Long-chain dicarboxylic acid and method for producing long-chain dicarboxylic acid through fermentation

Abstract

The invention discloses a long-chain dibasic acid and a method for producing the long-chain dibasic acid through fermentation, and the method comprises the following steps: inoculating a seed solution of a fermentation strain into a fermentation tank, adding a substrate for fermentation, and controlling the fermentation temperature to be 20-40 DEG C, the substrate comprising fatty acid ester and alkane, or comprising fatty acid ester and fatty acid. The fatty acid ester and the petroleum-based / bio-based alkane or the fatty acid ester and the fatty acid are taken as substrates, fossil energy such as the petroleum alkane can be partially or completely replaced, long-chain binary acid products with different bio-based contents can be obtained under the condition of ensuring higher yield and conversion rate, and the concept of sustainable development of material sources is met.

Description

[0001] This application claims the priority of the prior application filed with the State Intellectual Property Office of China on November 8, 2023, with patent application number 202311479510.7 and title “A long-chain dibasic acid and a method for producing long-chain dibasic acid by fermentation”. Technical Field

[0002] The invention belongs to the technical field of fermentation, and in particular relates to a long-chain dibasic acid and a method for producing the long-chain dibasic acid by fermentation. Background Art

[0003] Long-chain dibasic acid refers to a straight-chain dibasic acid with more than 10 carbon atoms in the carbon chain, and is an important chemical raw material. Nylon engineering plastics made of long-chain dibasic acid have extremely strong tensile resistance, wear resistance, heat resistance and flexibility. The service life of automobile tires modified with this is 5 to 10 times that of ordinary cars. Lubricating oils made of long-chain dibasic acids are not only resistant to high temperatures, but also resistant to ultra-low temperatures. High-end clothing made of long-chain dibasic acids is resistant to water washing and dry cleaning, and has a crisp style. High-end paints made with long-chain dibasic acids as raw materials have the characteristics of bright color, wear resistance, strong adhesion, flexibility, and anti-aging. They are widely used as surface paints for cars, defense tents, military vehicles and high-end luxury objects. Due to the excellent performance of downstream products of long-chain dibasic acids, it has directly promoted the rapid development of my country's fine chemical industry, and the demand for long-chain dibasic acids in downstream industries has continued to grow.

[0004] At present, most domestic production enterprises use long-chain alkanes separated from petroleum as raw materials to produce long-chain dibasic acids. However, petroleum is a non-renewable resource, and my country's petroleum is heavily dependent on imports and its price fluctuates greatly. Fatty acids are renewable raw materials with the cheapest cost. They are currently the best raw materials for producing corresponding long-chain dicarboxylic acids. However, the method of directly using fatty acids as raw materials to convert them into long-chain dibasic acids is not really used in production. On the one hand, fatty acids are solid and cannot be added in a flow manner. On the other hand, fermentation cannot be completed due to severe foaming. Fatty acid esters are obtained by esterifying fatty acids. This new type of raw material not only has the characteristics of green and renewable, but also can be added in a flow manner. It has significant advantages as an alternative substrate and has important practical significance in energy conservation, environmental protection and sustainable development. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for producing long-chain dibasic acids by fermentation, the method comprising: introducing seed liquid of a fermentation strain into a fermentation tank, adding a substrate for fermentation, and controlling the fermentation temperature to be 20-40°C, wherein the substrate comprises fatty acid esters and alkanes, or comprises fatty acid esters and fatty acids.

[0006] In one embodiment, from the start of fermentation to T hours, the substrate is added so that the viscosity η of the fermentation system and the fermentation time t satisfy the numerical relationship: η = A*e bt +C; wherein, e is a natural constant, 1.0≤A≤5.5, 0.02≤b≤0.043, 0≤C≤8, 135≤T≤165, the unit of the viscosity η is mPa·s, the unit of the fermentation time t is h, and t≤T in the present invention.

[0007] In the present invention, the natural constant e is a constant in mathematics, an infinite non-repeating decimal, and a transcendental number, and its value is approximately 2.718281828459045.

[0008] In one embodiment, 145≤T≤155.

[0009] In the present invention, those skilled in the art can understand that the range of the fermentation time t is greater than 0 and less than or equal to T.

[0010] In the present invention, the fermentation substrate is added to the fermentation system to maintain the viscosity of the fermentation system at an appropriate level, so that the strain maintains a high activity and acid production capacity, and there is a good mass transfer effect between the multiphases of the fermentation system.

[0011] In one embodiment, the mass ratio of fatty acid ester to alkane is controlled to be (0.01-30):1, further to be (0.1-20):1.

[0012] In one embodiment, the mass ratio of fatty acid ester to fatty acid is controlled to be 1:(0.01-40), further 1:(0.1-30).

[0013] In one embodiment, the type of the fatty acid ester is not particularly limited, including but not limited to any one of a deca-carbon fatty acid ester, an undine-carbon fatty acid ester, a lauric acid ester, a thirteen-carbon fatty acid ester, a tetradecyl fatty acid ester, a pentadecyl fatty acid ester, a hexadecyl fatty acid ester, a heptadecanyl fatty acid ester and an octadecyl fatty acid ester.

