A method for continuous fermentation production of long-chain dibasic acids

Abstract

This invention relates to the field of bio-fermentation technology and discloses a method for continuous fermentation to produce long-chain dicarboxylic acids. The method includes: after fermentation for 80-150 hours, continuously adding a substrate and a nutrient-containing solution to the fermentation broth, wherein the ratio of the flow rate of the nutrient-containing solution to the flow rate of the substrate is (0.83-300):1; and the dilution rate D of the fermentation system is 0.007-0.3 h. ‑1 This invention achieves an oversupply of nutrients by controlling the ratio of the flow rate of the nutrient-containing solution to the flow rate of the substrate within a specific range. This allows the metabolic activity of the cells to be maintained even at high dilution rates, thereby ensuring the continuous production of long-chain dicarboxylic acids and significantly improving the yield and productivity of these acids.

Description

[0001] This application is a divisional application. Its parent application has the application number 2021108509630, the application date is July 27, 2021, and the invention title is "Method for Continuous Fermentation to Produce Long-Chain Dicarboxylic Acids and Method for Producing Long-Chain Dicarboxylic Acids". Technical Field

[0002] This invention relates to the field of bio-fermentation technology, specifically to a method for continuous fermentation to produce long-chain dicarboxylic acids and a method for producing long-chain dicarboxylic acids. Background Technology

[0003] Due to their unique molecular structure and reactivity, long-chain dicarboxylic acids have a wide range of applications in many fields. They can be used as raw materials to synthesize special nylon (polyamide), high-grade fragrances, high-grade hot melt adhesives, cold-resistant plasticizers, high-grade lubricants, high-grade rust inhibitors, high-grade paints and coatings, etc.

[0004] Currently, the synthesis of long-chain dicarboxylic acids mainly involves two methods: chemical synthesis and bio-fermentation. Chemical synthesis is technically mature but has a long synthetic route; however, it requires high temperature and pressure conditions and places stringent demands on the catalyst, limiting its application to dicarboxylic acids of specific chain lengths. Bio-fermentation, on the other hand, uses long-chain alkanes or fatty acids as substrates, converting them through microbial fermentation to produce long-chain dicarboxylic acids. This process is carried out at ambient temperature and pressure and can be scaled up, such as for C9 to C6 dicarboxylic acids. 18 It produces a variety of long-chain dicarboxylic acids. Compared with chemical synthesis, bio-fermentation has more obvious advantages in terms of raw material availability, product diversity, production efficiency, production cost, and environmental benefits. In the past 20 years of industrial development, chemical synthesis has been gradually replaced by bio-fermentation.

[0005] CN106755146B discloses a method and apparatus for continuous fermentation to produce long-chain dicarboxylic acids. The continuous fermentation process is achieved by coupling a membrane filtration device with a seed tank and periodically replenishing fresh seed liquid. This not only increases the cost of membrane separation but also increases the risk of microbial contamination.

[0006] CN109868294A discloses a method with a low dilution rate (0.001-0.006h). -1 A continuous fermentation method for producing long-chain dicarboxylic acids involves continuously adding substrate and a nutrient-containing solution after 100-150 hours of fermentation, ensuring continuous production of the long-chain dicarboxylic acids. However, this method suffers from low dilution rates, resulting in high acid concentrations. Excessively high concentrations of material present significant challenges during transport, hindering industrial application. Furthermore, the yield and productivity of the long-chain dicarboxylic acids obtained by this method require further improvement. Summary of the Invention

[0007] The purpose of this invention is to further improve the yield and production rate of long-chain dicarboxylic acids through fermentation, and to solve the problems in existing continuous fermentation methods for producing long-chain dicarboxylic acids, such as high acid concentration due to low dilution rate and difficulties in material transportation in the later stages. This invention provides a method for continuous fermentation production of long-chain dicarboxylic acids and a production method for long-chain dicarboxylic acids.

[0008] To achieve the above objectives, the first aspect of the present invention provides a method for continuous fermentation to produce long-chain dicarboxylic acids, the method comprising: after fermentation for 80-150 h, continuously adding a substrate and a solution containing nutrients to the fermentation broth, wherein the ratio of the flow rate of the nutrient-containing solution to the flow rate of the substrate is (0.83-300):1; and the dilution rate D of the fermentation system is 0.007-0.3 h. -1 .

[0009] Preferably, the ratio of the flow acceleration of the nutrient-containing solution to the flow acceleration of the substrate is (1.5-150):1.

[0010] Preferably, the ratio of the flow acceleration of the nutrient-containing solution to the flow acceleration of the substrate is (1.88-40.54):1.

[0011] Preferably, the dilution rate D of the fermentation system is 0.007-0.15h. -1 .

[0012] Preferably, the dilution rate D of the fermentation system is 0.0076-0.15h. -1 .

[0013] A second aspect of the present invention provides a method for producing long-chain dicarboxylic acids, the method comprising the continuous fermentation method for producing long-chain dicarboxylic acids described in the first aspect of the present invention.

[0014] Preferably, before producing long-chain dicarboxylic acids using a continuous fermentation method, the method further includes: inoculating the fermentation strain into a fermentation medium for fermentation, and after fermentation for 10-100 hours, adding substrate to the fermentation broth to ensure that the substrate content in the fermentation broth is above 1% (v%).

[0015] Through the above technical solution, this invention continuously adds substrate and a nutrient-containing solution to the fermentation broth, ensuring that the flow rates of the substrate, the nutrient-containing solution, and the volume of the fermentation broth satisfy a specific stoichiometric relationship. Under high dilution conditions, the flow rates of the nutrient-containing solution and the substrate satisfy a synergistic relationship, thereby maintaining the activity of the cells in a high-speed metabolic environment, ensuring the continuous production of long-chain dicarboxylic acids, and significantly improving the yield and productivity of the long-chain dicarboxylic acid fermentation process. At the same time, the high dilution rate can also improve the problem of difficult material transportation in the later stages, which is beneficial for industrial application.

