A method for fermentative production of medium and long chain diacids

Abstract

The application belongs to the technical field of fermentation, and particularly relates to a method for producing medium and long chain dibasic acid by fermentation, which comprises the following steps: fermenting Candida, adding a fermentation substrate to perform fermentation, continuously adding carbon source and nitrogen source into the fermentation system when the fermentation lasts for 30-60 hours, and controlling the C / N ratio of the added carbon source and nitrogen source to be (20-30):1; the fermentation substrate is selected from any one or a combination of several of fatty acids with 9-11 carbon atoms, fatty acid esters, fatty acid salts or alkanes. By regulating the carbon-nitrogen ratio in the fermentation process, the application can not only promote the growth of the bacteria and ensure the stable activity of the bacteria, but also make the substrate be as much as possible and efficiently converted into medium and long chain dibasic acid, significantly improve the acid production of the target chain length dibasic acid, and has the potential for industrial scale production.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to a method for producing long-chain dicarboxylic acids through fermentation. Background Technology

[0002] Dicarboxylic acids are an important class of chemical raw materials. They usually refer to organic compounds with carboxyl groups at both ends of a straight carbon chain. Dicarboxylic acids with more than 10 carbon atoms are conventionally called long-chain dicarboxylic acids. Their structural formula is HOOC(CH2)nCOOH, where n is an integer greater than or equal to 8.

[0003] Azelaic acid is a very useful compound in industry, used as a plasticizer, in the synthesis of lubricants and polymers such as Nylon 69 and Nylon 9. It is also used in the production of pesticides, water-soluble coatings, and dielectric and heat transfer aids. Azelaic acid also has antibacterial properties and can be used as a food preservative. However, it does not exist naturally and is mainly synthesized through chemical methods. Currently, the industrial production and application of azelaic acid primarily uses oleic acid as a raw material, with ozone as an oxidant. The mixture is reacted countercurrently with oxygen (containing 2% ozone) to generate oleic acid ozonides. Then, in the presence of manganese salts, oxygen breaks the bonds, producing a mixture of nonanoic acid aldehyde and nonanoic acid, which is then oxidized back to azelaic acid and nonanoic acid. The resulting oxidized mixture is distilled to obtain nonanoic acid, and the remaining residue is extracted with hot water and then evaporated or crystallized to obtain the final product, azelaic acid. However, because the ozone oxidation of oleic acid uses catalysts, it is not only costly but also involves complex post-reaction processing.

[0004] Sebacic acid has a wide range of applications. It can be used as a raw material for plastics and cold-resistant rubber plasticizers, and can also be used to produce polyamides, polyurethanes, alkyd resins, synthetic lubricants, lubricant additives, fragrances, coatings, and cosmetics. It can also be used as a raw material for the production of Nylon 1010, Nylon 910, Nylon 810, and Nylon 610, as well as for the production of diethylhexyl ester, a high-temperature resistant lubricant. Currently, the main industrial production of sebacic acid uses the castor oil pyrolysis method: castor oil is heated and hydrolyzed under alkaline conditions to produce sodium ricinoleate soap, which is then acidified with sulfuric acid to produce ricinoleic acid. In the presence of the diluent cresol, alkali is added and heated to 260-280℃ for pyrolysis, producing disodium sebacic acid, 2-octanol, and hydrogen. The pyrolysis product is diluted with water, heated, and neutralized with acid to convert the disodium salt into a monosodium salt. Activated carbon is then used for decolorization, and the neutralized solution is boiled and acid is added to cause the monosodium sebacic acid to crystallize out as sebacic acid. After separation and drying, the finished product is obtained. Its production process is complex, and the alcohols and hydrogen produced are flammable and explosive. The diluents used in the production are toxic and pollute the environment, which seriously restricts the development of the sebacic acid industry.

[0005] Undecanoic acid (UDCA), used in advanced metalworking fluids, fragrance intermediates, high-grade lubricants, lubricant corrosion inhibitors, and high-end automotive coatings, is a key raw material for long-chain dicarboxylic acid diesters in polyamide engineering plastics, cutting fluids, hot melt adhesives, and hindered amine light stabilizers. UDCA can be produced through chemical synthesis. Traditional chemical methods for producing long-chain dicarboxylic acids require nine complex reaction steps, high temperature, high pressure, and catalysts; production necessitates fire prevention, explosion prevention, and poison prevention equipment; and suffers from low yield, high cost, and severe environmental pollution. To date, there is no economically feasible chemical synthesis method available domestically or internationally.

