Method for producing succinic acid

Abstract

The invention provides a method for producing succinic acid, and belongs to the field of succinic acid production. The method for producing succinic acid comprises the following steps: shake-flask culture, seed culture, contact fixation and batch continuous fermentation. According to the method for producing succinic acid disclosed by the invention, the production intensity and the conversion rate can be improved, and the generation of byproducts can be inhibited; the adaptability of the carrier to the strain is improved in a targeted manner, the immobilization stability of the carrier to the strain is improved, the carrier is prevented from adsorbing infectious microbes in the fermentation process, and the multi-batch continuous fermentation production performance is effectively maintained.

Description

Technical Field

[0001] The invention relates to the field of succinic acid production, in particular to a method for producing succinic acid. Background Art

[0002] Succinic acid, also known as succinic acid, is a common natural organic acid that is widely present in animals, plants and microorganisms. It is one of the intermediate products of the TCA cycle of organisms and one of the fermentation products of anaerobic metabolism. In the prior art, succinic acid is an important organic synthesis intermediate and an important raw material for the preparation of chemicals such as butanediol and tetrahydrofuran. It is widely used in the fields of food, medicine, and chemical industry. Specifically, succinic acid can be used as an acidulant, antibacterial agent, etc. in the food field; it can be used as a raw material for the preparation of antibiotics, vitamin A, sedatives, and diuretics in the pharmaceutical field; and it can be used as biodegradable plastics, surfactants, detergents, green solvents, etc. in the chemical field. In addition, succinic acid can also be used as an animal feed additive and a plant growth stimulant in the biological field.

[0003] At present, the production methods of succinic acid mainly include: paraffin oxidation, light oil oxidation, butane oxidation, succinonitrile hydrolysis, catalytic hydrogenation, and biological fermentation. In the process of producing succinic acid by chemical methods, a large amount of non-renewable petrochemical raw materials are consumed, the production cost is high, and the environmental pollution is serious, which directly limits the further application of succinic acid.

[0004] Since succinic acid is an important intermediate product of many strict anaerobes and facultative anaerobes, the bio-fermentation method is to use microorganisms to produce succinic acid through fermentation. Existing microorganisms that can produce succinic acid through fermentation include Escherichia coli, succinic acid-producing actinomycetes, succinic acid-producing anaerobic spirilla, etc.; at the same time, lactic acid bacteria, propionic acid-producing bacteria and fungi can also produce a small amount of succinic acid. Since the bio-fermentation method has the advantages of a wide source of raw materials, low production cost, low production energy consumption, renewable, and good environmental benefits, the production of succinic acid by bio-fermentation has become a hot research direction.

[0005] In the process of continuous fermentation of succinic acid by microorganisms, byproducts such as formic acid and acetic acid will be produced, which directly affect its production intensity and conversion rate. In the prior art, in order to suppress the production of byproducts and improve its production intensity and conversion rate, a method of producing succinic acid by fixing the strain is adopted, which mainly adopts fiber or porous material as a carrier, adsorbs and fixes the fermented strain, and then produces succinic acid by continuous fermentation. However, in the process of fixing the strain to produce succinic acid, the adaptability of the carrier to the strain is not good, and the immobilization stability of the carrier to the strain is poor; at the same time, during the fermentation process, it is easy to adsorb miscellaneous bacteria, which not only affects the continuous fermentation production performance of multiple batches, but also causes miscellaneous bacteria contamination in the fermentation process.

[0006] Therefore, how to inhibit the production of by-products while improving the production intensity and conversion rate in the production of succinic acid by fixed strain fermentation; and how to specifically improve the adaptability of the carrier to the strain, improve the immobilization stability of the carrier to the strain, avoid the adsorption of miscellaneous bacteria by the carrier during the fermentation process, and maintain the continuous fermentation production performance of multiple batches, has important technical significance and research value. Summary of the invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides a method for producing succinic acid, which can inhibit the generation of by-products while improving the production intensity and conversion rate; and specifically improve the adaptability of the carrier to the bacterial strain, improve the immobilization stability of the carrier to the bacterial strain, avoid the adsorption of miscellaneous bacteria by the carrier during the fermentation process, and effectively maintain the continuous fermentation production performance of multiple batches.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for producing succinic acid, comprising the following steps: shake flask culture, seed culture, contact fixation, and batch continuous fermentation; The shake flask culture comprises inoculating Pichia pastoris into a shake flask medium for shake flask culture to obtain a shake flask culture fluid; The Pichia pastoris is classified as Pichia sp., the deposit number is GDMCC No: 64916, deposited in Guangdong Microbiological Culture Collection Center, the deposit date is July 26, 2024, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; The seed culture comprises inoculating the shake flask culture solution into the seed culture medium for seed culture to obtain the seed culture solution; The contact fixation includes fixing the composite fiber cloth carrier to the stirring shaft of the fermentation tank, inoculating the seed culture solution into the liquid fermentation medium and then fermenting and culturing, fixing the Pichia pastoris on the composite fiber cloth carrier, and obtaining a fiber bed reactor with the Pichia pastoris fixed thereon; The composite fiber cloth carrier is prepared by the following steps: a first treatment, a second treatment; The first treatment comprises: using a first treatment liquid containing a silane coupling agent KH-550 to immerse the flax fiber cloth to obtain a modified flax fiber cloth; The second treatment comprises adding zinc nitrate hexahydrate into a solvent, dispersing the mixture evenly, adding the mixture into a modified flax fiber cloth, allowing the mixture to stand at room temperature for immersion, sequentially adding terephthalic acid and triethylamine, sealing the mixture and heating the mixture for reaction, taking out the treated modified flax fiber cloth, washing the mixture, and drying the mixture to obtain a composite fiber cloth carrier; The batch continuous fermentation uses a fiber bed reactor with Pichia yeast fixed thereon, and performs batch continuous fermentation by batch replacement of liquid fermentation medium and fermentation culture.

[0009] Preferably, in the shake flask culture, the shake flask culture temperature is 34-37°C, the shaking frequency is 180-200 rpm, and the culture time is 12-16 h; The shaking flask culture medium is composed of the following components: 7.4-8 g / L tryptone, 6.8-7.5 g / L yeast powder, 4-4.5 g / L NaCl, and a pH value of 7.0.