[0016] Furthermore, by controlling the ratio of the flow acceleration of the nutrient-containing solution to the flow acceleration of the substrate within a specific range, an excess supply of nutrients is achieved, thereby maintaining the metabolic activity of the cells under high dilution conditions and significantly improving the yield and productivity of the long-chain dicarboxylic acid fermentation process. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] One of the challenges in existing continuous fermentation production of long-chain dicarboxylic acids lies in maintaining cell activity and continuous acid production during long-term fermentation, while achieving high dicarboxylic acid yields and rates. Extensive research by the applicant has revealed that the feed rates of the substrate and the nutrient-containing solution significantly impact cell activity, dicarboxylic acid yield, and rate of production. Maintaining a balance between these two factors yields excellent results. Therefore, by continuously feeding the substrate and nutrient-containing solution into the fermentation broth, and further ensuring a specific stoichiometric relationship between the feed rates of these two solutions and the volume of the fermentation broth, a synergistic relationship is achieved between the feed rates of the nutrient-containing solution and the substrate under high dilution conditions. This maintains cell activity in a high-speed metabolic environment, ensuring continuous production of long-chain dicarboxylic acids and significantly improving the yield and rate of long-chain dicarboxylic acid fermentation.

[0019] Furthermore, by controlling the ratio of the flow acceleration of the nutrient-containing solution to the flow acceleration of the substrate within a specific range, an excess supply of nutrients is achieved, thereby maintaining the metabolic activity of the cells under high dilution conditions and significantly improving the yield and productivity of the long-chain dicarboxylic acid fermentation process.

[0020] As previously stated, the first aspect of this invention provides a method for continuous fermentation to produce long-chain dicarboxylic acids. The method includes: after fermentation for 80-150 hours, continuously adding a substrate and a nutrient-containing solution to the fermentation broth, wherein the ratio of the flow rate of the nutrient-containing solution to the flow rate of the substrate is (0.83-300):1; and the dilution rate D of the fermentation system is 0.007-0.3 h. -1 .

[0021] In some embodiments of the present invention, preferably, the ratio of the flow acceleration of the nutrient-containing solution to the flow acceleration of the substrate is (1.5-150):1.

[0022] In some embodiments of the present invention, preferably, the ratio of the flow acceleration of the nutrient-containing solution to the flow acceleration of the substrate is (1.88-40.54):1.

[0023] In some embodiments of the present invention, preferably, the flow acceleration of the substrate, the flow acceleration of the nutrient-containing solution, and the volume of the fermentation broth satisfy the following relationship:

[0024] 0.005h -1 *V (fermentation broth) ≤ Q (solution containing nutrients) ≤ 0.3h -1 *V (fermentation broth);

[0025] 0.001h -1 *V (fermentation broth) ≤ Q (substrate) ≤ 0.006h -1 *V (fermentation broth);

[0026] Wherein, V (fermentation broth) represents the volume of the fermentation broth, Q (nutrient-containing solution) represents the flow acceleration of the nutrient-containing solution, and Q (substrate) represents the flow acceleration of the substrate.

[0027] In some embodiments of the present invention, preferably, the flow acceleration of the substrate and the volume of the fermentation broth satisfy the following condition: 0.001 h⁻¹ -1 *V (fermentation broth) ≤ Q (substrate) ≤ 0.004h -1 *V (fermentation broth) This makes it easier to achieve a certain balance between the substrate flow rate and the fermentation broth volume, thereby ensuring the continuous production of long-chain dicarboxylic acids and improving the yield and productivity of long-chain dicarboxylic acids.

[0028] In some embodiments of the present invention, preferably, the flow rate of the nutrient-containing solution is related to the volume of the fermentation broth by 0.006 h⁻¹. -1 *V (fermentation broth) ≤ Q (solution containing nutrients) ≤ 0.15h -1 *V (fermentation broth). Under this preferred condition, the continuous production of long-chain dicarboxylic acids can be further guaranteed, and the yield and productivity of long-chain dicarboxylic acids can be improved.

[0029] According to a preferred embodiment of the present invention, for a 30L fermenter, the flow rate of the substrate is 15-150 mL / h, more preferably 15-100 mL / h; the flow rate of the nutrient-containing solution is 75-7500 mL / h, more preferably 75-3750 mL / h.

[0030] In some embodiments of the present invention, the dilution rate D also has a certain impact on cell activity, dicarboxylic acid yield, and production rate. The present invention, by optimizing the dilution rate, can further improve cell activity, dicarboxylic acid yield, and production rate. Simultaneously, compared to the low dilution rate used in the prior art, the present application, by using a high dilution rate, can effectively improve the acid concentration, facilitating subsequent material transport and thus promoting industrial application. Preferably, the dilution rate D of the fermentation system is 0.007-0.15 h. -1 Preferably, it is 0.0076-0.15h. -1 In this invention, the dilution rate D = [Q (solution containing nutrients) + Q (substrate)] / V (fermentation broth).

[0031] In some embodiments of the present invention, a nutrient-containing solution with specific components can ensure the viability of cell growth and a good fermentation state in the fermentation system, thereby achieving better technical results. Preferably, the nutrient-containing solution contains 0-2.5 (w / v)% glucose, 0.05-2.0 (w / v)% corn steep liquor, 0-1.0 (w / v)% yeast extract, 0.005-0.5 (w / v)% potassium dihydrogen phosphate, 0.005-0.05 (w / v)% urea, and 0.01-0.15 (w / v)% ammonium sulfate. In the present invention, the unit "(w / v)%" represents the mass-volume ratio, that is, the ratio of the mass of each component to the volume of the nutrient-containing solution, with mass expressed in grams and volume expressed in 100 mL.

[0032] The present invention has a wide range of substrate selection; preferably, the substrate is selected from C9-C. 18 At least one of n-alkanes, straight-chain saturated fatty acids, straight-chain saturated fatty acid esters, and straight-chain saturated fatty acid salts, preferably C14. 10 -C 16 The preferred method uses n-alkanes, including but not limited to undecane, dodecane, tridecane, and hexadecane. In this preferred case, the yield and productivity of long-chain dicarboxylic acids can be further improved.

[0033] In some embodiments of the present invention, preferably, the long-chain dicarboxylic acid is expressed as HOOC(CH2). n COOH, wherein n≥7, more preferably at least one of azelaic acid, sebacic acid, 1,11-undecanoic acid, 1,12-dodecanoic acid, 1,13-tetanedicarboxylic acid, 1,14-tetradecanoic acid, 1,15-pentadecanoic acid, 1,16-hexadecanoic acid, 1,17-heptadecanoic acid, and 1,18-octadecanoic acid.