[0006] Bio-fermentation can use petroleum byproducts such as wax oil, alkanes, fatty acids, or their derivatives as raw materials. Under low temperature and low pressure, it utilizes the unique oxidizing ability of microorganisms to produce dicarboxylic acids. Therefore, it has advantages such as a wide range of raw material sources, simple production processes, mild production conditions, and environmental friendliness. Thus, there is an urgent need to develop a microbial fermentation method to replace chemical synthesis for the production of environmentally friendly medium- and long-chain dicarboxylic acids. Summary of the Invention

[0007] To overcome the shortcomings of existing chemical synthesis methods for producing medium- and long-chain dicarboxylic acids, such as high cost, complex production process, low production efficiency, and environmental pollution, this invention provides a method for producing medium- and long-chain dicarboxylic acids through fermentation. By controlling the carbon-nitrogen ratio during fermentation, it can not only promote cell growth and ensure stable cell activity, but also enable the substrate to be converted into medium- and long-chain dicarboxylic acids as efficiently as possible, significantly increasing the yield of dicarboxylic acids of the target chain length, and has the potential for industrial-scale production.

[0008] The first aspect of the present invention provides a method for fermenting long-chain dicarboxylic acids, comprising: fermenting and culturing Candida albicans, adding fermentation substrate for fermentation, and continuously adding carbon source and nitrogen source to the fermentation system after 30-60 hours of fermentation, controlling the added C / N ratio to be (20-30):1;

[0009] The fermentation substrate is selected from any one or a combination of several of fatty acids, fatty acid esters, fatty acid salts or alkanes with 9-11 carbon atoms.

[0010] In some implementations, carbon and nitrogen sources are continuously added to the fermentation system starting at 40-60 hours of fermentation, and the C / N ratio of the added sources is controlled to be (25-30):1, such as 23:1, 25:1, 26:1, 28:1, etc.

[0011] In some embodiments, the flow rate of C in the added carbon source is controlled to be 0.01-0.5 g / L / h, preferably 0.01-0.3 g / L / h, such as 0.01 g / L / h, 0.03 g / L / h, 0.05 g / L / h, 0.1 g / L / h, 0.2 g / L / h, 0.25 g / L / h, 0.3 g / L / h, etc. This invention achieves the above-mentioned flow rate of C in the carbon source by adjusting the flow rate of the carbon source.

[0012] In some embodiments, the flow rate of N in the added nitrogen source is controlled to be 0.001-0.02 g / L / h, preferably 0.001-0.01 g / L / h, for example 0.001 g / L / h, 0.003 g / L / h, 0.005 g / L / h, 0.006 g / L / h, 0.008 g / L / h, 0.01 g / L / h, etc. This invention achieves the above-mentioned flow rate of N by adjusting the flow rate of the nitrogen source.

[0013] In some embodiments, the carbon source is selected from any one or more of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, more preferably glucose and / or sucrose. The present invention adds the carbon source by flowing over an aqueous sugar solution, the concentration of which is 10 wt%-70 wt%, preferably 30 wt%-50 wt%.

[0014] In some embodiments, the nitrogen source includes an inorganic nitrogen source and / or an organic nitrogen source.

[0015] In some embodiments, the inorganic nitrogen source is selected from any one or a combination of several of urea, potassium nitrate, ammonium sulfate, and ammonia water. The present invention adds the inorganic nitrogen source by flowing in an inorganic nitrogen source solution with a concentration of 1 wt%-5 wt%.

[0016] In some embodiments, the organic nitrogen source is selected from any one or a combination of several of corn steep liquor, yeast extract, and yeast powder. The present invention adds the organic nitrogen source by adding a solution of the organic nitrogen source in a flow-through manner, wherein the concentration of the organic nitrogen source solution is 1 wt%-5 wt%.

[0017] In some specific embodiments, the nitrogen source may be selected from any inorganic or organic nitrogen source; it may also be selected from any one or more of the following combinations: a combination of potassium nitrate and ammonium sulfate; a combination of urea and ammonium sulfate; a combination of urea and potassium nitrate; a combination of corn steep liquor and yeast extract; a combination of corn steep liquor and yeast powder; a combination of corn steep liquor and ammonium sulfate; a combination of corn steep liquor and potassium nitrate; a combination of corn steep liquor and urea; a combination of yeast extract and potassium nitrate; a combination of yeast extract and ammonium sulfate; a combination of yeast extract and urea; a combination of yeast powder and ammonium sulfate; a combination of yeast powder and potassium nitrate; a combination of yeast powder and urea.