[0010] Preferably, in the seed culture, the inoculation amount of the shake flask culture solution is 1.6-1.8%; The seed culture temperature is controlled at 34-37°C, the rotation speed is 180-200rpm, the seed culture pressure is 0.03-0.05Mpa, and the anaerobic seed culture is performed for 16-18h; The seed culture medium is composed of the following ingredients: glucose 12-12.5g / L, yeast powder 4-4.5g / L, diammonium phosphate 2.82-2.85g / L, diammonium phosphate 5.35-5.40g / L, magnesium sulfate 0.28-0.32g / L, betaine hydrochloride 0.2-0.25g / L, zinc chloride 0.3-0.35mg / L, manganese chloride 0.4-0.45mg / L, copper chloride 0.2-0.3μg / L, boric acid 0.08-0.09μg / L, vitamin B 1 1.5-1.6mg / L, biotin 0.1-0.2mg / L, solvent is water.

[0011] Preferably, in the contact fixation, the ratio of the total volume of the fermentation material in the fermenter to the total area of ​​the composite fiber cloth carrier is controlled to be 7-7.5 cm 3 :1cm 2 ; The inoculation amount of seed culture solution in liquid fermentation medium is 3.8-4%; The liquid fermentation medium is composed of the following ingredients: glucose 70-80 g / L, yeast powder 7.5-8 g / L, corn steep liquor 15-16 g / L, diammonium dihydrogen phosphate 2.82-2.85 g / L, diammonium hydrogen phosphate 5.35-5.40 g / L, magnesium sulfate 0.28-0.32 g / L, betaine hydrochloride 0.2-0.25 g / L, zinc chloride 0.2-0.25 mg / L, manganese chloride 0.4-0.45 mg / L, copper chloride 0.2-0.3 μg / L, boric acid 0.08-0.09 μg / L, vitamin B1 1.5-1.6 mg / L, biotin 0.1-0.2 mg / L, and the solvent is water.

[0012] Preferably, in the contact fixation, after the seed culture solution is inoculated into the liquid fermentation medium, the fermentation culture temperature is controlled to 37-38°C and the stirring speed is controlled to 50-60rpm under the condition of continuous introduction of nitrogen and carbon dioxide mixed gas, and the fermentation culture is carried out for 7-8h to complete the contact fixation.

[0013] Preferably, in the first treatment, the first treatment liquid is an ethanol solution containing a silane coupling agent KH-550; The immersion temperature is 50-55°C and the immersion time is 2-3h; The thickness of the linen fiber cloth is 1.2-1.8 mm and the porosity is 80-85%.

[0014] Preferably, in the second treatment, the immersion time at room temperature is 40-50 minutes; During the sealed temperature-raising reaction process, the temperature is raised to 90-95°C and kept for 5-6 hours.

[0015] Preferably, in the second treatment, the solvent is N,N-dimethylformamide; The weight ratio of the modified flax fiber cloth, zinc nitrate hexahydrate, terephthalic acid, triethylamine and N,N-dimethylformamide is 10-10.5:2.4-2.5:1.5-1.6:1.2-1.3:85-90.

[0016] Preferably, in the batch continuous fermentation, the ratio of the total volume of the fermentation material in each batch to the total area of ​​the composite fiber cloth carrier is controlled to be 7-7.5 cm 3 :1cm 2 ; The liquid fermentation medium is composed of the following ingredients: glucose 70-80 g / L, yeast powder 7.5-8 g / L, corn steep liquor 15-16 g / L, diammonium dihydrogen phosphate 2.82-2.85 g / L, diammonium hydrogen phosphate 5.35-5.40 g / L, magnesium sulfate 0.28-0.32 g / L, betaine hydrochloride 0.2-0.25 g / L, zinc chloride 0.2-0.25 mg / L, manganese chloride 0.4-0.45 mg / L, copper chloride 0.2-0.3 μg / L, boric acid 0.08-0.09 μg / L, vitamin B1 1.5-1.6 mg / L, biotin 0.1-0.2 mg / L, and the solvent is water.

[0017] Preferably, in the batch continuous fermentation, under the condition of continuous introduction of nitrogen and carbon dioxide mixed gas, the fermentation culture temperature is controlled to be 37-38°C, the stirring speed is 50-60rpm, and the fermentation culture is carried out to a residual sugar concentration of 4g / L, then the fermentation culture of the batch is stopped to obtain the fermentation culture liquid; after the fermentation culture liquid is discharged and collected, the liquid fermentation culture medium of the next batch is re-introduced to carry out the fermentation culture of the next batch.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The method for producing succinic acid of the present invention comprises the steps of shake flask culture, seed culture, contact fixation, and batch continuous fermentation, and the immobilization of Pichia pastoris on a composite fiber cloth carrier. The various technical means cooperate with each other and work synergistically, thereby improving the production intensity and conversion rate of succinic acid while inhibiting the production of by-products during the fermentation process; and specifically improving the adaptability of the carrier to the bacterial strain, improving the immobilization stability of the carrier to the bacterial strain, avoiding the adsorption of miscellaneous bacteria by the carrier during the fermentation process, and effectively maintaining the continuous fermentation production performance of multiple batches.

[0019] (2) The method for producing succinic acid of the present invention controls the ratio of the total volume of the fermentation material in each batch to the total area of ​​the composite fiber cloth carrier to be 7.2 cm 3 :1cm 2 And under the condition of glucose content of 80g / L, the succinic acid content in the fermentation culture medium prepared in the first batch was 80.2-80.8g / L, the acetic acid content was 0.92-0.93g / L, the lactic acid content was 0.09-0.11g / L, the succinic acid conversion rate was 1.003-1.01g / g, and the production intensity was 2.60-2.63g / L / h.

[0020] (3) In the method for producing succinic acid of the present invention, the ratio of the total volume of the fermented material in each batch to the total area of ​​the composite fiber cloth carrier is controlled to be 7.2 cm 3 :1cm 2 Under the condition of 80 g / L glucose dosage in each batch, the succinic acid content in the fermentation broth obtained in the 20th fermentation was 78.4 g / L, the acetic acid content was 0.96 g / L, the succinic acid conversion rate was 0.98 g / g, and the production intensity was 2.56 g / L / h.

