A method for preparing polymer-grade succinic acid

Abstract

The invention discloses a preparation method of polymer-grade succinic acid, belonging to the technical field of succinic acid preparation. The preparation method comprises: activating and expanding Pichia pastoris to obtain a seed liquid; inoculating the seed liquid into 5L of fermentation medium at an inoculum amount of 5% by volume, then adding 19-21g of a fermentation aid to the fermentation medium, and shaking culturing for 80-82 hours under the conditions of a temperature of 30-32°C, a stirring speed of 300-340r / m, and a ventilation volume of 0.1-0.11vvm; controlling the concentration of glucose to 20g / L by feeding a glucose aqueous solution during the shaking culture, and obtaining a fermentation liquid after the shaking culture. The preparation method of the invention can improve the output, yield and production intensity of succinic acid, and the output, yield and production intensity of the prepared succinic acid can be kept stable, and can also improve the purification yield of succinic acid and reduce the purification cost of succinic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of succinic acid preparation, and in particular to a method for preparing polymerization-grade succinic acid. Background Art

[0002] Succinic acid, also known as 1,4-butanedioic acid, occurs as colorless, odorless crystals at room temperature. The succinic acid molecule contains two active methylene groups, exhibiting important reactivity, including halogenation, dehydration, esterification, sulfonation, acylation, oxidation, and reduction. It can react with alkali, ammonia, and hydrogen to produce a variety of organic intermediates and pharmaceutical raw materials. It can also undergo esterification and polycondensation with alcohols to produce bioplastics such as polybutylene succinate and polybutylene succinate terephthalate copolyester.

[0003] Bioplastics such as polybutylene succinate and polybutylene succinate terephthalate copolyester are not only fully biodegradable, but also have excellent processing and mechanical properties. With the strengthening of environmental awareness, the market demand for bioplastics is increasing. As one of the upstream raw materials, succinic acid has also ushered in broad market development prospects.

[0004] The existing methods for preparing succinic acid include catalytic hydrogenation, electrochemical method, and biological method. Among them, the catalytic hydrogenation method is to use maleic anhydride as raw material, and prepare succinic acid through hydrogenation and hydrolysis. However, the catalytic hydrogenation method requires the use of expensive palladium catalysts, which increases the preparation cost of succinic acid; the electrochemical method is to use maleic anhydride as raw material, first hydrolyze maleic anhydride to obtain maleic acid, and then electrolytically reduce maleic acid to obtain succinic acid, but the electrochemical method has problems such as high power consumption, serious electrode corrosion, large sewage discharge, and large land occupation; the biological method includes biotransformation method and biofermentation method. Among them, the biotransformation method is to oxidize benzene to maleic acid in the presence of a catalyst, and then isomerize it to obtain fumaric acid, and then use grain Fumarate reductase produced by fermentation of Corynebacterium amineticum or Enterococcus faecalis converts fumarate into succinic acid, but the cost of the bioconversion method is high, which leads to an increase in the preparation cost of succinic acid; the biological fermentation method uses glucose, starch, corn and other substances existing or produced in nature and human production activities as raw materials, and produces succinic acid through microbial fermentation. Commonly used microorganisms include succinic acid-producing Anaerobic Spirillum, succinic acid-producing Actinobacillus, succinic acid-producing Mannheimia, Corynebacterium glutamicum, Escherichia coli and other bacteria. The biological fermentation method has the advantages of a wide source of raw materials and strong environmental protection. At present, foreign companies mainly use biological fermentation to produce succinic acid.

[0005] When using the biological fermentation method to prepare succinic acid, the following problems exist: First, when the microorganisms are fermenting, the pH value continues to decrease as the succinic acid content increases. At the same time, other acidic by-products such as formic acid, acetic acid, and fumaric acid are produced during the fermentation, which also causes the pH value to continue to decrease. The decrease in pH value will lead to a decrease in the activity of the microorganisms, and further, the production, yield and production intensity of succinic acid will be reduced; Second, the fermentation broth prepared by the biological fermentation method contains, in addition to succinic acid, a variety of impurities, including nitrogen sources used to cultivate microorganisms, acidic by-products, inorganic salts, etc., and the impurity content is high, making it difficult to remove the impurities. After being used in the preparation of polybutylene succinate and polybutylene succinate terephthalate copolyester bioplastics, nitrogen source impurities will affect the color of the bioplastics; formic acid and acetic acid impurities in the acidic by-product impurities will affect the mechanical strength and color of the bioplastics, and will also affect the esterification and polycondensation reaction, further affecting the relative molecular weight of the bioplastics, while fumaric acid impurities in the acidic by-product impurities will cause the relative molecular weight of the bioplastics to widen, further affecting the mechanical strength of the bioplastics; inorganic salt impurities will affect the mechanical strength of the bioplastics.

[0006] To address the above issues, the applicant has attempted the following solutions: First, a neutralizing agent is added to adjust the pH value during microbial fermentation; Second, yeast is used for fermentation. As a fungus, yeast has a high tolerance to low pH conditions. Commonly used yeasts include Yarrowia lipolytica, Saccharomyces cerevisiae, and Pichia pastoris; Third, the fermentation broth prepared by microbial fermentation is purified to obtain high-purity, low-impurity succinic acid, i.e., polymer-grade succinic acid. During purification, ceramic membrane filtration is first used to remove the bacteria and protein in the fermentation broth, and the filtrate is collected. Then, using the calcium salt method, a calcifying agent is added to the filtrate to calcify the succinic acid, converting the succinic acid into insoluble calcium succinate. The calcium succinate is collected by filtration, and then the calcium succinate is acid-hydrolyzed and decolorized. After that, succinic acid is obtained through ion exchange, concentration, vacuum distillation, low-temperature crystallization, and drying.