[0018] The inventors of this invention discovered in their research that by continuously adding carbon and nitrogen sources to the fermentation system and controlling the added C / N ratio within a certain range, stable cell activity can be ensured, enabling the substrate to be converted into the target long-chain dicarboxylic acid as efficiently as possible, and significantly increasing the acid production of the target long-chain dicarboxylic acid.

[0019] In some implementations, the feeding of carbon and nitrogen sources is stopped after 120-150 hours of fermentation.

[0020] In one specific embodiment, the fermentation substrate is selected from any one or a combination of several of nonanoic acid, nonanoic acid ester, fatty acid salts with 9 carbon atoms, and nonane, preferably nonane.

[0021] In this invention, the nonanoic acid ester is selected from at least one of methyl nonanoate, ethyl nonanoate, and butyl nonanoate. The fatty acid salt having 9 carbon atoms is selected from at least one of sodium, potassium, ammonium, and calcium salts of fatty acids having 9 carbon atoms. The nonane is preferably n-nonane, which can be a petroleum-based alkane or coal-derived alkane derived from petroleum or coal, or a bio-based alkane obtained through the processing of vegetable oils.

[0022] In one specific embodiment, the fermentation substrate is selected from any one or a combination of several of decacarbonylic acid, decacarbonyl ester, fatty acid salts with 10 carbon atoms, and decane, preferably decane.

[0023] In this invention, the decacarbonyl ester may be selected from at least one of methyl decacarbonate, ethyl decacarbonate, and butyl decacarbonate. The fatty acid salt having 10 carbon atoms may be selected from at least one of sodium, potassium, ammonium, and calcium salts of fatty acids having 10 carbon atoms. The decane is preferably n-decane, which may be a petroleum-based alkane or coal-derived alkane derived from petroleum or coal, or a bio-based alkane obtained through the processing of vegetable oils.

[0024] In one specific embodiment, the fermentation substrate is selected from any one or a combination of several of undecanoic acid, undecanoate, fatty acid salts with 11 carbon atoms, and undecane, preferably undecane.

[0025] In this invention, the undecanoate ester may be selected from at least one of methyl undecanoate, ethyl undecanoate, and butyl undecanoate. The fatty acid salt having 11 carbon atoms may be selected from at least one of sodium, potassium, ammonium, and calcium salts of fatty acids having 11 carbon atoms. The undecane is preferably n-undecane, which may be a petroleum-based alkane or coal-derived alkane derived from petroleum or coal, or a bio-based alkane obtained through the processing of vegetable oils.

[0026] In some embodiments, the C / N ratio of the fermentation medium is controlled to be (10-15):1, for example, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1. In this invention, Candida albicans is used to ferment the fermentation medium to convert the fermentation substrate into medium- and long-chain dicarboxylic acids. By controlling the C / N ratio of the fermentation medium within the above range, it is beneficial to the rapid growth of the cells.

[0027] In some embodiments, the fermentation medium contains at least a carbon source, a nitrogen source, inorganic salts, and / or growth factors.

[0028] In some embodiments, the carbon source is selected from any one or a combination of several of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, more preferably from any one or a combination of glucose, lactose, and sucrose.

[0029] In some embodiments, the nitrogen source is selected from any one or more combinations of yeast extract, peptone, yeast powder, corn steep liquor, urea, ammonium salts, and nitrates.

[0030] In some embodiments, the inorganic salt is selected from one or more combinations of sulfates, hydrochlorides, and phosphates, more preferably from one or more combinations of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, sodium chloride, and calcium chloride.

[0031] In some embodiments, the growth factor is selected from at least one of vitamin B1, vitamin B2, vitamin B5, vitamin C, and biotin.

[0032] In some embodiments, the fermentation medium further contains an antifoaming agent, which includes polyether antifoaming agents, silicone antifoaming agents, silicone-ether composite antifoaming agents, mineral oil antifoaming agents, or nonionic surfactants. Examples include commercially available polyether antifoaming agents, Dow DF-103 antifoaming agent, Sigma Aldrich antifoaming agent 204, polyethylene glycol monostearate, or dimethicone.

[0033] In some specific embodiments, the fermentation medium comprises 1%–6% glucose, 0.1%–1.5% corn steep liquor (containing 2–3 wt% nitrogen), 0.1%–0.9% yeast extract (containing 8–10 wt% nitrogen), 0.2%–1.0% potassium dihydrogen phosphate, 0.1%–0.5% magnesium sulfate, 0.001%–0.005% growth factors, and 0.01%–0.10% (w / v) defoamer.

