / L, and an acetic acid content of 0.89 g / L.

[0037] Comparative Example 1 Based on Example 1, the modifications are as follows: In one coating step, the modified chitosan was replaced with chitosan without any treatment; The rest of the operations are the same.

[0038] The fermentation broth prepared by the method of Comparative Example 1 had a succinic acid yield of 81.5 g / L, an acid production rate of 1.78 g / L / h, a malic acid content of 0.74 g / L, and an acetic acid content of 1.16 g / L.

[0039] Comparative Example 2 Based on Example 1, the modification is that in the step of preparing the immobilized microspheres, ① Omitting the steps of pretreatment, modification and primary coating of polyvinyl alcohol fiber; The secondary coating step is to add the wall material solution to the seed culture solution, stir evenly, then add 2wt% calcium chloride solution, stir at 120rpm for 1.2h, filter, wash, and dry to obtain the immobilized microspheres; The mass ratio of the seed culture solution, the wall material solution and the calcium chloride solution is 100:13:2.6; The preparation method of the wall material solution is the same as that of Example 1; ② In the fermentation culture step, niacinamide, vitamin B5 and active ingredients in the fermentation medium are omitted; The rest of the operations are the same.

[0040] The fermentation broth prepared by the method of Comparative Example 2 had a succinic acid yield of 74.3 g / L, an acid production rate of 1.63 g / L / h, a malic acid content of 0.97 g / L, and an acetic acid content of 1.25 g / L.

[0041] Application test The fermentation broths prepared in Example 1-3 and Comparative Example 1-2 were discharged respectively, and the immobilized microspheres were retained in the fermentation medium. After cleaning, fresh fermentation mediums identical to those in Example 1-3 and Comparative Example 1-2 were added respectively, and the next fermentation was carried out completely according to the fermentation conditions and parameters of Example 1-3 and Comparative Example 1-2; 25 batches were fermented according to the above method. After the single batch fermentation was completed, the succinic acid content (g / L) in the fermentation broth was as follows:

[0042] The present invention adopts a specific method to prepare modified polyvinyl alcohol fiber. In the step of pretreating the polyvinyl alcohol fiber, the hydroxyl groups of the polyvinyl alcohol fiber are carboxylated so that the surface of the pretreated polyvinyl alcohol fiber is rich in a large number of carboxyl groups. Then, the fiber is modified by grafting the amino groups of chitosan with the hydroxyl groups of the pretreated polyvinyl alcohol fiber so that the chitosan and the polyvinyl alcohol fiber can be firmly combined, thereby enhancing the stability of the chitosan. Then, the fiber is placed in a seed culture solution for a primary coating so that the outside of the strain is uniformly coated with chitosan with a three-dimensional network structure. Then, a secondary coating is performed to realize the immobilized microspheres with the strain as the microsphere core, chitosan as the inner wall material, and carrageenan as the outer wall material. The immobilized microspheres prepared by the present invention using the specific method have good mass transfer performance, high mechanical properties, and good stimulation to the strain. The invention has low activity, will not affect the activity of the strain, the strain has high activity and can maintain it for a long time, and has good permeability, which is convenient for the transportation of carbon dioxide gas, solves the product inhibition phenomenon, and is combined with specific fermentation culture and other technical means to improve the yield and production rate of succinic acid, reduce the generation of by-products such as malic acid, and when the immobilized microspheres are used for repeated batch production, the succinic acid yield is stable and can still be maintained at a high level; the invention adopts a specific method for fermentation culture, wherein the active agent acts on the protein group on the cell membrane, thereby increasing the permeability of the cell membrane, and vitamin B5 and nicotinamide can provide nutrients, promote the activity of the enzyme, and thus promote the growth of the bacteria; it is combined with the immobilized microspheres to promote the fermentation culture, improve the yield of the product, and reduce the content of by-products.