[0007] However, after trying the above two methods, the applicant encountered the following problems: First, in microbial fermentation, when using a neutralizer to adjust the pH value, commonly used neutralizers include sodium carbonate, magnesium carbonate, and calcium hydroxide. Sodium carbonate and magnesium carbonate will introduce inorganic salt impurities into the fermentation broth. Although calcium hydroxide can provide calcium ions for the subsequent calcium salt method, the high concentration of calcium ions introduced by calcium hydroxide will affect the activity of microorganisms during fermentation; Second, when preparing succinic acid by microbial fermentation, the synthesis pathways of succinic acid mainly include the oxidative tricarboxylic acid cycle pathway, the reductive tricarboxylic acid cycle pathway, and the glyoxylate cycle pathway. Among them, the maximum theoretical yield of succinic acid in the oxidative tricarboxylic acid cycle pathway is 1 mol succinic acid / 1 mol glucose, the maximum theoretical yield of succinic acid in the reductive tricarboxylic acid cycle pathway is 2 mol succinic acid / 1 mol glucose, and the maximum theoretical yield of succinic acid in the glyoxylate cycle pathway is 4 mol succinic acid / 3 mol glucose. The flux of the reductive tricarboxylic acid cycle pathway of yeast is small, and it cannot naturally accumulate succinic acid at a high flux, which further leads to a decrease in the output, yield and production intensity of succinic acid when succinic acid is prepared by yeast fermentation; thirdly, when using the calcium salt method and a calcifying agent for calcification, the succinic acid concentration in the fermentation broth is low, resulting in a low calcification yield, which further leads to a decrease in the purification yield of succinic acid.

[0008] In response to the above problems, the applicant has made the following improvements: in the microbial fermentation method, Pichia pastoris is used for fermentation, and microbial fermentation is carried out in a fiber bed bioreactor. The fiber bed bioreactor uses absorbent cotton gauze to fix the Pichia pastoris. The fiber bed bioreactor has the advantage of not being easily contaminated by foreign bacteria. It can also quickly acclimate and enrich the culture of Pichia pastoris to increase the density of Pichia pastoris, thereby increasing the yield, yield and production intensity of succinic acid. When purifying the fermentation broth, the fermentation broth is first concentrated to increase the succinic acid concentration in the fermentation broth, and then calcified using a calcifying agent to increase the calcification yield. However, when preparing succinic acid according to the above improvements, it was found that the following problems still exist: when using a fiber bed bioreactor, as the thickness and density of the bacterial layer formed by Pichia pastoris on the absorbent cotton gauze gradually increase, the mass transfer efficiency decreases. Further, in the continuous fermentation, the yield, yield and production intensity of succinic acid are unstable, showing a trend of first increasing and then decreasing; the energy consumption is large when concentrating the fermentation broth, which increases the purification cost of succinic acid. Summary of the Invention

[0009] In view of the shortcomings of the existing technology, the present invention provides a method for preparing polymerization-grade succinic acid, which can improve the output, yield and production intensity of succinic acid, and the output, yield and production intensity of the prepared succinic acid can be kept stable. It can also improve the purification yield of succinic acid and reduce the purification cost of succinic acid.

[0010] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0011] A method for preparing polymer-grade succinic acid comprises:

[0012] Step 1 is: activating and expanding the culture of Pichia pastoris to obtain a seed solution;

[0013] Furthermore, step 1 comprises: using a sterile inoculating loop to pick a loopful of bacteria from a frozen tube of Pichia pastoris, streaking the loopful onto 50-60 mL of activation culture medium, culturing the loopful at 30-32° C. for 48-50 hours, then using a sterile toothpick to pick a single colony, inoculating the colony into 180-200 mL of seed culture medium, and culturing the loopful at 30-32° C. and a stirring speed of 180-200 rpm for 24-26 hours to obtain a seed solution;

[0014] The Pichia pastoris used in step 1 was donated by Lao Yongmin's research group at the Frontier Technology Integration Innovation Platform of Traditional Chinese Medicine Resources and Synthetic Biology of the Guangdong Provincial Laboratory of Traditional Chinese Medicine.

[0015] The activation culture medium comprises: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast powder, 20 g / L agar powder, the solvent is purified water, and the pH value is 6;

[0016] The components of the seed culture medium are: glucose 20 g / L, tryptone 20 g / L, yeast powder 10 g / L, the solvent is purified water, and the pH value is 6;

[0017] Step 2 comprises inoculating the seed liquid into 5 L of fermentation medium at an inoculum size of 5% by volume, then adding 19-21 g of a fermentation aid to the fermentation medium, and shaking culturing the culture at a temperature of 30-32° C., a stirring speed of 300-340 rpm, and an aeration volume of 0.1-0.11 vvm for 80-82 hours, controlling the glucose concentration to 20 g / L by feeding a glucose aqueous solution during the shaking culture, and obtaining a fermentation broth after the shaking culture is completed.