[0034] In some specific embodiments, the fermentation medium comprises 1%–6% glucose, 0.1%–0.9% yeast extract (containing 8–10 wt% nitrogen), 0.1%–0.5% ammonium sulfate, 0.2%–1.0% potassium dihydrogen phosphate, 0.05%–0.40% sodium chloride, 0.001%–0.005% growth factors, and 0.01%–0.10% (w / v) antifoaming agent.

[0035] In some specific embodiments, the fermentation medium comprises 1%–6% glucose, 0.1%–1.0% corn steep liquor (containing 2–3 wt% nitrogen), 0.1%–0.9% yeast extract (containing 8–10 wt% nitrogen), 0.1%–0.8% potassium nitrate, 0.2%–1.0% potassium dihydrogen phosphate, 0.1%–0.5% ammonium sulfate, 0.05%–0.40% sodium chloride, and 0.01%–0.10% (w / v) defoamer.

[0036] In some specific embodiments, the fermentation medium comprises 1%–6% sucrose, 0.1%–0.8% potassium nitrate, 0.2%–1.0% potassium dihydrogen phosphate, 0.1%–0.5% ammonium sulfate, 0.02%–0.30% urea, 0.05%–0.40% sodium chloride, and 0.01%–0.10% (w / v) defoamer.

[0037] In some specific embodiments, the fermentation medium comprises 1%–6% glucose, 0.1%–0.5% ammonium sulfate, 0.2%–1.0% potassium dihydrogen phosphate, 0.05%–0.40% sodium chloride, 0.001%–0.005% growth factor, and 0.01%–0.10% (w / v) defoamer.

[0038] In some specific embodiments, the fermentation medium comprises 1%–6% glucose, 0.1%–0.8% potassium nitrate, 0.2%–1.0% potassium dihydrogen phosphate, 0.02%–0.30% urea, 0.05%–0.40% sodium chloride, and 0.01%–0.10% (w / v) defoamer.

[0039] In some specific embodiments, the fermentation medium comprises 1%–6% glucose, 0.1%–1.5% corn steep liquor (containing 2–3 wt% nitrogen), 0.1%–0.9% yeast extract (containing 10–12 wt% nitrogen), 0.2%–1.0% potassium dihydrogen phosphate, 0.1%–0.5% magnesium sulfate, 0.05%–0.40% sodium chloride, and 0.01%–0.10% (w / v) defoamer.

[0040] In some specific embodiments, the fermentation medium comprises 1%–6% glucose, 0.1%–0.9% yeast extract (containing 10–12 wt% nitrogen), 0.1%–0.8% potassium nitrate, 0.2%–1.0% potassium dihydrogen phosphate, 0.1%–0.5% magnesium sulfate, 0.05%–0.40% sodium chloride, 0.001%–0.005% growth factors, and 0.01%–0.10% (w / v) defoamer.

[0041] In some preferred embodiments, *Candida* is cultured by fermentation, and fermentation substrate is added for fermentation. The C / N ratio of the fermentation medium is controlled at (10-15):1. After 30-60 hours of fermentation, carbon and nitrogen sources are continuously fed into the fermentation system, with the fed-batch C / N ratio controlled at (20-30):1. This invention, by optimizing the fermentation medium and the fed-batch C / N ratio, promotes the utilization rate of the fermentation substrate by *Candida*, enabling more substrate to be fermented into dicarboxylic acids of the target chain length, and significantly increasing the production of dicarboxylic acids.

[0042] In some embodiments, *Candida* is first cultured in a seed culture medium, and then inoculated into a fermentation culture medium for further culture and fermentation. The seed culture medium contains the carbon source, the nitrogen source, and the inorganic salts.

[0043] In some specific embodiments, the seed culture medium comprises 1%–3.5% sucrose, 0.1%–1.0% corn steep liquor, 0.1%–0.6% yeast extract, 0.2%–1.2% potassium dihydrogen phosphate, 0.1%–0.5% urea, and 0.01%–0.10% (w / v) defoamer.

[0044] In some embodiments, when the optical density value OD of the strain 620 When diluted 30 times to reach 0.5-1.0, add the fermentation substrate to start fermentation.

[0045] In some embodiments, the fermentation substrate is added in a single addition, batch addition, or continuous feeding manner. Preferably, during fermentation culture, the concentration of the fermentation substrate is controlled to not exceed 10% (v / v), and more preferably not exceed 5% (v / v).