[0034] This invention does not impose any particular limitation on the equipment used for continuous fermentation production of long-chain dicarboxylic acids; any conventional equipment in the field can be used, with fermentation preferably carried out in a fermenter. In the continuous fermentation method described in this invention, the volume of the fermentation broth is preferably 50-85% of the fermenter volume. For example, when using a 30L fermenter for continuous fermentation, the volume of the fermentation broth in the fermenter is maintained between 15-25L by controlling the feed rate and discharge rate of the substrate and the nutrient-containing solution.

[0035] A second aspect of this invention provides a method for producing long-chain dicarboxylic acids, comprising the continuous fermentation method for producing long-chain dicarboxylic acids as described above. By employing the continuous fermentation method for producing long-chain dicarboxylic acids described in this invention, continuous production of long-chain dicarboxylic acids can be ensured, and the yield and productivity of long-chain dicarboxylic acids can be improved under conditions of higher dilution rates.

[0036] In some embodiments of the present invention, preferably, before producing long-chain dicarboxylic acids using a continuous fermentation method, the method further includes: inoculating fermentation strains into a fermentation culture medium for fermentation, and after fermentation for 10-100 hours, adding substrate to the fermentation broth to ensure that the substrate content in the fermentation broth is above 1% (v%).

[0037] The present invention does not impose any particular limitation on the method of adding the substrate. It can be added in batches or all at once. Those skilled in the art can choose according to actual needs.

[0038] In some embodiments of the present invention, preferably, the fermentation is carried out in a fermenter, and the initial volume of the fermentation is 30-70% of the total volume of the fermenter. For example, when the volume of the fermenter is 30L, the initial volume of the fermentation is 9-21L. In the present invention, the initial volume of the fermentation is the volume of the fermentation broth after inoculation.

[0039] In some embodiments of the present invention, preferably, the inoculation amount of the fermentation strain is 10-30 v%, based on the initial fermentation volume. Those skilled in the art should understand that before being inoculated into the fermentation medium, the fermentation strain is first cultured in a seed tank, and the cell concentration (OD) is measured. 620 When the concentration is diluted 30 times to 0.5-1.0, the culture is stopped. The seed liquid in the seed tank at this point is called mature seed liquid. Then, the mature seed liquid is inoculated into the fermentation medium. Therefore, in this invention, the inoculation amount of the fermentation strain is 10-30 v%, which means that the volume of the mature seed liquid inoculated into the fermentation medium accounts for 10-30% of the volume of the fermentation medium after inoculation.

[0040] In this invention, there is no particular limitation on the method of culturing the seed liquid; conventional seed liquid culturing methods in the art can be used, including but not limited to the following methods:

[0041] (i) Shake flask seed culture process: Take the inoculum and inoculate it into a 500mL Erlenmeyer flask containing seed culture medium (the liquid volume is 50-100mL). Under the conditions of 28-32℃ and optional initial pH value of 6.0-6.5, culture it on a shaker at a speed of 200-250rpm for 1-2 days.

[0042] (ii) Seed culture process: Take seeds from shake flasks and inoculate them into a 10L seed tank containing seed culture medium (5-8L liquid volume). Control the inoculation amount to 10-30 v%, and control the temperature to 28-32℃, pressure to 0.05-0.14MPa, and aeration rate to 0.3-0.7 vvm. Maintain the pH value to 3.0-7.5 by adding 10-40 (w / w)% liquid alkali. Keep a certain stirring speed to ensure that the dissolved oxygen (DO) is above 10% during seed culture. Culture for 15-30 hours to achieve the desired bacterial cell concentration (OD). 620 After diluting 30 times to a concentration of 0.5-1.0, it becomes mature seed liquid.

[0043] In some embodiments of the present invention, preferably, the method further includes: adding a substrate to the fermentation medium before inoculation, wherein the amount of substrate added is 0-10 v%, preferably 1-5 v%, relative to the volume of the fermentation medium; or

[0044] After inoculation, the substrate is added to the starting volume of fermentation, and the amount of substrate added is 0-10 v%, preferably 1-5 v%, relative to the starting volume of fermentation.

[0045] This invention does not impose particular limitations on the fermentation conditions, and any conventional selections in the art can be used. Preferably, the fermentation conditions include: a temperature of 28-32℃, a pressure of 0.05-0.14 MPa, a pH value of 5.5-7.5, and an aeration rate of 0.3-0.7 vvm. In this invention, the fermentation process is carried out under stirring conditions. This invention does not impose particular limitations on the stirring rate, and any conventional selections in the art can be used, preferably 500-700 rpm. In this invention, the pressure is gauge pressure. This invention does not impose particular limitations on the method of controlling the pH value of the fermentation broth, and any conventional selections in the art can be used, such as including but not limited to using an alkaline solution with a concentration of 10-40 (w / w)% for control.

[0046] In some embodiments of the present invention, preferably, the dissolved oxygen content during fermentation is above 10%. In this invention, the dissolved oxygen content during fermentation is measured using an online dissolved oxygen electrode.

[0047] In some embodiments of the present invention, preferably, the fermentation cell concentration OD 620 The concentration was 9-24, and after a 30-fold dilution, it was 0.3-0.8. In this invention, the bacterial concentration was determined using a spectrophotometer.

[0048] The present invention allows for a wide range of selection of fermentation strains, with *Candida tropicalis* or *Candida viswanathii* being preferred. For example, *Candida tropicalis* strain CAT H1614, with accession number CCTCC M2015303; and *Candida viswanathii* strain CAES2113, with accession number CCTCC M2020048.

[0049] In this invention, three types of culture media are mainly used: seed culture medium, fermentation culture medium, and fed-batch fermentation culture medium. This invention does not impose any particular limitations on the composition of each culture medium; commonly used culture medium components in the art can be used, including but not limited to the following three types of culture media:

[0050] Seed culture medium: sucrose content 1-3 (w / v)%, corn steep liquor content 0.15-1 (w / v)%, yeast extract content 0.2-1.5 (w / v)%, KH2PO4 content 0.4-1.5 (w / v)%, urea content 0.05-0.5 (w / v)%.

[0051] Fermentation medium: glucose content 1-5 (w / v)%, corn steep liquor content 0.1-0.9 (w / v)%, yeast extract content 0.1-0.5 (w / v)%, potassium nitrate content 0.05-1.2 (w / v)%, potassium dihydrogen phosphate content 0.05-1.0 (w / v)%, urea content 0.05-0.3 (w / v)%, ammonium sulfate content 0.05-0.3 (w / v)%, sodium chloride content 0.05-0.2 (w / v)%.