[0018] In step 2, the fermentation medium comprises the following components: 50 g / L glucose, 5 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium chloride dihydrate, 0.2 g / L urea, 0.4 mg / L biotin, 1 mg / L calcium pantothenate, 1 mg / L niacin, 25 mg / L inositol, 1 mg / L vitamin B1, 1 mg / L vitamin B6, 10 mL / L trace element mother solution, the solvent is purified water, and the pH value is natural;

[0019] The components of the trace element mother solution are: 0.05 g / L copper sulfate pentahydrate, 2 g / L ferrous sulfate heptahydrate, 0.2 g / L manganese chloride tetrahydrate, 0.5 g / L zinc chloride, 0.04 g / L sodium molybdate dihydrate, 0.03 g / L cobalt chloride hexahydrate, 0.1 g / L boric acid, 0.01 g / L potassium iodide, and the solvent is purified water;

[0020] The mass fraction of glucose in the glucose aqueous solution is 70%;

[0021] The preparation method of the fermentation aid comprises:

[0022] Step 2a comprises: mixing citric acid and purified water, stirring the mixture at a temperature of 20-40° C. and a stirring speed of 100-300 r / min for 10-30 minutes, maintaining the temperature and stirring speed constant, adding an aqueous solution of ferrous chloride, stirring the mixture for 10-30 minutes after the addition is complete, adjusting the pH to 11-12 by adding lime milk, stirring the mixture for 2-3 hours, standing the mixture at 20-40° C. for 5-6 hours, centrifuging the mixture at a centrifugal speed of 9000-10000 r / min for 15-20 minutes, collecting a precipitate, and drying the precipitate in a vacuum at 70-80° C. to obtain a complex;

[0023] In step 2a, the ratio of citric acid, purified water, and ferrous chloride aqueous solution is 9-11 g: 500-600 mL: 370-400 mL;

[0024] The mass fraction of ferrous chloride in the ferrous chloride aqueous solution is 2%;

[0025] The concentration of calcium oxide in the lime milk is 80g / L;

[0026] The addition rate of the ferrous chloride aqueous solution is 10-15 mL / min;

[0027] Step 2b comprises: mixing the complex and purified water, stirring at a temperature of 20-40° C. and a stirring speed of 100-300 r / min for 20-40 minutes, maintaining the temperature and stirring speed under a nitrogen atmosphere, adding an aqueous sodium borohydride solution, stirring for 30-50 minutes after the addition is complete, centrifuging at a centrifugal speed of 9000-10000 r / min for 15-20 minutes, collecting a precipitate, washing the precipitate 3-4 times with purified water, and then vacuum drying at 70-80° C. to obtain a fermentation aid;

[0028] The ratio of citric acid in step 2a to purified water and sodium borohydride aqueous solution in step 2b is 9-11 g:900-1000 mL:140-160 mL;

[0029] In step 2b, the mass fraction of sodium borohydride in the sodium borohydride aqueous solution is 5%;

[0030] The sodium borohydride aqueous solution is added at a rate of 4-5 mL / min;

[0031] Step 3 is: filtering the fermentation broth using a ceramic membrane with a filtration accuracy of 100 nm, collecting the filtrate, and using the collected filtrate as a primary filtrate. Under the conditions of a temperature of 75-80° C. and a stirring speed of 100-300 r / min, adding a calcium chloride aqueous solution to the primary filtrate, stirring for 30-40 minutes after the addition is completed, filtering, collecting the filtrate and the filter residue, and using the collected filtrate as a secondary filtrate, and using the collected filter residue as a primary filter residue; under the conditions of a temperature of 75-80° C. and a stirring speed of 100-300 r / min, adding a calcium chloride aqueous solution containing calcium carbonate to the secondary filtrate, stirring for 30-40 minutes after the addition is completed, filtering, collecting the filter residue, using the collected filter residue as a secondary filter residue, and mixing the primary filter residue and the secondary filter residue to obtain mixed calcium succinate;

[0032] In step 3, the mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%;

[0033] The calcium chloride aqueous solution is added at a rate of 20-30 mL / min;

[0034] The volume ratio of the primary filtrate to the calcium chloride aqueous solution is 1:1-1.2;

[0035] The calcium carbonate-containing calcium chloride aqueous solution has a particle size of 700 mesh, a mass fraction of calcium carbonate of 2%, and a mass fraction of calcium chloride of 30%;

[0036] The volume ratio of the secondary filtrate to the calcium chloride aqueous solution containing calcium carbonate is 10:1-1.2;

[0037] Step 4 comprises mixing calcium succinate, activated carbon, and aqueous sulfuric acid solution, stirring the mixture at a temperature of 75-80° C. and a stirring speed of 100-300 r / min for 30-40 minutes, filtering, and collecting the filtrate as a mixed succinic acid solution;

[0038] In step 4, the ratio of calcium succinate, activated carbon, and aqueous sulfuric acid solution is 300 g: 4-4.2 g: 240-250 mL;

[0039] The mass fraction of the sulfuric acid aqueous solution is 60%;

[0040] Step 5 is: using 732 strong acid cation exchange resin and 331 weak base anion exchange resin to exchange ions on the mixed succinic acid solution in sequence, concentrating under vacuum at 75-80° C. until crystallization occurs, then low-temperature crystallization at 4° C., filtering, collecting the filter residue, and vacuum drying at 60-70° C. to obtain polymer-grade succinic acid.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) The preparation method of polymer-grade succinic acid of the present invention uses a fermentation aid in the fermentation. When preparing the fermentation aid, citrate and ferrous ions are first mixed, and then mixed with lime milk. The citrate, ferrous ions, and calcium ions in the lime milk can be complexed with each other to form a complex. Then, sodium borohydride is used to reduce the ferrous ions in the complex to obtain a mixture of citrate, zero-valent iron, and calcium ions, i.e., the fermentation aid. During the fermentation, the zero-valent iron has reducing properties and can immobilize the Pichia pastoris and serve as a fermentation aid. Electron donor, promoting the reductive tricarboxylic acid cycle. As fermentation progresses, acid gradually accumulates. Zero-valent iron reacts with acid to generate ferrous ions. Ferrous ions can complex with citrate, reducing the inhibition of citrate on citrate synthase and promoting the production of succinic acid. It can also continue to serve as an electron donor. Furthermore, as acid gradually accumulates, the pH value gradually decreases, destroying the complexation between ferrous ions and citrate. However, ferrous ions can continue to promote the reductive tricarboxylic acid cycle, thereby increasing the yield of succinic acid.