[0046] In some embodiments, the total amount of fermentation substrate added is 20-50% (v / v) relative to the volume of the post-inoculation fermentation medium. In this invention, the volume of the post-inoculation fermentation medium is the sum of the volume of the seed culture and the volume of the initial fermentation medium.

[0047] In some embodiments, during fermentation culture, the inoculum size of Candida albicans is 5-50% (v / v) of the fermentation medium, that is, the volume ratio between the seed culture and the fermentation medium is (5-50):100.

[0048] In some embodiments, the Candida species includes at least one of Candida viswanathii, Candida albicans, Candida tropicalis, Candida sake, or Yarrowia lipolytica.

[0049] In some specific implementation schemes, the Candida species is Candida viswanathii CATH2402, with accession number CCTCC NO: M 20241167, which was deposited on June 6, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), and classified as Candida viswanathii.

[0050] In some implementations, the fermentation temperature is controlled at 27-30°C, for example, 27°C, 28°C, 29°C or 30°C.

[0051] In some implementations, the fermentation airflow is controlled to be 0.1-0.8 vvm, for example, 0.1 vvm, 0.2 vvm, 0.3 vvm, 0.4 vvm, 0.5 vvm, 0.6 vvm, 0.7 vvm or 0.8 vvm.

[0052] In some implementations, the fermentation pressure is controlled at 0.03-0.15 MPa during the fermentation process, for example, 0.03 MPa, 0.05 MPa, 0.08 MPa, 0.11 MPa, 0.12 MPa, or 0.15 MPa.

[0053] In some embodiments, during fermentation, the pH value of the fermentation is controlled to be 5.0-8.0, more specifically 5.0-7.0, for example 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.

[0054] In some embodiments, during fermentation, the dissolved oxygen is controlled to be above 10% (v / v), further to 10%-70% (v / v), and even further to 10%-40% (v / v), for example 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% (v / v).

[0055] In some implementations, after 120-150 hours of fermentation, the addition of fermentation substrate is stopped, and fermentation continues using the remaining substrate in the fermentation system until the fermentation substrate is completely consumed or no longer produces medium- or long-chain dicarboxylic acids. The total fermentation time is calculated.

[0056] In some implementations, the total fermentation time for producing DC9 is 130-150 hours.

[0057] In some implementations, the total fermentation time for producing DC10 and DC11 is 150-170 hours.

[0058] In some implementations, the fermentation broth produced by fermentation can be used directly as a product, or the medium- and long-chain dicarboxylic acids in the fermentation broth can be extracted as the final product.

[0059] A second aspect of the present invention provides a method for producing medium- and long-chain dicarboxylic acids, the method comprising: preparing a fermentation broth according to the above method, and extracting medium- and long-chain dicarboxylic acids from the obtained fermentation broth.

[0060] In some embodiments, the extraction includes: adjusting the pH of the fermentation broth to 2-5, performing solid-liquid separation, and obtaining a medium- to long-chain dicarboxylic acid. Specifically, dilute sulfuric acid or dilute hydrochloric acid can be used for pH adjustment.

[0061] In some embodiments, the solid-liquid separation can be achieved by centrifugation or filtration to separate the solid and liquid.

[0062] The technical solution of this invention has the following technical effects:

[0063] The method for producing medium- and long-chain dicarboxylic acids provided by this invention belongs to the bio-fermentation method, which solves the problems of high cost, complex production process, low production efficiency, and environmental pollution associated with traditional chemical synthesis methods for producing medium- and long-chain dicarboxylic acids. By controlling the C / N ratio during fermentation, the utilization rate of the fermentation substrate by Candida albicans is promoted, thereby significantly increasing the yield of the target long-chain dicarboxylic acid. Compared with existing production processes, this method not only has significant cost advantages but also effectively reduces the pressure on resources and the environment, aligning with the concept of sustainable material sourcing and possessing very obvious industrialization value advantages. Detailed Implementation

[0064] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are given for illustrative purposes only to make the features and advantages of the present invention clearer, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent; the scope of the present invention is not limited to the embodiments listed herein. Unless otherwise specified, the raw materials and ingredients used in the following examples are commercially available, and the methods and conditions used are known methods and conventional conditions in the art. Raw materials requiring sterilization are sterilized at 121°C for 20 minutes. Unless otherwise specified, the percentages used to characterize the component content in the culture medium of the present invention refer to general conventions in the fermentation field; percentages represent mass-volume ratios (w / v), i.e., % represents g / 100mL.