[0014] In one embodiment, the ten-carbon fatty acid ester includes any one or more of ten-carbon fatty acid formic acid, ten-carbon fatty acid acetic acid, and ten-carbon fatty acid butyric acid. The lauric acid ester includes any one or more of methyl laurate, ethyl laurate, and butyl laurate. The fourteen-carbon fatty acid ester includes any one or more of fourteen-carbon fatty acid methyl ester, fourteen-carbon fatty acid ethyl ester, and fourteen-carbon fatty acid butyl ester. The sixteen-carbon fatty acid ester includes any one or more of sixteen-carbon fatty acid methyl ester, sixteen-carbon fatty acid ethyl ester, and sixteen-carbon fatty acid butyl ester. Since the fatty acid ester is liquid at room temperature, it has a good dispersion effect and has characteristics similar to alkanes, and can replace the substrate alkanes currently used. Fatty acid esters mainly come from the seeds of tropical crops rich in oil such as palm trees and coconut trees. This new type of renewable resource can replace fossil raw materials such as petroleum alkanes, which is of great significance to the sustainable development of industrial processes.

[0015] In one embodiment, the alkane comprises a bio-based alkane and / or a petroleum-based alkane.

[0016] In one embodiment, the alkane comprises C9-C 22 Bio-based or petroleum-based normal alkanes, further including C 10 -C 18 Bio-based or petroleum-based n-alkanes, further including C 10 , C 11 , C 12 , C 13 , C 14 , C 15 or C 16 Bio-based or petroleum-based n-alkanes.

[0017] In one embodiment, the fatty acid includes a straight-chain monocarboxylic acid having more than 10 carbon atoms, and further a straight-chain monocarboxylic acid having 10 to 18 carbon atoms, including any one of decanoic acid, undecanoic acid, lauric acid (also known as dodecanoic acid), tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid (also known as hexadecanoic acid), heptadecanoic acid and octadecanoic acid.

[0018] In one embodiment, the fermentation strain comprises Candida viswanathii, Candida tropicalis, Candida sake, Candida albicans or Yarrowia lipolytica.

[0019] In one embodiment, the chemical formula of the long chain dibasic acid is HOOC(CH2) nCOOH, wherein n≥8, includes but is not limited to any one of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedicarboxylic acid, hexadecanedioic acid, heptadecanedioic acid and octadecanedicarboxylic acid.

[0020] In one embodiment, the inoculation amount of the fermentation strain seed solution is 10-50 v / v%.

[0021] In one embodiment, the fermentation temperature is controlled to be 27-33°C.

[0022] In one embodiment, the air volume during fermentation is controlled to be 0.2-0.8 vvm.

[0023] In one embodiment, the fermentation pressure is controlled to be 0.05-0.20 MPa.

[0024] In one embodiment, the pH value of the fermentation is controlled to be 4.5-6.5. Specifically, during the fermentation process, the pH value of the fermentation liquid can be maintained at 4.5-6.5, preferably 5.5-6.5, by adding a small amount of alkaline substances such as sodium hydroxide.

[0025] In one embodiment, the dissolved oxygen during the fermentation process is maintained at 5-80% by controlling the stirring speed.

[0026] In the present invention, the addition of substrate is stopped after a certain period of fermentation, and the fermentation is carried out using the residual substrate in the fermentation system until the substrate is completely consumed or no long-chain dibasic acid is produced. The total fermentation time is calculated. In one embodiment, the total fermentation time is 120-200 hours.

[0027] In one embodiment, when the optical density OD of the strain is diluted 30 times 620 When the pH reaches 0.5-1.0, preferably 0.7-1.0, the substrate is added to start fermentation. The substrate can be added or supplemented as a fermentation substrate in a one-time addition, batch addition or continuous flow addition manner.

[0028] In one embodiment, after fermentation for 100-180 h, preferably 120-170 h, more preferably 135-165 h, and even more preferably 145-155 h, the addition of fermentation substrate is stopped, and fermentation is carried out using the remaining substrate in the fermentation system.

[0029] In one embodiment, the method further comprises expanding the fermentation strain, wherein the process of expanding the culture comprises: activating the fermentation strain in a shake flask, and waiting for the optical density value OD of the shake flask seed when diluted 30 times 620When the optical density of the seed solution is 0.5 to 1.0, the seed is placed in a seed tank filled with a seed culture medium for seed culture until the optical density of the seed solution is OD 620 Reach 0.5~1.0.

[0030] In one embodiment, the activation culture conditions include: a temperature of 27-35° C., a shaker speed of 150-250 rpm, and a time of 38-42 h.

[0031] In one embodiment, during the seed culture process, the temperature is controlled at 27-35° C., the air volume is 0.2-0.8 vvm, the pressure is 0.05-0.20 MPa, and the dissolved oxygen is maintained at 5-50% by controlling the stirring rate.

[0032] In one embodiment, during the seed culture and fermentation, the components of the seed culture medium and fermentation medium used include a carbon source, a nitrogen source, an inorganic salt and nutritional factors.

[0033] In one embodiment, the carbon source comprises one or more of glucose, sucrose, maltose, molasses, fructose, rhamnose, arabinose, glycerol, methanol, ethanol and sorbitol.