[0021] (4) In the method for producing succinic acid of the present invention, the ratio of the total volume of the fermented material in each batch to the total area of ​​the composite fiber cloth carrier is controlled to be 7.2 cm 3 :1cm 2 Under the condition of 80 g / L glucose dosage in each batch, the succinic acid content in the fermentation broth obtained in the 40th fermentation was 72.8 g / L, the acetic acid content was 1.04 g / L, the succinic acid conversion rate was 0.91 g / g, and the production intensity was 2.40 g / L / h. DETAILED DESCRIPTION

[0022] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0023] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, "first", "second", etc. are used to distinguish similar objects, and are not used to describe a specific order or sequence. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0024] The embodiment of the present invention provides a method for producing succinic acid, which comprises the following steps: shake flask culture, seed culture, contact fixation, and batch continuous fermentation.

[0025] The shaking flask culture method comprises the following steps: inoculating the Pichia yeast cells on the plate into the shaking flask culture medium, controlling the shaking flask culture temperature to 34-37°C, the shaking frequency to 180-200 rpm, and the culture time to 12-16 hours, and performing shaking flask culture to obtain the shaking flask culture solution.

[0026] The Pichia yeast used in the embodiment of the present invention is classified and named Pichia sp., collected and isolated and purified in Nanning, Guangxi, China in April 2024, and preserved in Guangdong Microbiological Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 64916, and the preservation date is July 26, 2024.

[0027] The shaking flask culture medium is composed of the following components: 7.4-8 g / L tryptone, 6.8-7.5 g / L yeast powder, 4-4.5 g / L NaCl, and a pH value of 7.0.

[0028] The seed culture method comprises the following steps: inoculating the shake flask culture solution obtained by the shake flask culture into the seed culture medium at an inoculation rate of 1.6-1.8% to carry out anaerobic seed culture; controlling the seed culture temperature to be 34-37°C, the rotation speed to be 180-200rpm, and the seed culture pressure to be 0.03-0.05Mpa; at the same time, maintaining an anaerobic environment during the seed culture process, and not introducing any air into the seed culture medium; controlling the pH value to be maintained at 7.0 during the entire seed culture process, carrying out the seed culture for 16-18h, and obtaining the seed culture solution.

[0029] The seed culture medium is composed of the following ingredients: 12-12.5 g / L glucose, 4-4.5 g / L yeast powder, 2.82-2.85 g / L diammonium phosphate, 5.35-5.40 g / L diammonium hydrogen phosphate, 0.28-0.32 g / L magnesium sulfate, 0.2-0.25 g / L betaine hydrochloride, 0.3-0.35 mg / L zinc chloride, 0.4-0.45 mg / L manganese chloride, 0.2-0.3 μg / L copper chloride, 0.08-0.09 μg / L boric acid, and vitamin B 1 1.5-1.6mg / L, biotin 0.1-0.2mg / L, solvent is water.

[0030] The contact fixing method is to wind the composite fiber cloth carrier onto the stirring shaft of a fermentation tank with a volume of 3L, specifically, the number of winding layers of the composite fiber cloth carrier on the stirring shaft is 4-6 layers, and the ratio of the total volume of the fermentation material in the fermentation tank to the total area of ​​the composite fiber cloth carrier is controlled to be 7-7.5cm 3 :1cm 2 ; Then, a stirring paddle is used to limit the end of the stirring shaft, and a stainless steel wire mesh is used to fix it without affecting the rotation of the stirring shaft to form a composite fiber bed and then sterilize it; then, the liquid fermentation medium is introduced into the fermentation tank fiber bed reactor, and the seed culture liquid obtained by seed culture is inoculated into the liquid fermentation medium at an inoculation rate of 3.8-4%, and a nitrogen and carbon dioxide mixed gas with a volume ratio of 75-80:20-25 is continuously introduced, the fermentation culture temperature is controlled at 37-38°C, the stirring speed is 50-60rpm, and the fermentation culture is carried out for 7-8h to complete the contact fixation to obtain a fiber bed reactor with Pichia pastoris fixed.

[0031] In the contact fixation, the liquid fermentation medium is composed of the following ingredients: 70-80 g / L of glucose, 7.5-8 g / L of yeast powder, 15-16 g / L of corn steep liquor, 2.82-2.85 g / L of diammonium dihydrogen phosphate, 5.35-5.40 g / L of diammonium hydrogen phosphate, 0.28-0.32 g / L of magnesium sulfate, 0.2-0.25 g / L of betaine hydrochloride, 0.2-0.25 mg / L of zinc chloride, 0.4-0.45 mg / L of manganese chloride, 0.2-0.3 μg / L of copper chloride, 0.08-0.09 μg / L of boric acid, 1.5-1.6 mg / L of vitamin B1, and 0.1-0.2 mg / L of biotin, and the solvent is water.

[0032] The composite fiber cloth carrier is prepared by the following steps: a first treatment and a second treatment.

[0033] The first treatment method is to add silane coupling agent KH-550 into an ethanol solution with a volume concentration of 70-75%, stir for 20-30 minutes, and obtain a first treatment liquid; then immerse the flax fiber cloth in the first treatment liquid, heat it to 50-55°C, and keep it warm for 2-3 hours, then take out the treated flax fiber cloth, and dry it in a constant temperature box at 65-70°C to obtain a modified flax fiber cloth.

[0034] In the first treatment, the weight ratio of the flax fiber cloth, the silane coupling agent KH-550, and the ethanol solution is 10-11:2-2.3:100-110; The thickness of the linen fiber cloth is 1.2-1.8 mm and the porosity is 80-85%.

[0035] The second treatment method is as follows: zinc nitrate hexahydrate is added to N,N-dimethylformamide, stirred for 20-30 minutes, then modified flax fiber cloth is added, and after standing and impregnating at room temperature for 40-50 minutes, terephthalic acid is added, and after uniform dispersion, triethylamine is added, and the mixture is transferred into a polytetrafluoroethylene reactor, the reactor is sealed, and the temperature is raised to 90-95° C., and after the reaction is kept warm for 5-6 hours, the treated modified flax fiber cloth is taken out, and the modified flax fiber cloth is washed with N,N-dimethylformamide and deionized water in turn, and then dried to obtain a composite fiber cloth carrier.

[0036] In the second treatment, the weight ratio of the modified flax fiber cloth, zinc nitrate hexahydrate, terephthalic acid, triethylamine, and N,N-dimethylformamide is 10-10.5:2.4-2.5:1.5-1.6:1.2-1.3:85-90.