[0065] The strain used in the following examples and comparative examples is Candida viswanathii CATH2402, with accession number CCTCC NO: M 20241167, which was deposited on June 6, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), and classified as Candidaviswanathii.

[0066] Seed culture medium (w / v): 2.2% sucrose, 0.5% yeast extract, 0.9% corn steep liquor, 0.8% potassium dihydrogen phosphate, 0.2% urea, and 0.03% defoamer (polyether).

[0067] In the following examples and comparative examples, gas chromatography was used to determine the content of dicarboxylic acids in the fermentation broth.

[0068] Examples 1-6: Fermentation production of DC9 in fermenters

[0069] (1) Activation culture: Adjust the pH of 100 mL of malt extract (10 Baume degrees) to about 5.5, then inoculate it with glycerol seed tubes of Candida virescens (strain CATH2402), and activate the culture on a rotary shaker at 29℃ and 220 rpm. The cell OD 620 When the value reaches 0.75 (30-fold dilution), the activation culture ends and the shake flask seeds are obtained.

[0070] (2) Seed culture: The shake flask seeds obtained in step (1) were inoculated into a seed tank containing seed culture medium at an inoculation rate of 1.8% (v / v). The seed culture was carried out at a temperature of 30°C, an air flow rate of 0.3 vvm, a pressure of 0.12 MPa, and a dissolved oxygen content of 15% until the OD of the seed culture was 30 times diluted. 620 The culture was terminated when the value reached 0.8.

[0071] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into a fermenter containing fermentation medium at an inoculation rate of 20% (v / v). Fermentation culture was carried out under the conditions of 28℃, pH 5.6, air flow rate of 0.4 vvm, pressure of 0.06 MPa, and dissolved oxygen of 20%. When the OD of the strain... 620 When the value reaches 0.8 (30-fold dilution), the substrate n-nonane is added to the fermenter in batches for fermentation. The substrate concentration in the fermentation broth is controlled to not exceed 5.0% (v / v). After 125 hours of fermentation, the addition of substrate is stopped, and the remaining substrate in the fermentation system is used for fermentation until the substrate is completely consumed or no more azelaic acid is produced.

[0072] At 40 hours of fermentation, a carbon source with a concentration of 50 wt% and a nitrogen source with a concentration of 2 wt% were continuously added to the fermentation system. After 120 hours of fermentation, the addition of carbon and nitrogen sources was stopped.

[0073] The components of the fermentation medium in Examples 1-6, the C / N ratio of the fermentation medium, the types of continuously fed carbon and nitrogen sources, the flow acceleration rate of C in the fed carbon source, the flow acceleration rate of N in the fed nitrogen source, the C / N ratio of the fed carbon source, the total amount of substrate added, the amount of acid produced, and the total fermentation time are shown in Table 1 below. The amount of acid produced represents the concentration of azelaic acid detected in the fermentation broth.

[0074] Examples 7-9: Fermentation production of DC10 in fermenters

[0075] (1) Activation culture: Adjust the pH of 100 mL of malt extract (10 Baume degrees) to about 5.5, then inoculate it with glycerol seed tubes of Candida virescens (strain CATH2402), and activate the culture on a rotary shaker at 29℃ and 220 rpm. The cell OD 620 When the value reaches 0.75 (30-fold dilution), the activation culture ends and the shake flask seeds are obtained.

[0076] (2) Seed culture: The shake flask seeds obtained in step (1) were inoculated into a seed tank containing seed culture medium at an inoculation rate of 1.8% (v / v). The seed culture was carried out at a temperature of 30°C, an air flow rate of 0.3 vvm, a pressure of 0.12 MPa, and a dissolved oxygen content of 15% until the OD of the seed culture was 30 times diluted. 620 The culture was terminated when the value reached 0.8.

[0077] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into a fermenter containing fermentation medium at an inoculation rate of 15% (v / v). Fermentation culture was carried out under the conditions of 28℃, pH 6.0, air flow rate of 0.4 vvm, pressure of 0.06 MPa, and dissolved oxygen of 20%. When the OD of the strain... 620When the value reaches 0.8 (30-fold dilution), the substrate n-decane is added to the fermenter in batches for fermentation. The substrate concentration in the fermentation broth is controlled to not exceed 5.0% (v / v). After 150 hours of fermentation, the addition of substrate is stopped, and the remaining substrate in the fermentation system is used for fermentation until the substrate is completely consumed or sebacic acid is no longer produced.