[0034] In one embodiment, the amount of the carbon source added is 10-80 g / L.

[0035] In one embodiment, the nitrogen source includes one or more of yeast extract, peptone, corn steep liquor, urea, ammonium salt and nitrate.

[0036] In one embodiment, the amount of each nitrogen source added is 0-10 g / L.

[0037] In one embodiment, the inorganic salt includes one or more of sulfate, hydrochloride, nitrate and phosphate.

[0038] Preferably, it includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, sodium chloride and potassium nitrate.

[0039] In one embodiment, the amount of each inorganic salt added is 1-10 g / L.

[0040] In one embodiment, the growth factor comprises one or more of amino acids, citric acid and vitamins.

[0041] Preferably, the growth factors include one or more of vitamin B1, vitamin B2, vitamin C and biotin.

[0042] In one embodiment, the amount of the biological factor added is 0-1 g / L.

[0043] In a preferred embodiment, the seed culture medium includes the following ingredients: sucrose 5-35 g / L, corn steep liquor 2-8 g / L, yeast extract 1-10 g / L, potassium dihydrogen phosphate 2-10 g / L, urea 2-8 g / L, and defoaming agent 0.2-1 g / L.

[0044] In a preferred embodiment, the fermentation medium comprises the following ingredients: 0-10 g / L corn steep liquor, 20-60 g / L glucose, 0-8 g / L yeast extract, 4-10 g / L potassium dihydrogen phosphate, 2-6 g / L magnesium sulfate, 1-10 g / L ammonium sulfate, and 2-8 g / L potassium nitrate.

[0045] The second object of the present invention is to provide a method for preparing a long-chain dibasic acid, the method comprising:

[0046] S1: Obtaining a long-chain dibasic acid fermentation liquid according to the method for producing a long-chain dibasic acid by fermentation as described above;

[0047] S2: extracting and purifying the long-chain dibasic acid fermentation broth to obtain a long-chain dibasic acid product.

[0048] In one embodiment, the extraction and purification comprises: acidifying the fermentation broth, separating the solid, dissolving the solid in an organic solvent, crystallizing, and separating the solid from the liquid, washing and drying the separated solid to obtain a long-chain dibasic acid product.

[0049] In some embodiments, the pH of the acidification is 2 to 5, preferably 3.5 to 4.5. The long-chain dibasic acid in the fermentation broth is crystallized by acidification.

[0050] In some embodiments, the solid-liquid separation method comprises at least one of filtration or centrifugation.

[0051] In one embodiment, the mass ratio of the solid matter to the organic solvent is 1:3-6.

[0052] In some embodiments, the organic solvent includes one or more of an acid, an alcohol, an ester, and a ketone; wherein the alcohol includes one or more of methanol, ethanol, n-butanol, and isopropanol, the acid includes acetic acid, the ketone includes acetone, and the ester includes at least one of ethyl acetate and butyl acetate.

[0053] In some embodiments, after the solid is dissolved in the organic solvent, it is first subjected to a decolorization treatment before being crystallized.

[0054] In some embodiments, the amount of the decolorizing agent added during the decolorization process is 0.5% to 10% by mass of the long-chain dibasic acid contained in the decolorized solution, further 1% to 5%, further 1.2% to 4.8%.

[0055] In one embodiment, the decolorizing agent is activated carbon.

[0056] In one embodiment, the bleaching temperature is 80-110° C., and the bleaching time is 30-190 min.

[0057] In some embodiments, the crystallization is a temperature drop crystallization, and the temperature drop crystallization comprises the following steps: cooling to 50-80° C., keeping warm for 1-3 hours, cooling to 20-40° C., and finally crystallizing.

[0058] In some embodiments, before washing the separated solid, it is first treated with hot water, then washed and dried to obtain a long-chain dibasic acid product.

[0059] In one embodiment, the temperature of the hot water treatment is 70 to 150°C, and further 70 to 135°C.

[0060] In one embodiment, the hot water treatment comprises the following steps: contacting the separated solid with hot water to remove impurities, wherein the temperature of the hot water is 70 to 150°C, and further 70 to 135°C.

[0061] In one embodiment, the hot water treatment comprises the following steps: mixing solid and water in a mass ratio of 1:(1-10), keeping at 70-150° C. for 30-180 min, cooling to 25-60° C., separating the solid from the liquid, and obtaining a solid.

[0062] In one embodiment, the hot water treatment comprises the following steps: mixing solid and water in a mass ratio of 1:(1-10), keeping at 70-135° C. for 30-180 min, cooling to 25-60° C., separating the solid from the liquid, and obtaining a solid.

[0063] In one embodiment, the present invention also provides a long-chain dibasic acid product obtained by the above-mentioned preparation method, wherein the purity of the long-chain dibasic acid product is greater than 99wt%, and the bio-based content is 1-100%, further 5-98%, for example, it can be 95%, 98%, 90%, 80%, 70%, 50%, 40%, 30%, etc.