[0037] In view of the characteristics of the adopted Pichia pastoris and its binding characteristics with the carrier, in the preparation of the composite fiber cloth carrier in the embodiment of the present invention, the flax fiber cloth is first modified by using a first treatment liquid containing a silane coupling agent KH-550, and the amino group of the silane coupling agent KH-550 is retained as an active group on the fiber surface of the flax fiber cloth, which is conducive to forming a coordination bond with zinc ions in the subsequent second treatment, increasing the binding active sites, and improving the binding performance of the modified flax fiber cloth with MOF-5; then in the secondary treatment, it is first immersed in a zinc nitrate solution for immersion and adsorption, and then contacted with terephthalic acid and triethylamine, and MOF-5 crystals are generated in situ on the rough surface and gaps of the flax fiber through chemical bonding and physical anchoring, so as to obtain a composite fiber cloth carrier. Then, in the contact fixation step, after the composite fiber cloth carrier is fixed to the stirring shaft of the fermentation tank, the synergistic adsorption and fixation effect of flax fiber and MOF-5 on the bacteria is used to achieve effective fixation of Pichia pastoris, specifically improve the adaptability of the carrier to the bacteria, improve the immobilization stability of the carrier to the bacteria, and avoid the adsorption of miscellaneous bacteria by the carrier during the fermentation process.

[0038] The batch continuous fermentation method comprises: after the material in the fiber bed reactor fixed with Pichia yeast is emptied after the contact fixation is completed, fresh liquid fermentation medium is introduced, and the total volume of the fermentation material (i.e., the liquid fermentation medium) in each batch and the total area ratio of the composite fiber cloth carrier are controlled to be 7-7.5 cm 3 :1cm 2 ; Control the fermentation temperature to 37-38°C, the fermentation pressure to 0.03-0.05Mpa, the stirring speed to 80-100rpm, and continuously introduce a nitrogen and carbon dioxide mixed gas with a volume ratio of 75-80:20-25 to carry out fermentation culture; During the fermentation culture process, magnesium carbonate is used to control the pH in the range of 6-6.5, and when the residual sugar concentration reaches 4g / L, the fermentation culture of this batch is stopped to obtain a fermentation culture liquid; after discharging and collecting the fermentation culture liquid, fresh liquid fermentation culture medium is continuously introduced, and the next batch of fermentation culture is carried out under the aforementioned fermentation conditions; repeat the aforementioned process to achieve batch continuous fermentation.

[0039] In the batch continuous fermentation, the liquid fermentation medium is composed of the following ingredients: 70-80 g / L of glucose, 7.5-8 g / L of yeast powder, 15-16 g / L of corn steep liquor, 2.82-2.85 g / L of diammonium dihydrogen phosphate, 5.35-5.40 g / L of diammonium hydrogen phosphate, 0.28-0.32 g / L of magnesium sulfate, 0.2-0.25 g / L of betaine hydrochloride, 0.2-0.25 mg / L of zinc chloride, 0.4-0.45 mg / L of manganese chloride, 0.2-0.3 μg / L of copper chloride, 0.08-0.09 μg / L of boric acid, 1.5-1.6 mg / L of vitamin B1, 0.1-0.2 mg / L of biotin, and the solvent is water.

[0040] The present invention uses the aforementioned shake flask culture, seed culture, contact fixation, batch continuous fermentation steps, and is combined with a composite fiber cloth carrier. The various technical means cooperate with each other and work synergistically, which can inhibit the generation of by-products while improving production intensity and conversion rate; and specifically improve the adaptability of the carrier to the bacterial strain, improve the immobilization stability of the carrier to the bacterial strain, avoid the adsorption of miscellaneous bacteria by the carrier during the fermentation process, and effectively maintain the continuous fermentation production performance of multiple batches.

[0041] The present invention is further described below in conjunction with some specific embodiments.

[0042] Example 1 A method for producing succinic acid, specifically: 1. Shake flask culture The Pichia yeast cells on the plate were inoculated into a shake flask culture medium, and the shake flask culture temperature was controlled to be 34° C., the shaking frequency was 180 rpm, and the culture time was 14 h. Shake flask culture was performed to prepare a shake flask culture solution.

[0043] Among them, the Pichia yeast used was classified and named Pichia sp., collected and isolated and purified in Nanning, Guangxi, China in April 2024, and preserved in Guangdong Microbiological Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 64916, and the preservation date is July 26, 2024.

[0044] The shake flask medium consisted of the following ingredients: tryptone 7.6 g / L, yeast powder 7.2 g / L, NaCl 4.3 g / L, and pH 7.0.

[0045] 2. Seed cultivation The shake flask culture solution obtained by shake flask culture is inoculated into the seed culture medium at an inoculum rate of 1.7% to carry out anaerobic seed culture; the seed culture temperature is controlled to be 34°C, the rotation speed is 180rpm, and the seed culture pressure is 0.03Mpa; at the same time, the anaerobic environment is maintained during the seed culture process, and no air is introduced into the seed culture medium; the pH value is controlled to be maintained at 7.0 throughout the seed culture process, and the seed culture is carried out for 16 hours to obtain the seed culture solution.

[0046] The seed culture medium is composed of the following ingredients: glucose 12.5 g / L, yeast powder 4.2 g / L, diammonium phosphate 2.85 g / L, diammonium phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.3 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B 1 1.6mg / L, biotin 0.2mg / L, solvent is water.

[0047] 3. Contact fixation The composite fiber cloth carrier was wound onto the stirring shaft of a fermentation tank with a volume of 3 L. Specifically, the number of winding layers of the composite fiber cloth carrier on the stirring shaft was 4-6, and the total volume of the fermented material in the fermentation tank and the total area ratio of the composite fiber cloth carrier were controlled to be 7.2 cm 3 :1cm 2 ; Then, a stirring paddle is used to limit the end of the stirring shaft, and a stainless steel wire mesh is used to fix it without affecting the rotation of the stirring shaft to form a composite fiber bed and then sterilize it; then, the liquid fermentation medium is introduced into the fermentation tank fiber bed reactor, and the seed culture solution obtained by seed culture is inoculated into the liquid fermentation medium at an inoculation rate of 4%, and a nitrogen and carbon dioxide mixed gas with a volume ratio of 80:20 is continuously introduced, the fermentation culture temperature is controlled at 37°C, the stirring speed is 50rpm, and the fermentation culture is carried out for 7 hours to complete the contact fixation, and obtain a fiber bed reactor with Pichia pastoris fixed.