[0052] Feeding medium: This consists of substrate and a nutrient solution containing 0-2.5% (w / v) glucose, 0.05-2.0% (w / v) corn steep liquor, 0-1.0% (w / v) yeast extract, 0.005-0.5% (w / v) potassium dihydrogen phosphate, 0.005-0.05% (w / v) urea, and 0.01-0.15% (w / v) ammonium sulfate. All the above media are sterilized at 121°C for 20 min before use.

[0053] To clearly describe the production method of the long-chain dicarboxylic acid according to the present invention, a preferred embodiment is provided below:

[0054] (1) The seed liquid cultured in the seed tank is inoculated into a fermenter containing fermentation medium. The initial fermentation volume accounts for 30-70% of the fermenter volume. Based on the initial fermentation volume, the inoculation amount of the seed liquid is 10-30v%. Before or after inoculation, substrate is added to it. The amount of substrate added relative to the volume of fermentation medium or the initial fermentation volume is 1-5v%. Then, the temperature is controlled at 28-32℃, the pressure is 0.05-0.14MPa, the ventilation volume is 0.3-0.7vvm, and the pH value of the fermentation liquid is made to 5.5-7.5 by adding liquid alkali with a concentration of 10-40 (w / w)%. A certain stirring speed is maintained to control the dissolved oxygen content of fermentation to be above 10%. After fermentation for 10-100h, substrate is added to the fermentation liquid in batches to ensure that the substrate content in the fermentation liquid is above 1v%.

[0055] (2) After fermentation for 80-150 h, substrate and a solution containing nutrients are continuously added to the fermentation broth. The addition rate is controlled so that the addition rate of the substrate and the volume of the fermentation broth satisfy the following condition: 0.001 h -1 *V (fermentation broth) ≤ Q (substrate) ≤ 0.004h -1 *V (fermentation broth); The flow rate of the nutrient-containing solution and the volume of the fermentation broth satisfy: 0.006h -1 *V (fermentation broth) ≤ Q (solution containing nutrients) ≤ 0.15h -1 *V (fermentation broth), and control the cell concentration OD during fermentation. 620 After dilution 30 times, the concentration is 0.3-0.8, and the fermentation liquid volume is maintained at 15-25L by controlling the discharge rate.

[0056] In this invention, there are no particular limitations on the method for determining the concentration of dicarboxylic acid in the fermentation broth. Techniques well-known to those skilled in the art can be used, such as the method disclosed in Chinese Patent ZL95117436.3. Specifically, the pH of the fermentation broth is adjusted to 3.0 using hydrochloric acid solution, then 100 mL of diethyl ether is added to extract the dicarboxylic acid from the broth. The ether is then removed by evaporation to obtain dicarboxylic acid powder. The obtained dicarboxylic acid powder is dissolved in ethanol and titrated with 0.1 mol / L NaOH solution to finally obtain the titration amount of dicarboxylic acid in the fermentation broth.

[0057] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, all raw materials used are commercially available.

[0058] The yield of the long-chain dicarboxylic acid = amount of acid produced / initial fermentation volume / fermentation cycle, in g / L / h;

[0059] The yield of the long-chain dicarboxylic acid = (amount of acid produced / amount of substrate consumed) × 100%;

[0060] Dilution rate D = [Q (nutrient solution) + Q (substrate)] / V (fermentation broth), where V (fermentation broth) represents the volume of the fermentation broth, Q (nutrient solution) represents the flow acceleration of the nutrient solution, and Q (substrate) represents the flow acceleration of the substrate.

[0061] Example 1

[0062] 1 strain

[0063] A strain of Candida tropicalis, CAT H1614, with accession number CCTCC M2015303.

[0064] 2 Culture media

[0065] Seed culture medium: sucrose content 3.0 (w / v)%, corn steep liquor content 0.6 (w / v)%, yeast extract content 1.0 (w / v)%, KH2PO4 content 1.2 (w / v)%, urea content 0.30 (w / v)%.

[0066] Fermentation medium: glucose content 3.0 (w / v)%, corn steep liquor content 0.6 (w / v)%, yeast extract content 0.3 (w / v)%, potassium nitrate content 1.1 (w / v)%, potassium dihydrogen phosphate content 0.8 (w / v)%, urea content 0.2 (w / v)%, ammonium sulfate content 0.15 (w / v)%, sodium chloride content 0.1 (w / v)%.

[0067] Feeding medium: consists of a substrate and a nutrient solution, wherein the substrate is undecane; the nutrient solution contains 0.2 (w / v)% glucose, 0.15 (w / v)% corn steep liquor, 0.1 (w / v)% yeast extract, 0.012 (w / v)% potassium dihydrogen phosphate, 0.015 (w / v)% urea and 0.015 (w / v)% ammonium sulfate.

[0068] 3. Culture methods

[0069] (i) Shake flask seed culture process: Take the glycerol tube inoculum of Candida tropicalis and inoculate it into a 500mL Erlenmeyer flask containing seed culture medium (the liquid volume is 50mL). Under the conditions of initial pH value of 6.3 and temperature of 29℃, culture it in a shaker at 220rpm for 1.5 days.

[0070] (ii) Seed culture process: Seeds from shake flasks were inoculated into a 10L seed tank containing seed culture medium (6L liquid volume). The inoculum size was controlled at 20% v%, and the temperature was controlled at 29℃, pressure at 0.10MPa, and aeration rate at 0.6vvm. The pH was maintained at 5.7 by adding 25% (w / w) liquid alkali. A certain stirring speed was maintained to ensure that the dissolved oxygen (DO) during seed culture was above 10%. Culture was carried out for 20 hours to achieve the desired bacterial cell concentration (OD). 620 Diluted 30 times to 0.6, this is the mature seed liquid.