[0064] In one embodiment, the present invention uses fatty acid esters and petroleum-based / bio-based alkanes, or fatty acid esters and fatty acids as substrates to ferment and produce long-chain dibasic acids, which can completely or partially replace fossil energy such as petroleum alkanes. While ensuring a high yield and conversion rate, long-chain dibasic acid products with different bio-based contents can also be obtained, which is in line with the concept of sustainable development of material sources. DETAILED DESCRIPTION

[0065] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration, so that the features and advantages of the present invention are clearer, and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and the scope of the present invention is not limited to the examples listed herein.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0067] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0068] In the following examples, the fermentation strain used is Candida viswanathii CAES2113, which has been disclosed in patent CN111748480A, and was biologically deposited on February 24, 2020. The depository unit is China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), the deposit number is CCTCC M 2020048, and the classification name is Candida viswanathii.

[0069] In the following examples, the method for detecting the content of long-chain dibasic acid in the fermentation broth is: determined by gas chromatography GC, specifically, the fermentation broth is treated and then separated in a capillary column, and the content of dibasic acid in the fermentation broth is determined based on the peak area of ​​the compound.

[0070] The purity of the dibasic acid products in the following examples was detected by gas chromatography.

[0071] The bio-based content determination method of the dibasic acid products in the following examples is carried out by determining the radioactive C14 content, such as the standard ASTM-D6866 method of the American Society for Testing and Materials.

[0072] In the following examples, the n-dodecane of Examples 1-4 is derived from petroleum, i.e., a petroleum-based alkane, and has a purity of 99.92%; the n-dodecane of Example 5 is obtained by processing vegetable oils and fats as raw materials, and is a bio-based alkane, and has a purity of 99.94%; the decane of Example 12 is derived from petroleum, i.e., a petroleum-based alkane, and has a purity of 99.95%; the n-hexadecane of Example 13 is derived from petroleum, i.e., a petroleum-based alkane, and has a purity of 99.91%.

[0073] Example 1

[0074] Seed culture medium: corn syrup 5g / L, sucrose 23g / L, urea 3g / L, yeast extract 5g / L, potassium dihydrogen phosphate 6g / L, defoamer 0.5g / L.

[0075] Fermentation medium: 5 g / L potassium dihydrogen phosphate, 8 g / L corn steep liquor, 35 g / L glucose, 4 g / L ammonium sulfate, 4 g / L yeast extract, 5 g / L potassium nitrate, and 2 g / L magnesium sulfate.

[0076] The fermentation production process includes:

[0077] (1) The pH value of 10°B malt juice was adjusted to about 5.4, 100 mL was taken and placed in a conical flask, sterilized at 121°C for 20 min, and then inoculated with a seed of Candida viswanathii CAES2113 in a 2 mL glycerol tube stored in a -80°C refrigerator, and activated and cultured on a rotary shaker at 29°C and 220 rpm for 39 h. The OD of the strain was 0.184. 620 When the value reaches 0.8 (30-fold dilution), the activation culture is completed.

[0078] (2) The shake flask seeds obtained in step (1) were inoculated into a seed tank containing seed culture medium and cultured at an inoculum of 1.5% (v / v). The temperature during the culture process was controlled at 29°C, the air volume was 0.3 vvm, the pressure was 0.10 MPa, and the dissolved oxygen was 15%, until the OD of the obtained seed solution when diluted 30 times reached 0.04. 620 The value reaches 0.7.

[0079] (3) The seed solution obtained in step (2) was inoculated into a fermentation tank at an inoculation amount of 15% (v / v). The fermentation process temperature was controlled at 29° C., the air volume was 0.3 vvm, and the pressure was 0.10 MPa. The stirring rate was controlled to maintain the dissolved oxygen at 40%. A 30% (w / v) NaOH solution was added to control the pH value of the fermentation process to 6.4. When the strain OD 620 When the value reaches 0.85 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and petroleum-based n-dodecane, and the mass ratio of the two is 15.5:14.5. From the start of fermentation to 150 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=2.83, b=0.031, c=0.26, after 150h of fermentation, the substrate addition was stopped, and the fermentation was continued with the remaining substrate in the fermentation system until the substrate was completely consumed or no long-chain dodecanedioic acid was produced. The fermentation results are shown in Table 1.

[0080] Example 2

[0081] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 1, except that:

[0082] In step (3), when the strain OD 620 When the value reaches 0.77 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and petroleum-based n-dodecane, and the mass ratio of the two is 12:18. From the start of fermentation to 148 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=2.65, b=0.025, C=0.45, after 148h of fermentation, the addition of substrate was stopped. The rest was the same as in Example 1.

[0083] Example 3

[0084] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 1, except that:

[0085] In step (3), when the strain OD 620 When the value reaches 0.7 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and petroleum-based n-dodecane, and the mass ratio of the two is 27:3. From the start of fermentation to 155 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=3.05, b=0.04, C=0.05, after 155h of fermentation, the addition of substrate was stopped. The rest was the same as in Example 1.

[0086] Example 4

[0087] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 1, except that:

[0088] In step (3), when the strain OD 620 When the value reaches 0.8 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and petroleum-based n-dodecane, and the mass ratio of the two is 20:10. From the start of fermentation to 153 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=2.92, b=0.036, C=0.6, after 153h of fermentation, the addition of substrate was stopped. The rest was the same as in Example 1.