[0048] The liquid fermentation medium is composed of the following ingredients: glucose 80 g / L, yeast powder 7.5 g / L, corn steep liquor 16 g / L, diammonium dihydrogen phosphate 2.85 g / L, diammonium hydrogen phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.25 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B1 1.6 mg / L, biotin 0.2 mg / L, and the solvent is water.

[0049] The composite fiber cloth carrier used is prepared by the following steps: 1) First treatment The silane coupling agent KH-550 was added to an ethanol solution with a volume concentration of 70%, and stirred for 20 minutes to obtain a first treatment liquid; then the flax fiber cloth was immersed in the first treatment liquid, the temperature was raised to 50°C, and after heat preservation for 2 hours, the treated flax fiber cloth was taken out and dried at 65°C in a constant temperature box to obtain a modified flax fiber cloth.

[0050] Wherein, the weight ratio of the linen fiber cloth, the silane coupling agent KH-550 and the ethanol solution is 10:2.1:100.

[0051] The thickness of the linen fiber cloth is 1.5 mm and the porosity is 85%.

[0052] 2) Second treatment Zinc nitrate hexahydrate was added to N,N-dimethylformamide, stirred for 20 minutes, and then the modified flax fiber cloth was added. After standing and impregnating at room temperature for 40 minutes, terephthalic acid was added. After uniform dispersion, triethylamine was added, and the mixture was transferred into a polytetrafluoroethylene reactor. After the reactor was sealed, the temperature was raised to 90°C, and the reaction was kept warm for 5 hours. Then, the treated modified flax fiber cloth was taken out, and the modified flax fiber cloth was washed with N,N-dimethylformamide and deionized water in turn, and then dried to obtain a composite fiber cloth carrier.

[0053] The weight ratio of the modified flax fiber cloth, zinc nitrate hexahydrate, terephthalic acid, triethylamine and N,N-dimethylformamide is 10:2.4:1.5:1.2:85.

[0054] 4. Batch continuous fermentation After the materials in the fiber bed reactor with Pichia yeast fixed were emptied, fresh liquid fermentation medium was introduced to control the ratio of the total volume of the fermentation materials in each batch to the total area of ​​the composite fiber cloth carrier to be 7.2 cm 3 :1cm 2; Control the fermentation temperature to 38°C, the fermentation pressure to 0.03Mpa, the stirring speed to 80rpm, and continuously introduce a mixed gas of nitrogen and carbon dioxide with a volume ratio of 80:20 to carry out fermentation culture; During the fermentation culture process, magnesium carbonate is used to control the pH in the range of 6-6.5, and when the residual sugar concentration reaches 4g / L, the fermentation culture of this batch is stopped to obtain a fermentation culture liquid; after discharging and collecting the fermentation culture liquid, fresh liquid fermentation culture medium is continuously introduced, and the next batch of fermentation culture is carried out under the aforementioned fermentation conditions; repeat the aforementioned process to achieve batch continuous fermentation.

[0055] The liquid fermentation medium is composed of the following ingredients: glucose 80 g / L, yeast powder 7.5 g / L, corn steep liquor 16 g / L, diammonium dihydrogen phosphate 2.85 g / L, diammonium hydrogen phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.25 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B1 1.6 mg / L, biotin 0.2 mg / L, and the solvent is water.

[0056] Example 2 A method for producing succinic acid, specifically: 1. Shake flask culture The Pichia yeast cells on the plate were inoculated into a shake flask culture medium, and the shake flask culture temperature was controlled to be 35° C., the shaking frequency was 190 rpm, and the culture time was 15 h. Shake flask culture was performed to prepare a shake flask culture solution.

[0057] Among them, the Pichia yeast used was classified and named Pichia sp., collected and isolated and purified in Nanning, Guangxi, China in April 2024, and preserved in Guangdong Microbiological Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 64916, and the preservation date is July 26, 2024.

[0058] The shake flask medium consisted of the following ingredients: tryptone 7.6 g / L, yeast powder 7.2 g / L, NaCl 4.3 g / L, and pH 7.0.

[0059] 2. Seed cultivation The shake flask culture solution obtained by shake flask culture is inoculated into the seed culture medium at an inoculum rate of 1.7% to carry out anaerobic seed culture; the seed culture temperature is controlled to be 36°C, the rotation speed is 190rpm, and the seed culture pressure is 0.04Mpa; at the same time, the anaerobic environment is maintained during the seed culture process, and no air is introduced into the seed culture medium; the pH value is controlled to be maintained at 7.0 throughout the seed culture process, and the seed culture is carried out for 17 hours to obtain the seed culture solution.

[0060] The seed culture medium is composed of the following ingredients: glucose 12.5 g / L, yeast powder 4.2 g / L, diammonium phosphate 2.85 g / L, diammonium phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.3 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B 1 1.6mg / L, biotin 0.2mg / L, solvent is water.

[0061] 3. Contact fixation The composite fiber cloth carrier was wound onto the stirring shaft of a fermentation tank with a volume of 3 L. Specifically, the number of winding layers of the composite fiber cloth carrier on the stirring shaft was 4-6, and the total volume of the fermented material in the fermentation tank and the total area ratio of the composite fiber cloth carrier were controlled to be 7.2 cm 3 :1cm 2 ; Then, a stirring paddle is used to limit the end of the stirring shaft, and a stainless steel wire mesh is used to fix it without affecting the rotation of the stirring shaft to form a composite fiber bed and then sterilize it; then, the liquid fermentation medium is introduced into the fermentation tank fiber bed reactor, and the seed culture liquid obtained by seed culture is inoculated into the liquid fermentation medium at an inoculation rate of 4%, and a nitrogen and carbon dioxide mixed gas with a volume ratio of 80:20 is continuously introduced, the fermentation culture temperature is controlled at 38°C, the stirring speed is 55rpm, and the fermentation culture is carried out for 7.5h to complete the contact fixation, and a fiber bed reactor with Pichia pastoris fixed is obtained.