[0043] From the above comparison results, it can be seen that compared with Example 1, Comparative Example 1 only uses untreated chitosan and carrageenan for coating. The untreated chitosan has poor adsorption stability on the bacteria, and the bacteria are easy to fall off, which affects the permeability during the fermentation culture process, thereby leading to the occurrence of product inhibition, and ultimately reducing the production of succinic acid. The by-product content is high. In repeated tests, the bacteria gradually fall off, and in the subsequent fermentation process, the production of succinic acid gradually decreases, affecting the fermentation reaction.

[0044] Comparative Example 2 omitted one coating step and only used carrageenan for single-layer coating. In addition, the active agent, nicotinamide and vitamin B5 components were omitted in the fermentation medium. The cell membrane permeability was poor during the fermentation culture process, and sufficient nutrients could not be provided to the bacteria. In the later stage of fermentation, the nutrient supply was insufficient and the enzyme activity was reduced, which led to slow growth of the bacteria and inability to achieve complete fermentation culture of the strain, resulting in slow production rate of succinic acid, low yield and high by-product content. In repeated fermentation experiments, the strain coating was incomplete and easy to fall off, and the succinic acid production was unstable, showing a gradually declining trend.

[0045] Unless otherwise specified, all percentages used in the present invention are by mass.

[0046] Finally, it should be noted that the above 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 immobilized cells, characterized in that: The method comprises the steps of shake flask culture, seed culture, preparation of immobilized microspheres and fermentation culture; The shake flask culture step comprises: taking yeast and inoculating it into a shake flask medium for shake flask culture; The yeast is Pichia pastoris ( Pichia sp. ), the deposit number is GDMCC NO:64916; The steps of preparing the immobilized microspheres include pretreatment, modification, primary coating and secondary coating of polyvinyl alcohol fibers; The modification step comprises: mixing the pretreated polyvinyl alcohol fiber and deionized water, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and stirring to obtain a mixed solution; mixing chitosan and acetic acid solution, stirring to obtain a chitosan solution; dropping the mixed solution into the chitosan solution, reacting at 8.0-8.5° C. for 21-23 hours after the dropping is completed, and washing and drying to obtain modified chitosan; The first coating step is to place the modified chitosan in a seed culture solution for ultrasonic oscillation treatment, then add protocatechuic aldehyde, react for 7.3-7.8 hours, and prepare a first coating solution; The secondary coating step is to add the wall material solution to the primary coating solution, then add the calcium chloride solution, stir and react for 1.5-1.7 hours, filter, wash and dry to obtain the immobilized microspheres; The fermentation culture step comprises: adding the immobilized microspheres into a fermentation medium for fermentation culture to obtain a fermentation culture solution; The fermentation medium comprises nicotinamide, vitamin B5 and an active agent component, wherein the active agent is a mixture of Tween 80 and polyethylene glycol 600.

2. The method for producing succinic acid by immobilized cells according to claim 1, characterized in that: The shaking flask culture step comprises taking yeast from the plate and inoculating it into a shaking flask medium for shaking flask culture, wherein the culture time is 11-13 hours, the culture temperature is 33-35° C., the shaking frequency is 160-164 rpm, and after the culture is completed, a shaking flask culture solution is obtained; The shaking flask culture medium comprises the following components: 4.5-4.9 g / L tryptone, 4.3-4.7 g / L NaCl, 9.1-9.6 g / L yeast powder, and the rest is water.

3. The method for producing succinic acid by immobilized cells according to claim 1, characterized in that: The seed culture step comprises inoculating the shake flask culture solution into the seed culture medium at an inoculation rate of 1.8-2.2% by volume, culturing for 16-18 hours, at a temperature of 33-35° C., at a rotation speed of 173-185 rpm, and obtaining the seed culture solution after the culturing is completed; The seed culture medium is composed of the following ingredients: 9-11 g / L glucose, 4.0-4.4 g / L yeast powder, 7.8-8.3 g / L potassium hydrogen phosphate, 0.14-0.16 g / L manganese sulfate, 0.31-0.36 g / L magnesium sulfate heptahydrate, 0.14-0.17 g / L ferrous sulfate, 1.5-1.9 mg / L vitamin B1, 0.7-0.9 mg / L biotin, and the rest is water.