[0089] Example 5

[0090] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 1, except that:

[0091] In step (3), when the strain OD 620 When the value reaches 0.82 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and bio-based n-dodecane, and the mass ratio of the two is 6:24. From the start of fermentation to 145 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=2.1, b=0.021, C=1.02, after 145h of fermentation, the addition of substrate was stopped. The rest was the same as in Example 1.

[0092] Example 6

[0093] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 1, except that in step (3), the substrates were ethyl laurate and petroleum-based n-dodecane. The rest was the same as that of Example 1.

[0094] Example 7

[0095] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 1, except that in step (3), the substrates were butyl laurate and petroleum-based n-dodecane. The rest was the same as that of Example 1.

[0096] Example 8

[0097] Seed culture medium: corn syrup 6g / L, sucrose 30g / L, urea 5g / L, yeast extract 8g / L, potassium dihydrogen phosphate 6g / L, defoaming agent 1g / L.

[0098] Fermentation medium: 8 g / L potassium dihydrogen phosphate, 5 g / L corn steep liquor, 40 g / L glucose, 3 g / L ammonium sulfate, 3 g / L yeast extract, 3 g / L potassium nitrate, and 5 g / L magnesium sulfate.

[0099] The fermentation production process includes:

[0100] (1) The pH value of 10°B malt juice was adjusted to about 5.4, 100 mL was taken and placed in a conical flask, sterilized at 121°C for 20 min, and then inoculated with a seed of Candida viswanathii CAES2113 in a 2 mL glycerol tube stored in a -80°C refrigerator, and activated and cultured on a rotary shaker at 30°C and 230 rpm for 40 h. The OD of the strain was 0.184. 620When the value reaches 0.85 (30-fold dilution), the activation culture is completed.

[0101] (2) The shake flask seeds obtained in step (1) above were inoculated into a seed tank containing seed culture medium and cultured, with an inoculum amount of 2.0% (v / v), and the temperature during the culture process was controlled to be 32°C, the air volume to be 0.6 vvm, the pressure to be 0.1 MPa, and the dissolved oxygen to be 20%, until the OD of the obtained seed solution when diluted 30 times reached 620 The value reaches 0.8.

[0102] (3) The seed solution obtained in step (2) was inoculated into a fermentation tank at an inoculation amount of 20% (v / v). The fermentation process temperature was controlled at 32°C, the air volume was 0.5 vvm, and the pressure was 0.15 MPa. The stirring rate was controlled to maintain the dissolved oxygen at 45%. A 30% (w / v) NaOH solution was added to control the pH value of the fermentation process to 6.2. When the strain OD 620 When the value reaches 0.88 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and petroleum-based n-dodecane, and the mass ratio of the two is 15.5:14.5. From the start of fermentation to 151 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=4.5, b=0.035, C=0.1, after 151h of fermentation, the substrate addition was stopped, and the fermentation was continued using the remaining substrate in the fermentation system until the substrate was completely consumed or no more dodecanedioic acid was produced. The fermentation results are shown in Table 1.

[0103] Example 9

[0104] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 8, except that:

[0105] In step (3), when the strain OD 620 When the value reaches 0.72 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and petroleum-based n-dodecane, and the mass ratio of the two is 15.5:14.5. From the start of fermentation to 145 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=1.05, b=0.04, C=3.53, after 145h of fermentation, the addition of substrate was stopped. The rest was the same as in Example 8.

[0106] Example 10

[0107] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 8, except that:

[0108] In step (3), when the strain OD 620 When the value reaches 0.77 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and petroleum-based n-dodecane, and the mass ratio of the two is 15.5:14.5. From the start of fermentation to 150 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=2.36, b=0.028, C=2.25, after 150h of fermentation, the addition of substrate was stopped. The rest was the same as in Example 8.

[0109] Embodiment 11

[0110] The long-chain dibasic acid was produced by fermentation using a method almost identical to that of Example 8, except that:

[0111] In step (3), when the strain OD 620 When the value reaches 0.81 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and petroleum-based n-dodecane, and the mass ratio of the two is 15.5:14.5. From the start of fermentation to 153 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=3.4, b=0.022, C=1.2, after 153h of fermentation, the addition of substrate was stopped. The rest was the same as in Example 8.

[0112] Example 12

[0113] Seed culture medium: corn syrup 3g / L, sucrose 18g / L, urea 5g / L, yeast extract 6g / L, potassium dihydrogen phosphate 5g / L, defoamer 0.5g / L.

[0114] Fermentation medium: 6 g / L potassium dihydrogen phosphate, 5 g / L corn steep liquor, 30 g / L glucose, 5 g / L ammonium sulfate, 4 g / L yeast extract, 5 g / L potassium nitrate, and 4 g / L magnesium sulfate.

[0115] The fermentation production process includes:

[0116] (1) The pH value of 10°B malt juice was adjusted to about 5.4, 100 mL was taken and placed in a conical flask, sterilized at 121°C for 20 min, and then inoculated with a seed of Candida viswanathii CAES2113 in a 2 mL glycerol tube stored in a -80°C refrigerator, and activated and cultured on a rotary shaker at 29°C and 220 rpm for 40 h. The OD of the strain was 0.184. 620 When the value reaches 0.8 (30-fold dilution), the activation culture is completed.