[0062] The liquid fermentation medium is composed of the following ingredients: glucose 80 g / L, yeast powder 7.5 g / L, corn steep liquor 16 g / L, diammonium dihydrogen phosphate 2.85 g / L, diammonium hydrogen phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.25 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B1 1.6 mg / L, biotin 0.2 mg / L, and the solvent is water.

[0063] The composite fiber cloth carrier used is prepared by the following steps: 1) First treatment The silane coupling agent KH-550 was added to an ethanol solution with a volume concentration of 72%, and stirred for 25 minutes to obtain a first treatment liquid; then the linen fiber cloth was immersed in the first treatment liquid, the temperature was raised to 53°C, and after heat preservation treatment for 2.5 hours, the treated linen fiber cloth was taken out and dried at 70°C in a constant temperature box to obtain a modified linen fiber cloth.

[0064] Among them, the weight ratio of linen fiber cloth, silane coupling agent KH-550 and ethanol solution is 10.8:2.2:105.

[0065] The thickness of the linen fiber cloth is 1.5 mm and the porosity is 85%.

[0066] 2) Second treatment Zinc nitrate hexahydrate was added to N,N-dimethylformamide, stirred for 25 minutes, and then the modified flax fiber cloth was added. After standing and impregnating at room temperature for 45 minutes, terephthalic acid was added. After uniform dispersion, triethylamine was added, and then the mixture was transferred into a polytetrafluoroethylene reactor. After the reactor was sealed, the temperature was raised to 92°C, and the reaction was kept warm for 5.5 hours. Then, the treated modified flax fiber cloth was taken out, and the modified flax fiber cloth was washed with N,N-dimethylformamide and deionized water in turn, and then dried to obtain a composite fiber cloth carrier.

[0067] The weight ratio of the modified flax fiber cloth, zinc nitrate hexahydrate, terephthalic acid, triethylamine and N,N-dimethylformamide is 10.3:2.5:1.6:1.25:87.

[0068] 4. Batch continuous fermentation After the materials in the fiber bed reactor with Pichia yeast fixed were emptied, fresh liquid fermentation medium was introduced to control the ratio of the total volume of the fermentation materials in each batch to the total area of ​​the composite fiber cloth carrier to be 7.2 cm 3 :1cm 2 ; Control the fermentation temperature to 38°C, the fermentation pressure to 0.03Mpa, the stirring speed to 80rpm, and continuously introduce a mixed gas of nitrogen and carbon dioxide with a volume ratio of 80:20 to carry out fermentation culture; During the fermentation culture process, magnesium carbonate is used to control the pH in the range of 6-6.5, and when the residual sugar concentration reaches 4g / L, the fermentation culture of this batch is stopped to obtain a fermentation culture liquid; after discharging and collecting the fermentation culture liquid, fresh liquid fermentation culture medium is continuously introduced, and the next batch of fermentation culture is carried out under the aforementioned fermentation conditions; repeat the aforementioned process to achieve batch continuous fermentation.

[0069] The liquid fermentation medium is composed of the following ingredients: glucose 80 g / L, yeast powder 7.5 g / L, corn steep liquor 16 g / L, diammonium dihydrogen phosphate 2.85 g / L, diammonium hydrogen phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.25 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B1 1.6 mg / L, biotin 0.2 mg / L, and the solvent is water.

[0070] Example 3 A method for producing succinic acid, specifically: 1. Shake flask culture The Pichia yeast cells on the plate were inoculated into a shake flask culture medium, and the shake flask culture temperature was controlled to be 37° C., the shaking frequency was 200 rpm, and the culture time was 16 h. Shake flask culture was performed to prepare a shake flask culture solution.

[0071] Among them, the Pichia yeast used was classified and named Pichia sp., collected and isolated and purified in Nanning, Guangxi, China in April 2024, and preserved in Guangdong Microbiological Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 64916, and the preservation date is July 26, 2024.

[0072] The shake flask medium consisted of the following ingredients: tryptone 7.6 g / L, yeast powder 7.2 g / L, NaCl 4.3 g / L, and pH 7.0.

[0073] 2. Seed cultivation The shake flask culture solution obtained by shake flask culture is inoculated into the seed culture medium at an inoculum rate of 1.7% to carry out anaerobic seed culture; the seed culture temperature is controlled to be 37°C, the rotation speed is 200rpm, and the seed culture pressure is 0.05Mpa; at the same time, the anaerobic environment is maintained during the seed culture process, and no air is introduced into the seed culture medium; the pH value is controlled to be maintained at 7.0 throughout the seed culture process, and the seed culture is carried out for 18 hours to obtain the seed culture solution.

[0074] The seed culture medium is composed of the following ingredients: glucose 12.5 g / L, yeast powder 4.2 g / L, diammonium phosphate 2.85 g / L, diammonium phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.3 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B 1 1.6mg / L, biotin 0.2mg / L, solvent is water.

[0075] 3. Contact fixation The composite fiber cloth carrier was wound onto the stirring shaft of a fermentation tank with a volume of 3 L. Specifically, the number of winding layers of the composite fiber cloth carrier on the stirring shaft was 4-6, and the total volume of the fermented material in the fermentation tank and the total area ratio of the composite fiber cloth carrier were controlled to be 7.2 cm 3 :1cm 2; Then, a stirring paddle is used to limit the end of the stirring shaft, and a stainless steel wire mesh is used to fix it without affecting the rotation of the stirring shaft to form a composite fiber bed and then sterilize it; then, the liquid fermentation medium is introduced into the fermentation tank fiber bed reactor, and the seed culture solution obtained by seed culture is inoculated into the liquid fermentation medium at an inoculation rate of 4%, and a nitrogen and carbon dioxide mixed gas with a volume ratio of 80:20 is continuously introduced, the fermentation culture temperature is controlled at 38°C, the stirring speed is 60rpm, and the fermentation culture is carried out for 8 hours to complete the contact fixation, and obtain a fiber bed reactor with Pichia pastoris fixed.

[0076] The liquid fermentation medium is composed of the following ingredients: glucose 80 g / L, yeast powder 7.5 g / L, corn steep liquor 16 g / L, diammonium dihydrogen phosphate 2.85 g / L, diammonium hydrogen phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.25 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B1 1.6 mg / L, biotin 0.2 mg / L, and the solvent is water.