4. The method for producing succinic acid by using immobilized cells according to claim 1, characterized in that: The polyvinyl alcohol fiber pretreatment step comprises sterilizing the polyvinyl alcohol fiber, then putting it into a sodium hydroxide solution with a mass of 9-12 times and stirring it, adding chloroacetic acid after stirring evenly, placing it at room temperature for ultrasonic reaction, the ultrasonic time is 4.0-4.4h, the ultrasonic frequency is 16-20kHz, and the ultrasonic power is 137-142W. After the ultrasonic reaction is completed, filtering, washing, and drying are performed to obtain the pretreated polyvinyl alcohol fiber; The molecular weight of the polyvinyl alcohol fiber is 120,000-140,000 and the diameter is 0.8-1.2 mm; The mass concentration of the sodium hydroxide solution is 14-16%; The mass ratio of the sodium hydroxide solution to chloroacetic acid is 100:12-14.

5. The method for producing succinic acid by using immobilized cells according to claim 1, characterized in that: In the modification step, the drop rate of the mixed solution is 1.0-1.4 g / min; The mass ratio of the polyvinyl alcohol fiber, deionized water and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 15.1-15.5:2.0-2.3:127-132; The mass ratio of chitosan to acetic acid solution is 2.6-2.8:85-90; The mass concentration of the acetic acid solution is 1.0-1.2%; The mass ratio of the mixed solution to the chitosan solution is 2.0-2.2:

1.

6. The method for producing succinic acid by using immobilized cells according to claim 1, characterized in that: In the first coating step, the ultrasonic oscillation treatment has an ultrasonic time of 4.8-5.2h, an ultrasonic power of 117-124W, and an ultrasonic frequency of 11-15kHz; The mass ratio of the modified chitosan to the seed culture solution and protocatechuic aldehyde is 4.0-4.4:117-122:1.8-2.

0.

7. The method for producing succinic acid by using immobilized cells according to claim 1, characterized in that: In the secondary coating step, the mass ratio of the primary coating solution, the wall material solution and the calcium chloride solution is 97-104:12-14:2.5-2.7; The mass concentration of the calcium chloride solution is 1.8-2.2%; The preparation method of the wall material solution is as follows: adding carrageenan to deionized water, then heating it to 80-84° C., stirring it evenly, and sterilizing it at 0.04-0.06 MPa for 22-28 minutes to obtain the wall material solution; The mass ratio of the carrageenan to deionized water is 4-6:81-87.

8. The method for producing succinic acid by using immobilized cells according to claim 1, characterized in that: The fermentation culture step comprises: aseptically treating the immobilized microspheres, and then putting them into a fermentation medium for fermentation culture, wherein the volume of the fermentation medium is 18-22 times the volume of the immobilized microspheres, the fermentation temperature is controlled to be 36-38° C., and carbon dioxide is continuously introduced at a ventilation flow rate of 0.14-0.16 vvm, and the pressure is maintained at 0.04-0.06 MPa. When the residual sugar concentration is reduced to 8-12 g / L, glucose is added to 17-22 g / L, and sodium carbonate is added during the fermentation process to control the pH to 6.5-6.

6. The fermentation time is 45.7-46.0 h, and a fermentation culture solution is obtained; The fermentation medium comprises the following components: 50-54 g / L glucose, 8-10 g / L corn steep liquor, 5.7-5.9 g / L potassium dihydrogen phosphate, 6.3-6.5 g / L potassium hydrogen phosphate, 0.17-0.22 mg / L calcium chloride, 1.67-1.73 mg / L cobalt chloride hexahydrate, 0.46-0.50 mg / L zinc sulfate heptahydrate, 1.6-1.8 mg / L biotin, 6.0-6.5 mg / L nicotinamide, 8.2-8.5 mg / L vitamin B5, 0.32-0.34 g / L active agent, and the pH is 6.7-6.9; In the active agent, the mass ratio of Tween 80 to polyethylene glycol 600 is 1:1.1-1.3.

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