[0043] (2) In the preparation method of polymer-grade succinic acid of the present invention, when a calcifying agent is added to calcify succinic acid, considering the low calcium ion concentration, a secondary calcification is performed after the primary calcification. In the secondary calcification, calcium carbonate and calcium hydroxide are used in conjunction as calcifying agents. During the experiment, it was found that when calcium carbonate is used alone as a calcifying agent, the effect is very poor. However, after calcium carbonate and calcium hydroxide are used in conjunction, in a low-concentration succinic acid aqueous solution, the calcium ions on the surface of calcium carbonate can adsorb and fix succinate radicals, promote the combination between calcium hydroxide and succinic acid, thereby improving the purification yield. In addition, compared with concentration, the purification cost of succinic acid can be reduced;

[0044] (3) The preparation method of polymer-grade succinic acid of the present invention can improve the production, yield and production intensity of succinic acid. The production of succinic acid can reach 74.1-79.3 g / L, the yield can reach 1.159-1.185 mol / 1 mol glucose, and the production intensity can reach 0.926-0.967 g / (L•h);

[0045] (4) The method for preparing polymer-grade succinic acid of the present invention can maintain stable yield, rate and production intensity of the prepared succinic acid. According to the method of the present invention, 20 batches of succinic acid were continuously produced, and the difference between the maximum yield and the minimum yield was 0.8-1.1 g / L, and the difference between the maximum yield and the minimum yield was 0.5-0.6 mol / 1 mol glucose.

[0046] (5) The method for preparing polymer-grade succinic acid of the present invention can improve the purification yield of succinic acid. The liquid phase purity of the obtained succinic acid is 99.17-99.25%, and the purification yield is 65.7-66.2%. DETAILED DESCRIPTION

[0047] 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.

[0048] The culture medium and its components used in the Examples and Comparative Examples are as follows:

[0049] The components of the activation culture medium are: glucose 20 g / L, tryptone 20 g / L, yeast powder 10 g / L, agar powder 20 g / L, the solvent is purified water, and the pH value is 6.

[0050] The components of the seed culture medium are: glucose 20 g / L, tryptone 20 g / L, yeast powder 10 g / L, the solvent is purified water, and the pH value is 6.

[0051] The fermentation medium consists of 50 g / L glucose, 5 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium chloride dihydrate, 0.2 g / L urea, 0.4 mg / L biotin, 1 mg / L calcium pantothenate, 1 mg / L niacin, 25 mg / L inositol, 1 mg / L vitamin B1, 1 mg / L vitamin B6, 10 mL / L trace element stock solution. The solvent is purified water, and the pH value is natural.

[0052] The components of the trace element mother solution are: 0.05 g / L of copper sulfate pentahydrate, 2 g / L of ferrous sulfate heptahydrate, 0.2 g / L of manganese chloride tetrahydrate, 0.5 g / L of zinc chloride, 0.04 g / L of sodium molybdate dihydrate, 0.03 g / L of cobalt chloride hexahydrate, 0.1 g / L of boric acid, and 0.01 g / L of potassium iodide, and the solvent is purified water.

[0053] The Pichia pastoris used in the examples was donated by Lao Yongmin's research group at the Frontier Technology Integration Innovation Platform for Traditional Chinese Medicine Resources and Synthetic Biology of the Guangdong Provincial Laboratory of Traditional Chinese Medicine.

[0054] Example 1

[0055] A method for preparing polymer-grade succinic acid is as follows:

[0056] Step 1. Use a sterile inoculating loop to pick a loopful of Pichia from a frozen vial, streak the loopful onto 50 mL of activation medium, and incubate at 30°C for 48 hours. Then, use a sterile toothpick to pick a single colony and inoculate it into 180 mL of seed medium. Incubate the culture at 30°C with shaking and 180 rpm for 24 hours to obtain a seed solution.

[0057] Step 2. The seed liquid was inoculated into 5 L of fermentation medium at an inoculum size of 5% by volume. 19 g of a fermentation aid was then added to the fermentation medium. The culture was shaken at 30° C., a stirring speed of 300 rpm, and an aeration volume of 0.1 vvm for 80 hours. During the shaking culture, the glucose concentration was constantly controlled to 20 g / L by feeding a glucose aqueous solution. After the shaking culture, a fermentation broth was obtained.

[0058] The mass fraction of glucose in the glucose aqueous solution is 70%;

[0059] The preparation method of the fermentation aid is:

[0060] Step 2a. 9 g of citric acid and 500 mL of purified water were mixed and stirred at a temperature of 20 ° C and a stirring speed of 100 r / min for 10 minutes. Keeping the temperature and stirring speed constant, 370 mL of ferrous chloride solution was added at a rate of 10 mL / min. After the addition was completed, the mixture was stirred for 10 minutes, and the pH was adjusted to 11 by adding lime milk. The mixture was stirred for 2 hours and allowed to stand at 20 ° C for 5 hours. The mixture was centrifuged at 9000 r / min for 15 minutes, the precipitate was collected, and the precipitate was dried in vacuo at 70 ° C to obtain a complex;

[0061] The mass fraction of ferrous chloride in the ferrous chloride aqueous solution is 2%;

[0062] The concentration of calcium oxide in the lime milk is 80g / L;

[0063] Step 2b. After all the complexes in step 2a were mixed with 900 mL of purified water, the mixture was stirred at a temperature of 20 ° C and a stirring speed of 100 r / min for 20 minutes. Under a nitrogen atmosphere, the temperature and stirring speed were kept constant, 140 mL of sodium borohydride aqueous solution was added at a rate of 4 mL / min, and stirred for 30 minutes after the addition was completed. The mixture was centrifuged at a speed of 9000 r / min for 15 minutes, and the precipitate was collected. After washing the precipitate three times with purified water, the mixture was dried in vacuo at 70 ° C to obtain a fermentation aid.