[0077] The composite fiber cloth carrier used is prepared by the following steps: 1) First treatment The silane coupling agent KH-550 was added to an ethanol solution with a volume concentration of 75%, and stirred for 30 minutes to obtain a first treatment liquid; then the flax fiber cloth was immersed in the first treatment liquid, the temperature was raised to 55°C, and after heat preservation for 3 hours, the treated flax fiber cloth was taken out and dried at 70°C in a constant temperature box to obtain a modified flax fiber cloth.

[0078] Among them, the weight ratio of linen fiber cloth, silane coupling agent KH-550 and ethanol solution is 11:2.3:110.

[0079] The thickness of the linen fiber cloth is 1.5 mm and the porosity is 85%.

[0080] 2) Second treatment Zinc nitrate hexahydrate was added to N,N-dimethylformamide, stirred for 30 minutes, and then the modified flax fiber cloth was added. After standing and impregnating at room temperature for 50 minutes, terephthalic acid was added. After uniform dispersion, triethylamine was added, and then the mixture was transferred into a polytetrafluoroethylene reactor. After the reactor was sealed, the temperature was raised to 95°C, and the reaction was kept warm for 6 hours. Then, the treated modified flax fiber cloth was taken out, and the modified flax fiber cloth was washed with N,N-dimethylformamide and deionized water in turn, and then dried to obtain a composite fiber cloth carrier.

[0081] Wherein, the weight ratio of modified flax fiber cloth, zinc nitrate hexahydrate, terephthalic acid, triethylamine and N,N-dimethylformamide is 10.5:2.5:1.6:1.3:90.

[0082] 4. Batch continuous fermentation After the materials in the fiber bed reactor with Pichia yeast fixed were emptied, fresh liquid fermentation medium was introduced to control the ratio of the total volume of the fermentation materials in each batch to the total area of ​​the composite fiber cloth carrier to be 7.2 cm 3 :1cm 2 ; Control the fermentation temperature to 38°C, the fermentation pressure to 0.03Mpa, the stirring speed to 80rpm, and continuously introduce a mixed gas of nitrogen and carbon dioxide with a volume ratio of 80:20 to carry out fermentation culture; During the fermentation culture process, magnesium carbonate is used to control the pH in the range of 6-6.5, and when the residual sugar concentration reaches 4g / L, the fermentation culture of this batch is stopped to obtain a fermentation culture liquid; after discharging and collecting the fermentation culture liquid, fresh liquid fermentation culture medium is continuously introduced, and the next batch of fermentation culture is carried out under the aforementioned fermentation conditions; repeat the aforementioned process to achieve batch continuous fermentation.

[0083] The liquid fermentation medium is composed of the following ingredients: glucose 80 g / L, yeast powder 7.5 g / L, corn steep liquor 16 g / L, diammonium dihydrogen phosphate 2.85 g / L, diammonium hydrogen phosphate 5.40 g / L, magnesium sulfate 0.3 g / L, betaine hydrochloride 0.25 g / L, zinc chloride 0.25 mg / L, manganese chloride 0.45 mg / L, copper chloride 0.25 μg / L, boric acid 0.09 μg / L, vitamin B1 1.6 mg / L, biotin 0.2 mg / L, and the solvent is water.

[0084] Comparative Example 1 For comparative experiments, Comparative Example 1 adopts the technical solution of Example 3, with the difference that in the preparation of the composite fiber cloth carrier, the first treatment liquid is not used to treat the flax fiber cloth, and the untreated flax fiber cloth is directly used in the second treatment.

[0085] Comparative Example 2 For comparative experiments, Comparative Example 2 adopts the technical solution of Example 3, with the difference that no composite fiber cloth carrier is prepared, and untreated flax fiber cloth is directly used as the carrier material to be wound and fixed to the stirring shaft.

[0086] The first batch of fermentation broth obtained in the batch continuous fermentation steps of Examples 1-3 and Comparative Examples 1-2 was tested respectively, specifically the succinic acid content, formic acid content, acetic acid content, and lactic acid content in the fermentation broth were tested, and the conversion rate (g / g) and production intensity were calculated. The specific results are shown below:

[0087] Further, the batch continuous fermentation methods of Example 3 and Comparative Example 1 were used to carry out batch continuous fermentation respectively; after the fermentation culture of the previous batch was completed, the fermentation culture medium was discharged and collected, and then fresh liquid fermentation culture medium was continuously introduced to carry out the fermentation culture of the next batch; the succinic acid content and acetic acid content in the fermentation culture medium of the 20th and 40th times were detected respectively, and the conversion rate (g / g) and production intensity were calculated. The specific results are shown as follows:

[0088] It can be seen that after the first treatment is omitted in Comparative Example 1, the binding performance of the flax fiber in the composite fiber cloth carrier and MOF-5 is reduced. During the batch continuous fermentation, the immobilization stability of the carrier on the strain is reduced, and the inhibitory effect on the by-products is reduced; specifically, the succinic acid content, conversion rate, and production intensity in the 20th and 40th fermentation broth are significantly reduced compared with Example 3, and the by-product acetic acid content is increased. Comparative Example 2 directly uses flax fiber cloth as a carrier, the compatibility of the carrier and the strain is poor, the immobilization effect on the bacteria is reduced, and the carrier cannot avoid the adsorption of miscellaneous bacteria during the fermentation process; specifically, the succinic acid content, conversion rate, and production intensity in the first batch of fermentation broth are significantly reduced, the by-product content is increased, and the formic acid content of the miscellaneous bacteria metabolites is increased.

[0089] Unless otherwise specified, all percentages used in the present invention are by mass.