[0071] 4. Methods for producing long-chain dicarboxylic acids

[0072] (1) The seed liquid cultured in the seed tank was inoculated into a 30L fermenter containing fermentation medium. The initial fermentation volume was 15L. Based on the initial fermentation volume, the inoculation amount of the seed liquid was 25v%. After inoculation, undecane was added to it. The amount of undecane added was 5v% relative to the initial fermentation volume. Then, the temperature was controlled at 29℃, the pressure at 0.10MPa, and the ventilation volume at 0.5vvm. The pH of the fermentation liquid was adjusted to 5.7 by adding 30 (w / w)% liquid alkali. The stirring speed was kept at 600rpm to control the dissolved oxygen content of the fermentation to be above 10%. After 25h of fermentation, undecane was added in batches to ensure that the content of undecane in the fermentation liquid was above 10v%.

[0073] (2) After 125 h of fermentation, undecane was continuously added to the fermentation broth at a flow rate of 35.2 mL / h, while the above-mentioned nutrient salt solution was continuously added to the fermentation broth at a flow rate of 132 mL / h to control the cell concentration OD of the fermentation. 620 After being diluted 30 times, the concentration is 0.65. By controlling the discharge rate, the volume of the fermentation liquid is maintained at 22L, thereby continuously producing 1,11-undecanoic acid.

[0074] Based on records and calculations, the continuous fermentation period was 300 hours, and the total fermentation period was 425 hours; the yield of 1,11-undecanoic acid was 1.6 g / L / h, the total yield was 93%, and the dilution rate D was 0.0076 h. -1 .

[0075] Comparative Example 1

[0076] Long-chain dicarboxylic acids are produced in the same manner as in Example 1, except that there is no supplemental culture medium; in the method of producing long-chain dicarboxylic acids in 4, in step (1), after fermentation for 25 hours, undecane is added to the fermentation broth in batches so that the content of undecane in the fermentation broth is above 2v%, and there is no step (2).

[0077] That is, after 160 hours of fermentation, the cell activity decreased significantly, and the fermentation was stopped after 165 hours. The yield of 1,11-undecanoic acid was 1.2 g / L / h, and the total yield was 89%.

[0078] As can be seen from Example 1 and Comparative Example 1, compared with the prior art, the present invention, by continuously adding substrate and nutrient-containing solution to the fermentation broth and ensuring that the flow rate of the substrate, the flow rate of the nutrient-containing solution and the volume of the fermentation broth satisfy a specific stoichiometric relationship, can increase the acid production rate from 1.2 g / L / h to 1.6 g / L / h, an increase of 33%; and increase the total yield from 89% to 93%.

[0079] Example 2

[0080] 1 strain

[0081] A strain of Candida tropicalis, CAT H1614, with accession number CCTCC M2015303.

[0082] 2 Culture media

[0083] Seed culture medium: sucrose content 1.0 (w / v)%, corn steep liquor content 0.30 (w / v)%, yeast extract content 0.6 (w / v)%, KH2PO4 content 0.8 (w / v)%, urea content 0.25 (w / v)%.

[0084] Fermentation medium: glucose content 2.0 (w / v)%, corn steep liquor content 0.20 (w / v)%, yeast extract content 0.25 (w / v)%, potassium nitrate content 0.05 (w / v)%, potassium dihydrogen phosphate content 0.07 (w / v)%, urea content 0.15 (w / v)%, ammonium sulfate content 0.20 (w / v)%, sodium chloride content 0.12 (w / v)%.

[0085] Feeding medium: consists of a substrate and a nutrient solution, wherein the substrate is C16 dodecane; the nutrient solution contains 0.3 (w / v)% corn steep liquor, 0.2 (w / v)% yeast extract, 0.008 (w / v)% potassium dihydrogen phosphate, 0.05 (w / v)% urea and 0.05 (w / v)% ammonium sulfate.

[0086] 3. Culture methods

[0087] (i) Shake flask seed culture process: Take the glycerol tube inoculum of Candida tropicalis and inoculate it into a 500mL Erlenmeyer flask containing seed culture medium (60mL of liquid). Culture it in a shaker at 250rpm for 1 day at a temperature of 32℃.

[0088] (ii) Seed culture process: Seeds from shake flasks were inoculated into a 10L seed tank containing seed culture medium (5L liquid volume). The inoculation rate was controlled at 10v%, and the temperature, pressure, and ventilation rate were controlled at 32℃, 0.10MPa, and 0.5vvm. The pH was maintained at 6.2 by adding 40% (w / w) liquid alkali. A certain stirring speed was maintained to ensure that the dissolved oxygen (DO) during seed culture was above 10%. Culture was carried out for 15 hours to achieve the desired bacterial cell concentration (OD). 620 Diluted 30 times to 0.8, this is the mature seed liquid.

[0089] 4. Methods for producing long-chain dicarboxylic acids

[0090] (1) The seed liquid cultured in the seed tank was inoculated into a 30L fermenter containing fermentation medium. The initial fermentation volume was 10L. Based on the initial fermentation volume, the inoculation amount of the seed liquid was 20v%. Before inoculation, C16 hydrocarbons were added to it. The amount of C16 hydrocarbons added was 5v% relative to the volume of the fermentation medium. Then, the temperature was controlled at 32℃, the pressure at 0.05MPa, and the ventilation rate at 0.7vvm. The pH of the fermentation liquid was adjusted to 6.2 by adding 40% (w / w)% liquid alkali. The stirring speed was kept at 600rpm to control the dissolved oxygen content of the fermentation to be above 10%. After 10h of fermentation, C16 hydrocarbons were added to the fermentation liquid in batches to ensure that the content of C16 hydrocarbons in the fermentation liquid was above 5v%.

[0091] (2) After 100 h of fermentation, C6 dodecane was continuously added to the fermentation broth at a flow rate of 64 mL / h, while the above-mentioned nutrient salt solution was continuously added to the fermentation broth at a flow rate of 120 mL / h to control the cell concentration OD of the fermentation. 620 After being diluted 30 times, the concentration is 0.55. By controlling the discharge rate, the volume of the fermentation liquid is maintained at 20L, thereby continuously producing 1,12-dodecanoic acid.

[0092] Based on records and calculations, the continuous fermentation period was 400 hours, and the total fermentation period was 510 hours; the yield of 1,12-dodecanoic acid was 2.3 g / L / h, the total yield was 100%, and the dilution rate D was 0.009 h. -1 .

[0093] Comparative Example 2

[0094] Long-chain dicarboxylic acids were produced using the same method as in Example 2, except that no supplemental culture medium was used; and step (2) was omitted in the method for producing long-chain dicarboxylic acids in Example 4.