[0117] (2) The shake flask seeds obtained in step (1) above were inoculated into a seed tank containing seed culture medium and cultured, with an inoculum amount of 1.5% (v / v), and the temperature during the culture process was controlled to be 30°C, the air volume to be 0.4 vvm, the pressure to be 0.1 MPa, and the dissolved oxygen to be 18%, until the OD of the obtained seed solution when diluted 30 times reached 620 The value reaches 0.85.

[0118] (3) The seed solution obtained in step (2) was inoculated into a fermentation tank at an inoculation amount of 20% (v / v). The fermentation process temperature was controlled at 30°C, the air volume was 0.5 vvm, and the pressure was 0.12 MPa. The stirring rate was controlled to maintain the dissolved oxygen at 40%. A 30% (w / v) NaOH solution was added to control the pH value of the fermentation process to 6.3. When the strain OD 620 When the value reaches 0.8 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is ten-carbon fatty acid methyl ester and petroleum-based decane, and the mass ratio of the two is 15.5:14.5. From the start of fermentation to 145 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=2.0, b=0.021, C=0.26, after 145h of fermentation, the substrate addition was stopped, and the fermentation was continued using the remaining substrate in the fermentation system until the substrate was completely consumed or no more sebacic acid was produced. The fermentation results are shown in Table 1.

[0119] Embodiment 13

[0120] Seed culture medium: corn syrup 6g / L, sucrose 20g / L, urea 5g / L, yeast extract 6g / L, potassium dihydrogen phosphate 3g / L, defoamer 0.5g / L.

[0121] Fermentation medium: 5 g / L potassium dihydrogen phosphate, 8 g / L corn steep liquor, 30 g / L glucose, 5 g / L ammonium sulfate, 4 g / L yeast extract, 5 g / L potassium nitrate, and 2 g / L magnesium sulfate.

[0122] The fermentation production process includes:

[0123] (1) The pH value of 10°B malt juice was adjusted to about 5.4, 100 mL was taken and placed in a conical flask, sterilized at 121°C for 20 min, and then inoculated with a seed of Candida viswanathii CAES2113 in a 2 mL glycerol tube stored in a -80°C refrigerator, and activated and cultured on a rotary shaker at 29°C and 220 rpm for 40 h. The OD of the strain was 0.184. 620 When the value reaches 0.8 (30-fold dilution), the activation culture is completed.

[0124] (2) The shake flask seeds obtained in step (1) above were inoculated into a seed tank containing seed culture medium and cultured, with an inoculum amount of 1.5% (v / v), and the temperature during the culture process was controlled to be 30°C, the air volume to be 0.3 vvm, the pressure to be 0.1 MPa, and the dissolved oxygen to be 15%, until the OD of the obtained seed solution when diluted 30 times reached 620 The value reaches 0.8.

[0125] (3) The seed solution obtained in step (2) was inoculated into a fermentation tank at an inoculation amount of 18% (v / v). The fermentation process temperature was controlled at 30°C, the air volume was 0.5 vvm, and the pressure was 0.10 MPa. The stirring rate was controlled to maintain the dissolved oxygen at 40%. A 30% (w / v) NaOH solution was added to control the pH value of the fermentation process to 6.0. When the strain OD 620 When the value reaches 0.82 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is 16-carbon fatty acid methyl ester and petroleum-based n-16-carbon alkane, and the mass ratio of the two is 3:27. From the start of fermentation to 150 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=2.5, b=0.031, C=0.66, after 150h of fermentation, the substrate addition was stopped, and the fermentation was continued with the remaining substrate in the fermentation system until the substrate was completely consumed or no more hexadecane dibasic acid was produced. The fermentation results are shown in Table 1.

[0126] Embodiment 14

[0127] The long-chain dibasic acid was produced by fermentation using a method almost identical to that in Example 1, except that:

[0128] In step (2), the dissolved oxygen is controlled to be 50%;

[0129] In step (3), when the strain OD 620When the value reaches 0.75 (diluted 30 times), the substrate is added into the fermentation tank to start fermentation. The substrate is methyl laurate and lauric acid, and the mass ratio of the two is 14:16. From the start of fermentation to 150 hours, the viscosity η of the fermentation system is detected in real time by an online viscosity detection device, and the amount of substrate added is regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfies: η=A*e bt +C, A=2.8, b=0.03, C=0.55, after 150h of fermentation, the addition of substrate was stopped. The rest was the same as in Example 1.

[0130] Comparative Example 1

[0131] The long-chain dibasic acid was fermented and produced by a method almost the same as that in Example 1, except that the viscosity η of the fermentation system was detected in real time by an online viscosity detection device, and the amount of substrate added was regulated according to the feedback of the viscosity η so that the numerical relationship between the viscosity η of the fermentation system and the fermentation time t satisfied: η = A*e bt +C, A=2.83, b=0.063, C=0.26. Others are the same as in Example 1.