[0078] At 30 hours of fermentation, a carbon source with a concentration of 50 wt% and a nitrogen source with a concentration of 2 wt% were continuously added to the fermentation system. After 145 hours of fermentation, the addition of carbon and nitrogen sources was stopped.

[0079] The components of the fermentation medium in Examples 7-9, the C / N ratio of the fermentation medium, the types of continuously fed carbon and nitrogen sources, the flow acceleration rate of C in the fed carbon source, the flow acceleration rate of N in the fed nitrogen source, the C / N ratio of the fed carbon source, the total amount of substrate added, the amount of acid produced, and the total fermentation time are shown in Table 1 below. The amount of acid produced represents the concentration of sebacic acid detected in the fermentation broth.

[0080] Examples 10-11: Fermentation production of DC11 in fermenters

[0081] (1) Activation culture: Adjust the pH of 100 mL of malt extract (10 Baume degrees) to about 5.5, then inoculate it with glycerol seed tubes of Candida virescens (strain CATH2402), and activate the culture on a rotary shaker at 29℃ and 220 rpm. The cell OD 620 When the value reaches 0.75 (30-fold dilution), the activation culture ends and the shake flask seeds are obtained.

[0082] (2) Seed culture: The shake flask seeds obtained in step (1) were inoculated into a seed tank containing seed culture medium at an inoculation rate of 1.8% (v / v). The seed culture was carried out at a temperature of 30°C, an air flow rate of 0.3 vvm, a pressure of 0.12 MPa, and a dissolved oxygen content of 15% until the OD of the seed culture was 30 times diluted. 620 The culture was terminated when the value reached 0.8.

[0083] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into a fermenter containing fermentation medium at an inoculation rate of 15% (v / v). Fermentation culture was carried out under the conditions of 28℃, pH 6.3, air flow rate of 0.5vvm, pressure of 0.08MPa, and dissolved oxygen of 20%. When the OD of the strain... 620 When the value reaches 0.8 (30-fold dilution), the substrate n-undecane is added to the fermenter in batches for fermentation. The substrate concentration in the fermentation broth is controlled to not exceed 5.0% (v / v). After 155 hours of fermentation, the addition of substrate is stopped, and the remaining substrate in the fermentation system is used for fermentation until the substrate is completely consumed or no more undecane dicarboxylic acid is produced.

[0084] At 30 hours of fermentation, a carbon source with a concentration of 50 wt% and a nitrogen source with a concentration of 2 wt% were continuously added to the fermentation system. After 150 hours of fermentation, the addition of carbon and nitrogen sources was stopped.

[0085] The components of the fermentation medium in Examples 10-11, the C / N ratio of the fermentation medium, the types of continuously fed carbon and nitrogen sources, the flow rate of C in the fed carbon source, the flow rate of N in the fed nitrogen source, the C / N ratio of the fed carbon source, the total amount of substrate added, the amount of acid produced, and the total fermentation time are shown in Table 1 below. The amount of acid produced represents the concentration of sebacic acid detected in the fermentation broth.

[0086] Comparative Example 1

[0087] The method for fermenting DC9 is the same as in Example 1, except that the feed rate of C and N in the fed carbon and nitrogen sources is adjusted to achieve a C / N ratio of 35:1. The results are shown in Table 1 below.

[0088] Comparative Example 2

[0089] The method for fermenting DC9 is the same as in Example 1, except that the feed rate of C and N in the fed carbon and nitrogen sources is adjusted to achieve a C / N ratio of 16:1. The results are shown in Table 1 below.

[0090] Comparative Example 3

[0091] The method for producing DC9 by fermentation is the same as in Example 1, except that the C / N ratio of the fermentation medium is controlled at 7:1. The results are shown in Table 1 below.

[0092] Comparative Example 4

[0093] The method for producing DC9 by fermentation is the same as in Example 1, except that no nitrogen source is added. The results are shown in Table 1 below.

[0094] Comparative Example 5

[0095] The method for producing DC10 by fermentation is the same as in Example 7, except that the flow rates of C and N in the fed-batch carbon and nitrogen sources are adjusted to achieve a C / N ratio of 15:1. The results are shown in Table 1 below.

[0096] Comparative Example 6

[0097] The method for producing DC11 by fermentation is the same as in Example 10, except that the flow rates of C and N in the fed-batch carbon and nitrogen sources are adjusted so that the C / N ratio is 15:1. The results are shown in Table 1 below.