[0090] 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, characterized in that, The method consists of the following steps: shake flask culture, seed culture, contact fixation, and batch continuous fermentation; The shake flask culture comprises inoculating Pichia pastoris into a shake flask medium for shake flask culture to obtain a shake flask culture fluid; The Pichia pastoris is classified as Pichia sp., the deposit number is GDMCC No: 64916, deposited in Guangdong Microbiological Culture Collection Center, the deposit date is July 26, 2024, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; The seed culture comprises inoculating the shake flask culture solution into the seed culture medium for seed culture to obtain the seed culture solution; The contact fixation includes fixing the composite fiber cloth carrier to the stirring shaft of the fermentation tank, inoculating the seed culture solution into the liquid fermentation medium and then fermenting and culturing, fixing the Pichia pastoris on the composite fiber cloth carrier, and obtaining a fiber bed reactor with the Pichia pastoris fixed thereon; The composite fiber cloth carrier is prepared by the following steps: a first treatment, a second treatment; The first treatment comprises: impregnating the flax fiber cloth with a first treatment liquid containing a silane coupling agent KH-550 to obtain a modified flax fiber cloth; The second treatment comprises adding zinc nitrate hexahydrate into a solvent, dispersing the mixture evenly, adding the mixture into a modified flax fiber cloth, allowing the mixture to stand at room temperature for immersion, sequentially adding terephthalic acid and triethylamine, sealing the mixture and heating the mixture for reaction, taking out the treated modified flax fiber cloth, washing the mixture, and drying the mixture to obtain a composite fiber cloth carrier; The batch continuous fermentation uses a fiber bed reactor with Pichia yeast fixed thereon, and performs batch continuous fermentation by batch replacement of liquid fermentation medium and fermentation culture.

2. The method for producing succinic acid according to claim 1, characterized in that In the shake flask culture, the shake flask culture temperature is 34-37°C, the shaking frequency is 180-200 rpm, and the culture time is 12-16 hours; The shaking flask culture medium is composed of the following components: 7.4-8 g / L tryptone, 6.8-7.5 g / L yeast powder, 4-4.5 g / L NaCl, and a pH value of 7.

0.

3. The method for producing succinic acid according to claim 1, characterized in that In the seed culture, the inoculation amount of the shake flask culture solution is 1.6-1.8%; The seed culture temperature is controlled at 34-37°C, the rotation speed is 180-200rpm, the seed culture pressure is 0.03-0.05Mpa, and the anaerobic seed culture is performed for 16-18h; The seed culture medium is composed of the following ingredients: glucose 12-12.5 g / L, yeast powder 4-4.5 g / L, diammonium phosphate 2.82-2.85 g / L, diammonium hydrogen phosphate 5.35-5.40 g / L, magnesium sulfate 0.28-0.32 g / L, betaine hydrochloride 0.2-0.25 g / L, zinc chloride 0.3-0.35 mg / L, manganese chloride 0.4-0.45 mg / L, copper chloride 0.2-0.3 μg / L, boric acid 0.08-0.09 μg / L, vitamin B1 1.5-1.6 mg / L, biotin 0.1-0.2 mg / L, and the solvent is water.

4. The method for producing succinic acid according to claim 1, characterized in that In the contact fixation, the ratio of the total volume of the fermentation material in the fermenter to the total area of ​​the composite fiber cloth carrier is controlled to be 7-7.5 cm 3 :1cm 2 ; The inoculation amount of seed culture solution in liquid fermentation medium is 3.8-4%; The liquid fermentation medium is composed of the following ingredients: glucose 70-80 g / L, yeast powder 7.5-8 g / L, corn steep liquor 15-16 g / L, diammonium dihydrogen phosphate 2.82-2.85 g / L, diammonium hydrogen phosphate 5.35-5.40 g / L, magnesium sulfate 0.28-0.32 g / L, betaine hydrochloride 0.2-0.25 g / L, zinc chloride 0.2-0.25 mg / L, manganese chloride 0.4-0.45 mg / L, copper chloride 0.2-0.3 μg / L, boric acid 0.08-0.09 μg / L, vitamin B1 1.5-1.6 mg / L, biotin 0.1-0.2 mg / L, and the solvent is water.

5. The method for producing succinic acid according to claim 1, characterized in that In the contact fixation, after the seed culture solution is inoculated into the liquid fermentation medium, the fermentation culture temperature is controlled to 37-38° C. and the stirring speed is controlled to 50-60 rpm under the condition of continuous introduction of nitrogen and carbon dioxide mixed gas, and the fermentation culture is carried out for 7-8 hours to complete the contact fixation.

6. The method for producing succinic acid according to claim 1, characterized in that In the first treatment, the first treatment liquid is an ethanol solution containing a silane coupling agent KH-550; The immersion temperature is 50-55°C and the immersion time is 2-3h; The thickness of the linen fiber cloth is 1.2-1.8 mm and the porosity is 80-85%.

7. The method for producing succinic acid according to claim 1, characterized in that In the second treatment, the immersion time at room temperature is 40-50 minutes; During the sealed temperature-raising reaction process, the temperature is raised to 90-95°C and kept for 5-6 hours.

8. The method for producing succinic acid according to claim 1, characterized in that In the second treatment, the solvent is N,N-dimethylformamide; The weight ratio of the modified flax fiber cloth, zinc nitrate hexahydrate, terephthalic acid, triethylamine and N,N-dimethylformamide is 10-10.5:2.4-2.5:1.5-1.6:1.2-1.3:85-90.

9. The method for producing succinic acid according to claim 1, characterized in that In the batch continuous fermentation, the ratio of the total volume of the fermentation material in each batch to the total area of ​​the composite fiber cloth carrier is controlled to be 7-7.5 cm 3 :1cm 2 ; The liquid fermentation medium is composed of the following ingredients: glucose 70-80 g / L, yeast powder 7.5-8 g / L, corn steep liquor 15-16 g / L, diammonium dihydrogen phosphate 2.82-2.85 g / L, diammonium hydrogen phosphate 5.35-5.40 g / L, magnesium sulfate 0.28-0.32 g / L, betaine hydrochloride 0.2-0.25 g / L, zinc chloride 0.2-0.25 mg / L, manganese chloride 0.4-0.45 mg / L, copper chloride 0.2-0.3 μg / L, boric acid 0.08-0.09 μg / L, vitamin B1 1.5-1.6 mg / L, biotin 0.1-0.2 mg / L, and the solvent is water.

10. The method for producing succinic acid according to claim 1, characterized in that In the batch continuous fermentation, under the condition of continuous introduction of nitrogen and carbon dioxide mixed gas, the fermentation culture temperature is controlled to be 37-38°C, the stirring speed is 50-60rpm, and the fermentation culture is carried out to a residual sugar concentration of 4g / L, then the fermentation culture of the batch is stopped to obtain the fermentation culture liquid; after the fermentation culture liquid is discharged and collected, the liquid fermentation culture medium of the next batch is reintroduced to carry out the fermentation culture of the next batch.

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