[0132] Table 1

[0133]

[0134] Extraction and purification of long-chain dibasic acid fermentation broth

[0135] The fermentation broth obtained in the above Examples 1-7 and 14 was extracted and purified, specifically, sulfuric acid was added to the fermentation broth to adjust the pH to 3.5, acidification and crystallization were performed, and solids were separated to obtain a crude product of dodecanedioic acid.

[0136] The crude product of the above-mentioned dodecanedioic acid is placed in a decolorizing tank, acetic acid is added, the mass ratio of the crude dodecanedioic acid to acetic acid is controlled at 1:4.5, activated carbon is added, and the amount of activated carbon added is 3.8% of the mass of the dodecanedioic acid in the decolorized liquid, the above-mentioned materials are heated to 90°C, decolorized for 60 minutes, and filtered through a plate and frame filter press to obtain a dodecanedioic acid clear liquid; the obtained clear liquid is placed in a crystallization tank, cooled to 60°C, kept warm for 2 hours, and then cooled to 30°C, after crystals are precipitated, solid-liquid separation is performed by a centrifuge, and the obtained solid is added to water, the mass ratio of solid to water is 1:10, kept warm at 110°C for 90 minutes, cooled to 30°C, filtered, and a solid is obtained, and the solid is washed and dried to obtain a dodecanedioic acid product. The results are shown in Table 2.

[0137] The fermentation broth obtained in Example 12 was extracted and purified, specifically, sulfuric acid was added to the fermentation broth to adjust the pH to 4, acidification and crystallization were performed, and solids were separated to obtain a crude product of sebacic acid.

[0138] The crude product of sebacic acid is placed in a decolorizing tank, acetic acid is added, the mass ratio of the crude product of sebacic acid to acetic acid is controlled at 1:4, activated carbon is added, and the amount of activated carbon added is 4% of the mass of sebacic acid in the decolorized liquid, the above materials are heated to 95°C, decolorized for 50 minutes, and filtered through a plate and frame filter press to obtain a clear solution of sebacic acid; the obtained clear solution is placed in a crystallization tank, cooled to 60°C, kept warm for 2 hours, and then cooled to 30°C, after crystals are precipitated, solid-liquid separation is performed by a centrifuge, and the obtained solid is added to water, the mass ratio of solid to water is 1:10, kept warm at 108°C for 90 minutes, cooled to 30°C, filtered, and a solid is obtained, and the solid is washed and dried to obtain a sebacic acid product. The results are shown in Table 2.

[0139] The fermentation broth obtained in Example 13 was extracted and purified, specifically, sulfuric acid was added to the fermentation broth to adjust the pH to 3.8, acidification and crystallization were performed, and solids were separated to obtain a crude product of hexadecanedioic acid.

[0140] The crude product of the above-mentioned 16-carbon dicarboxylic acid is placed in a decolorizing tank, acetic acid is added, and the mass ratio of the crude 16-carbon dicarboxylic acid to acetic acid is controlled at 1:4. Activated carbon is added, and the amount of activated carbon added is 3.5% of the mass of the 16-carbon dicarboxylic acid in the decolorized liquid. The above-mentioned materials are heated to 95°C, decolorized for 40 minutes, and filtered through a plate and frame filter press to obtain a 16-carbon dicarboxylic acid clear liquid; the obtained clear liquid is placed in a crystallization tank, cooled to 60°C, kept warm for 2 hours, and then cooled to 30°C. After the crystals are precipitated, they are separated into solid and liquid by a centrifuge, and the obtained solid is added to water, and the mass ratio of the solid to water is 1:8. After keeping warm at 108°C for 90 minutes, the temperature is lowered to 30°C, filtered, and a solid is obtained. The solid is washed and dried to obtain a 16-carbon dicarboxylic acid product. The results are shown in Table 2

[0141] Table 2

[0142] Product purity wt% Product Biobased Content % Example 1-DC12 99.69 45％ Example 2-DC12 99.83 34％ Example 3-DC12 99.78 87.5％ Example 4-DC12 99.81 61％ Example 5-DC12 99.73 98％ Example 6-DC12 99.67 44％ Example 7-DC12 99.7 41.5％ Example 12-DC10 99.88 45％ Example 13-DC16 99.75 8.5％ Example 14-DC12 99.8 98.8％

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing long-chain dibasic acids by fermentation, characterized in that: The method comprises: introducing the seed liquid of the fermentation strain into a fermentation tank, adding a substrate for fermentation, and controlling the fermentation temperature to be 20-40° C. The substrate comprises fatty acid esters and alkanes, or fatty acid esters and fatty acids.

2. The method for producing long-chain dibasic acid by fermentation according to claim 1, characterized in that: From the start of fermentation to T hours, the viscosity η of the fermentation system is made to satisfy the numerical relationship between the fermentation time t and the following equation: η=A*e bt +C; wherein, e is a natural constant, 1.0≤A≤5.5, 0.02≤b≤0.043, 0≤C≤8, 135≤T≤165, the unit of the viscosity η is mPa·s, and the unit of the fermentation time t is h.