[0095] That is, after 170 hours of fermentation, the cell activity gradually decreased, and the fermentation was stopped after 180 hours. The yield of 1,12-dodecanoic acid was calculated to be 1.5 g / L / h, and the total yield was 87%.

[0096] As can be seen from Example 2 and Comparative Example 2, compared with the prior art, the present invention, by continuously adding substrate and nutrient solution to the fermentation broth and ensuring that the flow rate of the substrate, the flow rate of the nutrient solution and the volume of the fermentation broth satisfy a specific stoichiometric relationship, can increase the acid production rate from 1.5 g / L / h to 2.3 g / L / h, an increase of 53%, and the total yield from 87% to 100%.

[0097] Example 3

[0098] 1 strain

[0099] A strain of Candida tropicalis, CAT H1614, with accession number CCTCC M2015303.

[0100] 2 Culture media

[0101] Seed culture medium: sucrose content 3.0 (w / v)%, corn steep liquor content 1.0 (w / v)%, yeast extract content 0.2 (w / v)%, KH2PO4 content 0.9 (w / v)%, urea content 0.5 (w / v)%.

[0102] Fermentation medium: glucose content 5.0 (w / v)%, corn steep liquor content 0.7 (w / v)%, yeast extract content 0.30 (w / v)%, potassium nitrate content 0.55 (w / v)%, potassium dihydrogen phosphate content 0.60 (w / v)%, urea content 0.20 (w / v)%, ammonium sulfate content 0.2 (w / v)%, sodium chloride content 0.15 (w / v)%.

[0103] Supplemental culture medium: comprising substrate and nutrient solution, wherein the substrate is C3tridecane; the nutrient solution contains 0.5 (w / v)% glucose, 0.10 (w / v)% corn steep liquor, 0.008 (w / v)% potassium dihydrogen phosphate, 0.007 (w / v)% urea and 0.15 (w / v)% ammonium sulfate.

[0104] 3. Culture methods

[0105] (i) Shake flask seed culture process: Take the glycerol tube inoculum of Candida tropicalis and inoculate it into a 500mL Erlenmeyer flask containing seed culture medium (the liquid volume is 100mL). Under the conditions of initial pH value of 6 and temperature of 30℃, culture it on a shaker at 200rpm for 1 day.

[0106] (ii) Seed culture process: Seeds from shake flasks were inoculated into a 10L seed tank containing seed culture medium (8L liquid volume). The inoculation rate was controlled at 30% v%, and the temperature was controlled at 30℃, pressure at 0.14MPa, and aeration rate at 0.3vvm. The pH was controlled to 7 by adding 10% (w / w) liquid alkali. A certain stirring speed was maintained to ensure that the dissolved oxygen (DO) during seed culture was above 10%. Culture was carried out for 30 hours to achieve the desired bacterial cell concentration (OD). 620 Diluted 30 times to 1.0, this is the mature seed liquid.

[0107] 4. Methods for producing long-chain dicarboxylic acids

[0108] (1) The seed culture in the seed tank was inoculated into a 30L fermenter containing fermentation medium. The initial fermentation volume was 15L. Based on the initial fermentation volume, the inoculation amount of the seed culture was 10v%. Before inoculation, C13 hydrocarbons were added to it. The amount of C13 hydrocarbons added was 3v% relative to the volume of the fermentation medium. Then, the temperature was controlled at 30℃, the pressure at 0.14Mpa, and the ventilation rate at 0.3vvm. The pH of the fermentation liquid was made up by adding 10% (w / w)% liquid alkali. The stirring speed was kept at 600rpm to control the dissolved oxygen content of the fermentation to be above 10%. After 80h of fermentation, C13 hydrocarbons were added to the fermentation liquid in batches to make the content of C13 hydrocarbons in the fermentation liquid above 2v%.

[0109] (2) After 140 h of fermentation, C13 hydrocarbons were continuously added to the fermentation broth at a flow rate of 39.2 mL / h, while the above-mentioned nutrient salt solution was continuously added to the fermentation broth at a flow rate of 198 mL / h to control the cell concentration OD of the fermentation. 620 After being diluted 30 times, the concentration is 0.65, and the fermentation liquid volume is maintained at 22L by controlling the discharge rate, thereby continuously producing 1,13-tridecanoic acid.

[0110] Recorded or calculated, the continuous fermentation period was 250 hours, and the total fermentation period was 400 hours; the yield of 1,13-tetratedicarboxylic acid was 1.8 g / L / h, the total yield was 90%, and the dilution rate D was 0.01 h. -1 .

[0111] Comparative Example 3

[0112] Long-chain dicarboxylic acids were produced in the same manner as in Example 3, except that no supplemental culture medium was used; and step (2) was omitted in the method for producing long-chain dicarboxylic acids in Example 4.

[0113] That is, after 160 hours of fermentation, the cell activity gradually decreased, and the fermentation was stopped at 175 hours. The yield of 1,13-tetanedicarboxylic acid was calculated to be 1.3 g / L / h, and the total yield was 85%.

[0114] As can be seen from Example 3 and Comparative Example 3, compared with the prior art, the present invention, by continuously adding substrate and nutrient solution to the fermentation broth and ensuring that the flow rate of the substrate, the flow rate of the nutrient solution and the volume of the fermentation broth satisfy a specific stoichiometric relationship, can increase the acid production rate from 1.3 g / L / h to 1.8 g / L / h, an increase of 38%, and the total yield from 85% to 90%.

[0115] Example 4

[0116] 1 strain

[0117] A strain of Candida tropicalis, CAT H1614, with accession number CCTCC M2015303.

[0118] 2 Culture media

[0119] Seed culture medium: sucrose content 1.3 (w / v)%, corn steep liquor content 0.35 (w / v)%, yeast extract content 0.45 (w / v)%, KH2PO4 content 1.1 (w / v)%, urea content 0.5 (w / v)%.

[0120] Fermentation medium: glucose content 4.5 (w / v)%, corn steep liquor content 0.1 (w / v)%, yeast extract content 0.15 (w / v)%, potassium nitrate content 1.2 (w / v)%, potassium dihydrogen phosphate content 0.75 (w / v)%, urea content 0.3 (w / v)%, ammonium sulfate content 0.18 (w / v)%, sodium chloride content 0.09 (w / v)%.