[0064] The mass fraction of sodium borohydride in the sodium borohydride aqueous solution is 5%;

[0065] Step 3. The fermentation broth was filtered using a ceramic membrane with a filtration accuracy of 100 nm, and the filtrate was collected. The collected filtrate was used as a primary filtrate. At a temperature of 75 ° C. and a stirring speed of 100 r / min, a calcium chloride aqueous solution was added to the primary filtrate at a rate of 20 mL / min, and the volume ratio of the primary filtrate to the calcium chloride aqueous solution was controlled to be 1: 1. After the addition was completed, the mixture was stirred for 30 minutes, filtered, and the filtrate and filter residue were collected. The collected filtrate was used as a secondary filtrate, and the collected filter residue was used as a primary filter residue; at a temperature of 75 ° C. and a stirring speed of 100 r / min, a calcium chloride aqueous solution containing calcium carbonate was added to the secondary filtrate, and the volume ratio of the secondary filtrate to the calcium chloride aqueous solution containing calcium carbonate was controlled to be 10: 1. After the addition was completed, the mixture was stirred for 30 minutes, filtered, and the filter residue was collected. The collected filter residue was used as a secondary filter residue, and the primary filter residue and the secondary filter residue were mixed to obtain mixed calcium succinate;

[0066] The mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%;

[0067] The calcium carbonate-containing calcium chloride aqueous solution has a particle size of 700 mesh, a mass fraction of calcium carbonate of 2%, and a mass fraction of calcium chloride of 30%;

[0068] Step 4. Calcium succinate, activated carbon, and aqueous sulfuric acid solution were mixed in a ratio of 300 g:4 g:240 mL, stirred at 75° C. and 100 rpm for 30 minutes, filtered, and the filtrate was collected as a mixed succinic acid solution;

[0069] The mass fraction of the sulfuric acid aqueous solution is 60%;

[0070] Step 5. After ion exchange of the mixed succinic acid solution using 732 strong acid cation exchange resin and 331 weak base anion exchange resin, the mixture was concentrated under vacuum at 75°C until crystallization occurred, and then low-temperature crystallization was performed at 4°C. The mixture was filtered, the filter residue was collected, and vacuum dried at 60°C to obtain polymer-grade succinic acid.

[0071] Comparative Example 1

[0072] Based on the technical solution of Example 1, in step 2, the addition of the fermentation aid is omitted, and steps 2a and 2b are omitted.

[0073] The remaining operations are the same as those in Example 1.

[0074] Comparative Example 2

[0075] Based on the technical solution of Example 1, in step 3, the treatment of the secondary filtrate is omitted, and step 3 is specifically changed to:

[0076] The fermentation broth was filtered using a ceramic membrane with a filtration accuracy of 100 nm, and the filtrate was collected. The collected filtrate was used as the primary filtrate. Under the conditions of a temperature of 75° C. and a stirring speed of 100 r / min, a calcium chloride aqueous solution was added to the primary filtrate at a rate of 20 mL / min, and the volume ratio of the primary filtrate to the calcium chloride aqueous solution was controlled to be 1:1. After the addition was completed, the mixture was stirred for 30 minutes, filtered, and the filter residue was collected as mixed calcium succinate;

[0077] The mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%.

[0078] The remaining operations are the same as those in Example 1.

[0079] Comparative Example 3

[0080] On the basis of the technical solution of Example 1, in step 3, an equal volume of calcium chloride aqueous solution is used to replace the calcium chloride aqueous solution containing calcium carbonate, and specifically step 3 is changed to:

[0081] The fermentation broth was filtered using a ceramic membrane with a filtration accuracy of 100 nm, and the filtrate was collected. The collected filtrate was used as a primary filtrate. At a temperature of 75° C. and a stirring speed of 100 r / min, a calcium chloride aqueous solution was added to the primary filtrate at a rate of 20 mL / min, and the volume ratio of the primary filtrate to the calcium chloride aqueous solution was controlled to be 1:1. After the addition was completed, the mixture was stirred for 30 minutes, filtered, and the filtrate and filter residue were collected. The collected filtrate was used as a secondary filtrate, and the collected filter residue was used as a primary filter residue. At a temperature of 75° C. and a stirring speed of 100 r / min, a calcium chloride aqueous solution was added to the secondary filtrate, and the volume ratio of the secondary filtrate to the calcium chloride aqueous solution was controlled to be 10:1. After the addition was completed, the mixture was stirred for 30 minutes, filtered, and the filter residue was collected. The collected filter residue was used as a secondary filter residue, and the primary filter residue and the secondary filter residue were mixed to obtain mixed calcium succinate;

[0082] The mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%.

[0083] The remaining operations are the same as those in Example 1.

[0084] Comparative Example 4

[0085] On the basis of the technical solution of Comparative Example 2, a concentration step was added in step 3, and step 3 was specifically changed to:

[0086] The fermentation broth was filtered using a ceramic membrane with a filtration accuracy of 100 nm, and the filtrate was collected. The collected filtrate was used as the primary filtrate. Under the conditions of a temperature of 75° C. and a stirring speed of 100 r / min, a calcium chloride aqueous solution was added to the primary filtrate at a rate of 20 mL / min, and the volume ratio of the primary filtrate to the calcium chloride aqueous solution was controlled to be 1:1. After the addition was completed, the mixture was stirred for 30 minutes, and vacuum concentrated at 75° C. to 50% of the original volume. The mixture was filtered and the filter residue was collected as mixed calcium succinate.