3. The method for producing long-chain dibasic acid by fermentation according to claim 1, characterized in that: The mass ratio of fatty acid ester to alkane is (0.01-30):1, further (0.1-20):1; and / or, the mass ratio of fatty acid ester to fatty acid is 1:(0.01-40), further 1:(0.1-30); And / or, the fatty acid ester includes any one of a deca-carbon fatty acid ester, an undica-carbon fatty acid ester, a lauric acid ester, a thirteen-carbon fatty acid ester, a tetradeca-carbon fatty acid ester, a penta-carbon fatty acid ester, a hexa-carbon fatty acid ester, a heptadeca-carbon fatty acid ester and an octa-carbon fatty acid ester; And / or, the alkane comprises bio-based alkane and / or petroleum-based alkane, further comprising C9-C 22 The normal alkanes further include C 10 -C 18 n-alkanes, further including C 10 , C 11 , C 12 , C 13 , C 14 , C 15 or C 16 of n-alkanes; And / or, the fatty acid includes any one of decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid and octadecanoic acid.

4. The method for producing long-chain dibasic acid by fermentation according to claim 1, characterized in that: The fermentation strain comprises Candida viswanathii, Candida tropicalis, Candida sake, Candida albicans or Yarrowia lipolytica; and / or, The chemical formula of the long chain dibasic acid is HOOC(CH2) n COOH, wherein n≥8, includes but is not limited to any one of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedicarboxylic acid, hexadecanedioic acid, heptadecanedioic acid and octadecanedicarboxylic acid.

5. The method for producing long-chain dibasic acid by fermentation according to claim 1, characterized in that: The inoculation amount of the fermentation strain seed liquid is 10-50 v / v%; and / or, Control the fermentation air volume to 0.2-0.8vvm; and / or, The fermentation pressure is controlled to be 0.05-0.20 MPa; and / or, Control the pH of the fermentation to 4.5-6.5; and / or, Controlling dissolved oxygen to 5-80%; and / or, Control the total fermentation time to 120-200h.

6. The method for producing long-chain dibasic acid by fermentation according to claim 1, characterized in that: When the strain was diluted 30 times, the optical density OD 620 When the pH reaches 0.5-1.0, the substrate is added to start fermentation.

7. The method for producing long-chain dibasic acid by fermentation according to claim 1, characterized in that: The components of the fermentation medium include carbon sources, nitrogen sources, inorganic salts and nutritional factors; Further, the carbon source includes one or more of glucose, sucrose, maltose, molasses, fructose, rhamnose, arabinose, glycerol, methanol, ethanol and sorbitol, and the added amount of the carbon source is 10-80 g / L; Further, the nitrogen source includes one or more of yeast extract, peptone, corn steep liquor, urea, ammonium salt and nitrate, and the addition amount of each nitrogen source is 0-10g / L; Further, the inorganic salt includes one or more of sulfate, hydrochloride, nitrate and phosphate; preferably, includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, sodium chloride and potassium nitrate, and the addition amount of each inorganic salt is 1-10g / L; Further, the growth factor includes one or more of amino acids, citric acid and vitamins; preferably, the growth factor includes one or more of vitamin B1, vitamin B2, vitamin C and biotin, and the added amount of the biological factor is 0-1g / L; Preferably, the fermentation medium comprises the following ingredients: corn steep liquor 0-10 g / L, glucose 20-60 g / L, yeast extract 0-8 g / L, potassium dihydrogen phosphate 4-10 g / L, magnesium sulfate 2-6 g / L, ammonium sulfate 1-10 g / L, and potassium nitrate 2-8 g / L.

8. The method for producing long-chain dibasic acid by fermentation according to any one of claims 1 to 7, characterized in that: The method further comprises expanding the fermentation strain, wherein the expanding culture process comprises: activating the fermentation strain in a shaking bottle, and waiting for the optical density value OD of the shaking bottle seeds when diluted 30 times 620 When the optical density of the seed solution is 0.5 to 1.0, the seed is placed in a seed tank filled with a seed culture medium for seed culture until the optical density of the seed solution is OD 620 Reach 0.5~1.

0.

9. A method for preparing a long-chain dibasic acid, characterized in that: The method comprises: S1: obtaining a long-chain dibasic acid fermentation liquid according to the method for producing a long-chain dibasic acid by fermentation as described in any one of claims 1 to 8; S2: extracting and purifying the long-chain dibasic acid fermentation broth to obtain a long-chain dibasic acid product.

10. The preparation method according to claim 9, characterized in that: The extraction and purification comprises: acidifying the fermentation liquid, separating the solid, dissolving the solid in an organic solvent, crystallizing, and separating the solid from the liquid, washing and drying the separated solid to obtain a long-chain dibasic acid product.

11. The preparation method according to claim 10, characterized in that: Before washing the separated solid, it is first treated with hot water, then washed and dried; The temperature of the hot water treatment is 70-150°C; The hot water treatment comprises the following steps: mixing solid and water in a mass ratio of 1:(1-10), keeping the temperature at 70-150° C. for 30-180 min, cooling to 25-60° C., separating the solid from the liquid, and obtaining a solid.

12. The long-chain dibasic acid product obtained by the preparation method according to any one of claims 9 to 11, wherein the purity of the long-chain dibasic acid product is greater than 99 wt%; and / or the biobased content is 1-100%, further 5-98%.

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