[0121] Feeding medium: consists of a substrate and a nutrient solution, wherein the substrate is hexadecane; the nutrient solution contains 0.15 (w / v)% corn steep liquor, 0.015 (w / v)% potassium dihydrogen phosphate, 0.015 (w / v)% urea and 0.15 (w / v)% ammonium sulfate.

[0122] 3. Culture methods

[0123] (i) Shake flask seed culture process: Take the glycerol tube inoculum of Candida tropicalis and inoculate it into a 500mL Erlenmeyer flask containing seed culture medium (the liquid volume is 50mL). Under the conditions of initial pH value of 6.3 and temperature of 28℃, culture it in a shaker at 240rpm for 1.5 days.

[0124] (ii) Seed culture process: Seeds from shake flasks were inoculated into a 10L seed tank containing seed culture medium (5L liquid volume). The inoculation rate was controlled at 30% v%, and the temperature was controlled at 28℃, pressure at 0.08MPa, and aeration rate at 0.6vvm. The pH was maintained at 7.4 by adding 15% (w / w) liquid alkali. A certain stirring speed was maintained to ensure that the dissolved oxygen (DO) during seed culture was above 10%. Culture was carried out for 28 hours to achieve the desired bacterial cell concentration (OD). 620 After diluting 30 times, the concentration becomes 0.9, which is the mature seed liquid.

[0125] 4. Methods for producing long-chain dicarboxylic acids

[0126] (1) The seed liquid cultured in the seed tank was inoculated into a 30L fermenter containing fermentation medium. The initial fermentation volume was 13L. Based on the initial fermentation volume, the inoculation amount of the seed liquid was 30v%. Before inoculation, hexadecane was added to it. The amount of hexadecane added was 2v% relative to the volume of the fermentation medium. Then, the temperature was controlled at 28℃, the pressure at 0.08MPa, and the ventilation rate at 0.6vvm. The pH of the fermentation liquid was adjusted to 7.4 by adding 15% (w / w)% liquid alkali. The stirring speed was kept at 600rpm to control the dissolved oxygen content of the fermentation to be above 10%. After 60h of fermentation, hexadecane was added to the fermentation liquid in batches to make the content of hexadecane in the fermentation liquid above 3v%.

[0127] (2) After fermentation for 130 h, hexadecane was continuously added to the fermentation broth at a flow rate of 27.2 mL / h, and the above-mentioned nutrient salt solution was continuously added to the fermentation broth at a flow rate of 170 mL / h to control the cell concentration OD of the fermentation. 620 After being diluted 30 times, the concentration is 0.65, and the fermentation liquid volume is maintained at 17L by controlling the discharge rate, thereby continuously producing 1,16-hexadecanoic acid.

[0128] Based on records and calculations, the continuous fermentation period was 320 hours, and the total fermentation period was 450 hours; the yield of 1,16-hexadecanoic acid was 1.85 g / L / h, the total yield was 75%, and the dilution rate D was 0.012 h. -1 .

[0129] Comparative Example 4

[0130] Long-chain dicarboxylic acids were produced using the same method as in Example 4, except that no feeding medium was used; and step (2) was omitted in the method for producing long-chain dicarboxylic acids in Example 4.

[0131] That is, after 150 hours of fermentation, the cell activity gradually decreased, and the fermentation was stopped after 165 hours. The yield of 1,16-hexadecanoic acid was calculated to be 1.3 g / L / h, and the total yield was 68%.

[0132] As can be seen from Example 4 and Comparative Example 4, compared with the prior art, the present invention, by continuously adding substrate and nutrient solution to the fermentation broth and ensuring that the flow rate of the substrate, the flow rate of the nutrient solution and the volume of the fermentation broth satisfy a specific stoichiometric relationship, can increase the acid production rate from 1.3 g / L / h to 1.85 g / L / h, an increase of 42%, and the total yield from 68% to 75%.

[0133] Example 5

[0134] Long-chain dicarboxylic acids were produced using the same method as in Example 2, except that step (2) was changed to: after 140 h of fermentation, dodecane was continuously added to the fermentation broth at a flow rate of 80.5 mL / h, while a nutrient solution was continuously added to the fermentation broth at a flow rate of 690 mL / h, controlling the cell concentration OD of the fermentation. 620 After being diluted 30 times, the concentration is 0.65, and the fermentation liquid volume is maintained at 23L by controlling the discharge rate, thereby continuously producing 1,12-dodecanoic acid.

[0135] Based on records and calculations, the continuous fermentation period was 500 hours, and the total fermentation period was 610 hours; the yield of 1,12-dodecanoic acid was 2.4 g / L / h, the total yield was 100%, and the dilution rate D was 0.033 h. -1 .

[0136] Similarly, by comparing Example 5 and Comparative Example 2, it can be seen that, compared with the prior art, the present invention can increase the acid production rate from 1.5 g / L / h to 2.4 g / L / h, which is an increase of 60%, and the total yield from 87% to 100%.

[0137] Example 6

[0138] Long-chain dicarboxylic acids were produced using the same method as in Example 2, except that step (2) was changed to: after 140 h of fermentation, dodecane was continuously added to the fermentation broth at a flow rate of 85.1 mL / h, while a nutrient solution was continuously added to the fermentation broth at a flow rate of 3450 mL / h, controlling the cell concentration OD of the fermentation. 620 After being diluted 30 times, the concentration was 0.68, and the fermentation liquid volume was maintained at 23L by controlling the discharge rate, thereby continuously producing 1,12-dodecanoic acid.

[0139] Based on records and calculations, the continuous fermentation period was 500 hours, and the total fermentation period was 610 hours; the yield of 1,12-dodecanoic acid was 2.45 g / L / h, the total yield was 99%, and the dilution rate D was 0.15 h. -1 .

[0140] Similarly, by comparing Example 6 and Comparative Example 2, it can be seen that, compared with the prior art, the present invention can increase the acid production rate of 1,12-dodecanoic acid from 1.5 g / L / h to 2.45 g / L / h, which is an increase of 63%, and the total yield is increased from 87% to 99%.

[0141] In Examples 1-6, the flow rate of the substrate, the flow rate of the nutrient-containing solution, the ratio of the flow rate of the nutrient-containing solution to the flow rate of the substrate, the dilution rate, the fermentation cycle, the acid production rate, and the total yield are shown in Table 1.