[0087] The mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%.

[0088] The remaining operations are the same as those in Comparative Example 2.

[0089] Example 2

[0090] A method for preparing polymer-grade succinic acid is as follows:

[0091] Step 1. Use a sterile inoculating loop to pick a loopful of Pichia pastoris from a frozen tube, streak the loopful onto 60 mL of activation medium, and incubate at 32°C for 50 hours. Then, use a sterile toothpick to pick a single colony and inoculate it into 200 mL of seed medium. Incubate the culture at 32°C with shaking and 200 rpm for 26 hours to obtain a seed solution.

[0092] Step 2. The seed liquid was inoculated into 5 L of fermentation medium at an inoculum size of 5% by volume. 21 g of a fermentation aid was then added to the fermentation medium. The medium was shake-cultured at a temperature of 32° C., a stirring speed of 340 rpm, and a ventilation volume of 0.11 vvm for 82 hours. During the shaking culture, the glucose concentration was constantly controlled to 20 g / L by feeding a glucose aqueous solution. After the shaking culture, a fermentation broth was obtained.

[0093] The mass fraction of glucose in the glucose aqueous solution is 70%;

[0094] The preparation method of the fermentation aid is:

[0095] Step 2a. 11 g of citric acid and 600 mL of purified water were mixed and stirred at a temperature of 40 ° C and a stirring speed of 300 r / min for 30 minutes. Keeping the temperature and stirring speed constant, 400 mL of ferrous chloride solution was added at a rate of 15 mL / min. After the addition, the mixture was stirred for 30 minutes, and the pH was adjusted to 12 by adding lime milk. The mixture was stirred for 3 hours and allowed to stand at 40 ° C for 6 hours. The mixture was centrifuged at a speed of 10000 r / min for 20 minutes, and the precipitate was collected and dried in vacuo at 80 ° C to obtain a complex.

[0096] The mass fraction of ferrous chloride in the ferrous chloride aqueous solution is 2%;

[0097] The concentration of calcium oxide in the lime milk is 80g / L;

[0098] Step 2b. After all the complexes in step 2a were mixed with 1000 mL of purified water, the mixture was stirred at a temperature of 40 ° C and a stirring speed of 300 r / min for 40 minutes. Under a nitrogen atmosphere, the temperature and stirring speed were kept constant, 160 mL of sodium borohydride aqueous solution was added at a rate of 5 mL / min, and stirred for 50 minutes after the addition was completed. The mixture was centrifuged at a speed of 10000 r / min for 20 minutes, and the precipitate was collected and washed four times with purified water. After that, the precipitate was dried in vacuo at 80 ° C to obtain a fermentation aid;

[0099] The mass fraction of sodium borohydride in the sodium borohydride aqueous solution is 5%;

[0100] Step 3. The fermentation broth was filtered using a ceramic membrane with a filtration accuracy of 100 nm, and the filtrate was collected. The collected filtrate was used as a primary filtrate. At a temperature of 80 ° C and a stirring speed of 300 r / min, a calcium chloride aqueous solution was added to the primary filtrate at a rate of 30 mL / min, and the volume ratio of the primary filtrate to the calcium chloride aqueous solution was controlled to be 1: 1.2. After the addition was completed, the mixture was stirred for 40 minutes, filtered, and the filtrate and filter residue were collected. The collected filtrate was used as a secondary filtrate, and the collected filter residue was used as a primary filter residue; At a temperature of 80 ° C and a stirring speed of 300 r / min, a calcium chloride aqueous solution containing calcium carbonate was added to the secondary filtrate, and the volume ratio of the secondary filtrate to the calcium chloride aqueous solution containing calcium carbonate was controlled to be 10: 1.2. After the addition was completed, the mixture was stirred for 40 minutes, filtered, and the filter residue was collected. The collected filter residue was used as a secondary filter residue, and the primary filter residue and the secondary filter residue were mixed to obtain mixed calcium succinate;

[0101] The mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%;

[0102] The calcium carbonate-containing calcium chloride aqueous solution has a particle size of 700 mesh, a mass fraction of calcium carbonate of 2%, and a mass fraction of calcium chloride of 30%;

[0103] Step 4. Calcium succinate, activated carbon, and aqueous sulfuric acid solution were mixed in a ratio of 300 g:4.2 g:250 mL, stirred at 80° C. and 300 rpm for 40 minutes, filtered, and the filtrate was collected as a mixed succinic acid solution;

[0104] The mass fraction of the sulfuric acid aqueous solution is 60%;

[0105] Step 5. After ion exchange of the mixed succinic acid solution using 732 strong acid cation exchange resin and 331 weak base anion exchange resin, the mixture was concentrated under vacuum at 80°C until crystallization occurred, and then low-temperature crystallization was performed at 4°C. The mixture was filtered, the filter residue was collected, and vacuum dried at 70°C to obtain polymer-grade succinic acid.

[0106] Comparative Example 5

[0107] Based on the technical solution of Example 2, in step 2, the addition of the fermentation aid is omitted, and steps 2a and 2b are omitted.

[0108] The remaining operations are the same as those in Example 2.

[0109] Comparative Example 6

[0110] Based on the technical solution of Example 2, in step 3, the treatment of the secondary filtrate is omitted, and step 3 is specifically changed to:

[0111] The fermentation broth was filtered using a ceramic membrane with a filtration accuracy of 100 nm, and the filtrate was collected. The collected filtrate was used as the primary filtrate. Under the conditions of a temperature of 80° C. and a stirring speed of 300 r / min, a calcium chloride aqueous solution was added to the primary filtrate at an addition rate of 30 mL / min, and the volume ratio of the primary filtrate to the calcium chloride aqueous solution was controlled to be 1:1.2. After the addition was completed, the mixture was stirred for 40 minutes, filtered, and the filter residue was collected as mixed calcium succinate;

[0112] The mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%.

[0113] The remaining operations are the same as those in Example 2.

[0114] Comparative Example 7

[0115] On the basis of the technical solution of Example 2, in step 3, an equal volume of calcium chloride aqueous solution is used to replace the calcium chloride aqueous solution containing calcium carbonate, and specifically step 3 is changed to:

[0116] The fermentation broth was filtered using a ceramic membrane with a filtration accuracy of 100 nm, and the filtrate was collected. The collected filtrate was used as a primary filtrate. At a temperature of 80° C. and a stirring speed of 300 r / min, a calcium chloride aqueous solution was added to the primary filtrate at a rate of 30 mL / min, and the volume ratio of the primary filtrate to the calcium chloride aqueous solution was controlled to be 1:1.2. After the addition was completed, the mixture was stirred for 40 minutes, filtered, and the filtrate and filter residue were collected. The collected filtrate was used as a secondary filtrate, and the collected filter residue was used as a primary filter residue. At a temperature of 80° C. and a stirring speed of 300 r / min, a calcium chloride aqueous solution was added to the secondary filtrate, and the volume ratio of the secondary filtrate to the calcium chloride aqueous solution was controlled to be 10:1.2. After the addition was completed, the mixture was stirred for 40 minutes, filtered, and the filter residue was collected. The collected filter residue was used as a secondary filter residue, and the primary filter residue and the secondary filter residue were mixed to obtain mixed calcium succinate;

[0117] The mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%.

[0118] The remaining operations are the same as those in Example 2.

[0119] Comparative Example 8

[0120] Based on the technical solution of Comparative Example 6, a concentration step was added in step 3, and step 3 was specifically changed to:

[0121] The fermentation broth was filtered using a ceramic membrane with a filtration accuracy of 100 nm, and the filtrate was collected. The collected filtrate was used as the primary filtrate. Under the conditions of a temperature of 80° C. and a stirring speed of 300 r / min, a calcium chloride aqueous solution was added to the primary filtrate at a rate of 30 mL / min, and the volume ratio of the primary filtrate to the calcium chloride aqueous solution was controlled to be 1:1.2. After the addition was completed, the mixture was stirred for 40 minutes, and vacuum concentrated at 80° C. to 50% of the original volume. The mixture was filtered and the filter residue was collected as mixed calcium succinate;

[0122] The mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%.

[0123] The remaining operations are the same as those in Comparative Example 6.

[0124] Test Example 1

[0125] Based on the total amount of glucose fed in Examples 1-2, Comparative Example 1, and Comparative Example 4, the total glucose consumption was calculated based on the total amount of glucose fed, the decrease in glucose concentration in the fermentation medium, and the volume change of the fermentation medium. The yield, rate, and production intensity of succinic acid were then calculated. The results are as follows:

[0126]

[0127] From the above results, it can be seen that the yield, yield and production intensity of succinic acid in Comparative Example 1 are worse than those in Example 1, and the yield, yield and production intensity of succinic acid in Comparative Example 2 are worse than those in Example 2. This shows that the fermentation aid can improve the yield, yield and production intensity of succinic acid.

[0128] Test Example 2

[0129] According to the methods of Examples 1-2, Comparative Example 1, and Comparative Example 4, 20 batches of succinic acid were continuously produced. The yield, yield, and production intensity of each batch of succinic acid were calculated, and the difference between the maximum yield and the minimum yield, and the difference between the maximum yield and the minimum yield were calculated. The results are as follows:

[0130]

[0131] It can be seen from the above results that the output, yield and production intensity of succinic acid in Examples 1-2, Comparative Example 1 and Comparative Example 4 can all be kept stable.

[0132] Test Example 3

[0133] The liquid phase purity and weight of the succinic acid prepared in Examples 1-2 and Comparative Examples 1-6 were detected and counted, and then the purification yield was calculated based on the weight of the succinic acid. The results are as follows:

[0134]

[0135] It can be seen from the above results that the purification yields of succinic acid in Comparative Examples 2 and 3 are worse than that in Example 1, the purification yields of Comparative Examples 6 and 7 are worse than that in Example 2, the purification yield of succinic acid in Comparative Example 4 is better than that in Example 1, and the purification yield of Comparative Example 8 is better than that in Example 2. However, Comparative Examples 4 and 8 require concentration, which consumes a lot of energy.