[0142] Table 1

[0143] Example Substrate flow acceleration (mL / h) Flow rate (mL / h) of nutrient-containing solutions The ratio of the flow velocity of the nutrient-containing solution to the flow velocity of the substrate. <![CDATA[Dilution rate (h -1 )]]> Fermentation cycle (h) Acid production rate (g / L / h) Overall success rate (%) Example 1 32.5 132 3.75：1 0.0076 425 1.6 93 Example 2 64 120 1.88：1 0.009 510 2.3 100 Example 3 39.2 198 5.05：1 0.001 400 1.8 90 Example 4 27.2 170 6.25：1 0.012 450 1.85 75 Example 5 80.5 690 8.57：1 0.033 610 2.4 100 Example 6 85.1 3450 40.54：1 0.15 610 2.45 99

[0144] Table 1 shows that at dilution rates of 0.0076-0.15h... -1 Under high dilution conditions, by controlling the ratio of the flow rate of the nutrient-containing solution to the flow rate of the substrate within the range of (1.88-40.54):1, cell activity can be maintained, thereby achieving an acid production rate of 1.6-2.45 g / L / h, a total yield of 75-100%, and a fermentation cycle extended to 400-610h under high dilution conditions.

[0145] In addition, Examples 2, 5, and 6 further verified the technical effects under different dilution ratios. The results showed that as the dilution ratio increased, the nutrient / substrate ratio needed to be increased accordingly to maintain cell activity and acid production capacity. That is, a higher acid production rate was achieved under a higher nutrient / substrate ratio.

[0146] In summary, this invention achieves high dilution rates and long-cycle fermentation under high nutrient salt supply conditions, thereby achieving a higher acid production rate.

[0147] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for continuous fermentation to produce long-chain dicarboxylic acids, characterized in that, The method includes: after fermentation for 80-150 hours, continuously adding substrate and a solution containing nutrients to the fermentation broth, wherein the ratio of the flow rate of the solution containing nutrients to the flow rate of the substrate is (0.83-300):1; The dilution rate D of the fermentation system is 0.007-0.3h. -1 .

2. The method according to claim 1, characterized in that, The ratio of the flow rate of the nutrient-containing solution to the flow rate of the substrate is (1.5-150):1; Preferably, the ratio of the flow acceleration of the nutrient-containing solution to the flow acceleration of the substrate is (1.88-40.54):

1.

3. The method according to claim 1 or 2, characterized in that, The dilution rate D of the fermentation system is 0.007-0.15h. -1 Preferably, it is 0.0076-0.15h. -1 .

4. The method according to any one of claims 1-3, characterized in that, The flow rate of the substrate, the flow rate of the nutrient-containing solution, and the volume of the fermentation broth satisfy the following relationship: 0.005h -1 *V (fermentation broth) ≤ Q (solution containing nutrients) ≤ 0.3h -1 *V (fermentation broth); 0.001h -1 *V (fermentation broth) ≤ Q (substrate) ≤ 0.006h -1 *V (fermentation broth); Wherein, V (fermentation broth) represents the volume of the fermentation broth, Q (nutrient-containing solution) represents the flow acceleration of the nutrient-containing solution, and Q (substrate) represents the flow acceleration of the substrate.

5. The method according to claim 4, characterized in that, The flow rate of the substrate and the volume of the fermentation broth satisfy the following condition: 0.001 h⁻¹ -1 *V (fermentation broth) ≤ Q (substrate) ≤ 0.004h -1 *V (fermentation broth); and / or The flow rate of the nutrient-containing solution is related to the volume of the fermentation broth by 0.006 h. -1 *V (fermentation broth) ≤ Q (solution containing nutrients) ≤ 0.15h -1 *V (fermentation broth).

6. The method according to any one of claims 1-5, characterized in that, The nutrient-containing solution contains 0-2.5 (w / v)% glucose, 0.05-2.0 (w / v)% corn steep liquor, 0-1.0 (w / v)% yeast extract, 0.005-0.5 (w / v)% potassium dihydrogen phosphate, 0.005-0.05 (w / v)% urea, and 0.01-0.15 (w / v)% ammonium sulfate; and / or, The substrate is selected from C9-C. 18 At least one of n-alkanes, straight-chain saturated fatty acids, straight-chain saturated fatty acid esters, and straight-chain saturated fatty acid salts, preferably C14. 10 -C 16 n-Alkanes; and / or The long-chain dicarboxylic acid is expressed as HOOC(CH2). n COOH, wherein n≥7, is preferably at least one of azelaic acid, sebacic acid, 1,11-undecanoic acid, 1,12-dodecanoic acid, 1,13-tetanedicarboxylic acid, 1,14-tetradecanoic acid, 1,15-pentadecanoic acid, 1,16-hexadecanoic acid, 1,17-heptadecanoic acid, and 1,18-octadecanoic acid.

7. A method for producing a long-chain dicarboxylic acid, characterized in that, The method includes the method for producing long-chain dicarboxylic acids by continuous fermentation as described in any one of claims 1-6.

8. The method according to claim 7, characterized in that, Before using continuous fermentation to produce long-chain dicarboxylic acids, the method also includes: inoculating fermentation strains into fermentation medium for fermentation, and after fermentation for 10-100 hours, adding substrate to the fermentation broth to ensure that the substrate content in the fermentation broth is above 1%; The fermentation is carried out in a fermenter, and the initial volume of the fermentation is 30-70% of the total volume of the fermenter; and / or Based on the initial volume of the fermentation, the inoculum size of the fermentation strain is 10-30 v.

9. The method according to claim 8, characterized in that, The method further includes: adding a substrate to the fermentation medium before inoculation, wherein the amount of substrate added is 0-10 v%, preferably 1-5 v%, relative to the volume of the fermentation medium; or After inoculation, the substrate is added to the starting volume of fermentation, and the amount of substrate added is 0-10 v%, preferably 1-5 v%, relative to the starting volume of fermentation.

10. The method according to claim 8 or 9, characterized in that, The fermentation conditions include: a temperature of 28-32℃, a pressure of 0.05-0.14 MPa, a pH of 5.5-7.5, and an aeration rate of 0.3-0.7 vvm; and / or The dissolved oxygen content during fermentation is above 10%; and / or The fermentation cell concentration OD 620 The value is 9-24, and after dilution 30 times, it becomes 0.3-0.8.