Claims

1. A method for preparing polymer-grade succinic acid, characterized in that: include: Step 1 is: activating and expanding the culture of Pichia pastoris to obtain a seed solution; Step 2 comprises inoculating the seed liquid into 5 L of fermentation medium at an inoculum size of 5% by volume, then adding 19-21 g of a fermentation aid to the fermentation medium, and shaking culturing the culture at a temperature of 30-32° C., a stirring speed of 300-340 rpm, and an aeration volume of 0.1-0.11 vvm for 80-82 hours, controlling the glucose concentration to 20 g / L by feeding a glucose aqueous solution during the shaking culture, and obtaining a fermentation broth after the shaking culture is completed. The preparation method of the fermentation aid comprises: Step 2a comprises: mixing citric acid and purified water, stirring at a temperature of 20-40° C., maintaining the temperature and stirring speed constant, adding an aqueous solution of ferrous chloride, stirring, adjusting the pH to 11-12 by adding lime milk, stirring, standing at 20-40° C., centrifuging, collecting a precipitate, and vacuum drying to obtain a complex; Step 2b comprises: mixing the complex and purified water, stirring at a temperature of 20-40° C., adding a sodium borohydride aqueous solution under a nitrogen atmosphere, stirring, centrifuging, collecting the precipitate, washing, and vacuum drying to obtain a fermentation aid; The fermentation aid is a mixture of citrate, zero-valent iron, and calcium ions; Step 3 comprises: filtering the fermentation broth using a ceramic membrane with a filtration accuracy of 100 nm, collecting the filtrate, using the collected filtrate as the primary filtrate, adding a calcium chloride aqueous solution to the primary filtrate at a temperature of 75-80° C., stirring for 30-40 minutes after the addition is completed, filtering, collecting the filtrate and filter residue, using the collected filtrate as the secondary filtrate, and using the collected filter residue as the primary filter residue; adding a calcium chloride aqueous solution containing calcium carbonate to the secondary filtrate at a temperature of 75-80° C., stirring for 30-40 minutes after the addition is completed, filtering, collecting the filter residue, using the collected filter residue as the secondary filter residue, and mixing the primary filter residue and the secondary filter residue to obtain mixed calcium succinate.

2. The preparation method of polymer-grade succinic acid according to claim 1, wherein Step 1 is: using a sterile inoculating loop to pick a loopful of bacteria from a frozen tube of Pichia pastoris, streaking it onto 50-60 mL of activation culture medium, culturing it at a temperature of 30-32° C. for 48-50 hours, then using a sterile toothpick to pick a single colony, inoculating it into 180-200 mL of seed culture medium, and culturing it with shaking at a temperature of 30-32° C. and a stirring speed of 180-200 rpm for 24-26 hours to obtain a seed solution.

3. The preparation method of polymer-grade succinic acid according to claim 2, wherein The activation culture medium comprises: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast powder, 20 g / L agar powder, the solvent is purified water, and the pH value is 6; The components of the seed culture medium are: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast powder, the solvent is purified water, and the pH value is 6.

4. The preparation method of polymer-grade succinic acid according to claim 1, wherein In step 2, the fermentation medium comprises the following components: 50 g / L glucose, 5 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium chloride dihydrate, 0.2 g / L urea, 0.4 mg / L biotin, 1 mg / L calcium pantothenate, 1 mg / L niacin, 25 mg / L inositol, 1 mg / L vitamin B1, 1 mg / L vitamin B6, 10 mL / L trace element mother solution, the solvent is purified water, and the pH value is natural; The components of the trace element mother solution are: 0.05 g / L copper sulfate pentahydrate, 2 g / L ferrous sulfate heptahydrate, 0.2 g / L manganese chloride tetrahydrate, 0.5 g / L zinc chloride, 0.04 g / L sodium molybdate dihydrate, 0.03 g / L cobalt chloride hexahydrate, 0.1 g / L boric acid, 0.01 g / L potassium iodide, and the solvent is purified water; The mass fraction of glucose in the glucose aqueous solution is 70%.

5. The preparation method of polymer-grade succinic acid according to claim 1, wherein In step 2a, the ratio of citric acid, purified water, and ferrous chloride aqueous solution is 9-11 g: 500-600 mL: 370-400 mL; The mass fraction of ferrous chloride in the ferrous chloride aqueous solution is 2%; The concentration of calcium oxide in the lime milk is 80g / L; The addition rate of the ferrous chloride aqueous solution is 10-15 mL / min.

6. The preparation method of polymer-grade succinic acid according to claim 1, wherein The ratio of citric acid in step 2a to purified water and sodium borohydride aqueous solution in step 2b is 9-11 g:900-1000 mL:140-160 mL; In step 2b, the mass fraction of sodium borohydride in the sodium borohydride aqueous solution is 5%; The sodium borohydride aqueous solution is added at a rate of 4-5 mL / min.

7. The method for preparing polymer-grade succinic acid according to claim 1, wherein In step 3, the mass fraction of calcium chloride in the calcium chloride aqueous solution is 30%; The calcium chloride aqueous solution is added at a rate of 20-30 mL / min; The volume ratio of the primary filtrate to the calcium chloride aqueous solution is 1:1-1.2; The calcium carbonate-containing calcium chloride aqueous solution has a particle size of 700 mesh, a mass fraction of calcium carbonate of 2%, and a mass fraction of calcium chloride of 30%; The volume ratio of the secondary filtrate to the calcium chloride aqueous solution containing calcium carbonate is 10:1-1.

2.

8. The method for preparing polymer-grade succinic acid according to claim 1, wherein Step 4 is: mixing calcium succinate, activated carbon and sulfuric acid aqueous solution, stirring at a temperature of 75-80° C. and a stirring speed of 100-300 r / min for 30-40 minutes, filtering, and collecting the filtrate as a mixed succinic acid solution.

9. The method for preparing polymer-grade succinic acid according to claim 8, wherein In step 4, the ratio of calcium succinate, activated carbon, and aqueous sulfuric acid solution is 300 g: 4-4.2 g: 240-250 mL; The mass fraction of the sulfuric acid aqueous solution is 60%.

10. The method for preparing polymer-grade succinic acid according to claim 1, wherein Step 5 is: using 732 strong acid cation exchange resin and 331 weak base anion exchange resin to exchange ions on the mixed succinic acid solution in sequence, concentrating under vacuum at 75-80° C. until crystallization occurs, then low-temperature crystallization at 4° C., filtering, collecting the filter residue, and vacuum drying at 60-70° C. to obtain polymer-grade succinic acid.

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