[0098] Table 1

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] As shown in Table 1, under the same fermentation conditions, compared with Comparative Examples 1 and 2, this invention significantly increased the yield of azelaic acid by continuously adding carbon and nitrogen sources to the fermentation system and controlling the C / N ratio within the range of (20-30):1. Similarly, compared with Comparative Example 5, this invention significantly increased the yield of sebacic acid by controlling the C / N ratio of the added carbon and nitrogen sources within a specific range in Example 7. Similarly, compared with Comparative Example 6, this invention significantly increased the yield of undecanoic acid by controlling the C / N ratio of the added carbon and nitrogen sources within a specific range in Example 10.

[0105] As can be seen from Table 1, under the same fermentation conditions, compared with Example 1, Comparative Example 3, which used a fermentation medium with a C / N ratio of 7:1, showed a significant decrease in the production of azelaic acid and a significant extension in the total fermentation time.

[0106] As can be seen from Table 1, under the same fermentation conditions, compared with Comparative Example 4, the present invention significantly improved the production of azelaic acid by continuously adding carbon and nitrogen sources to the fermentation system and controlling the added C / N ratio within a specific range.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 fermenting long-chain dicarboxylic acids, characterized in that, It includes: fermenting and culturing Candida albicans, adding fermentation substrate for fermentation, and continuously adding carbon and nitrogen sources to the fermentation system after 30-60 hours of fermentation, controlling the added C / N ratio to be (20-30):1; The fermentation substrate is selected from any one or a combination of several of fatty acids, fatty acid esters, fatty acid salts or alkanes with 9-11 carbon atoms.

2. The method for producing long-chain dicarboxylic acids by fermentation according to claim 1, characterized in that, When fermentation has been going on for 40-60 hours, carbon and nitrogen sources are continuously added to the fermentation system, and the C / N ratio of the added sources is controlled to be (25-30):

1.

3. The method for producing long-chain dicarboxylic acids by fermentation according to claim 1, characterized in that, The controlled-flow acceleration rate of C in the carbon source is 0.01-0.5 g / L / h, preferably 0.01-0.3 g / L / h; and / or, The flow rate of N in the controlled-flow nitrogen source is 0.001-0.02 g / L / h, preferably 0.001-0.01 g / L / h.

4. The method for producing long-chain dicarboxylic acids by fermentation according to claim 1, characterized in that, The carbon source is selected from any one or more of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, more preferably glucose and / or sucrose; The nitrogen source includes inorganic nitrogen source and / or organic nitrogen source; the inorganic nitrogen source is selected from any one or a combination of several of urea, potassium nitrate, ammonium sulfate and ammonia water; the organic nitrogen source is selected from any one or a combination of several of corn steep liquor, yeast extract and yeast powder.

5. The method for producing long-chain dicarboxylic acids by fermentation according to claim 1, characterized in that, The C / N ratio of the fermentation medium was controlled to be (10-15):

1.

6. The method for producing long-chain dicarboxylic acids by fermentation according to claim 5, characterized in that, The fermentation medium contains at least a carbon source, a nitrogen source, inorganic salts and / or growth factors; The carbon source is selected from any one or a combination of several of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, more preferably from any one or a combination of glucose, lactose, and sucrose; The nitrogen source is selected from any one or more combinations of yeast extract, peptone, yeast powder, corn steep liquor, urea, ammonium salts, and nitrates; The inorganic salt is selected from one or more combinations of sulfates, hydrochlorides and phosphates, more preferably from one or more combinations of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, sodium chloride and calcium chloride; The growth factor is selected from at least one of vitamin B1, vitamin B2, vitamin B5, vitamin C, and biotin.

7. The method for producing long-chain dicarboxylic acids by fermentation according to claim 1, characterized in that, The fermentation culture temperature is 27-30℃, and / or the dissolved oxygen is 10-70%, and / or the air volume is 0.1-0.8 vvm, and / or the pressure is 0.03-0.15 MPa, and / or the pH value is 5.0-8.

0.

8. The method for producing long-chain dicarboxylic acids by fermentation according to claim 1, characterized in that, When the optical density value OD of the strain 620 When diluted 30 times to reach a concentration of 0.5-1.0, add the fermentation substrate to begin fermentation; and / or, During fermentation, the concentration of the fermentation substrate should be controlled to not exceed 10% (v / v), preferably not exceeding 5% (v / v).

9. A method for producing a medium- to long-chain dicarboxylic acid, characterized in that, The method includes: preparing a fermentation broth according to any one of claims 1-8, and extracting medium- and long-chain dicarboxylic acids from the obtained fermentation broth.

10. The method for producing medium- to long-chain dicarboxylic acids according to claim 9, characterized in that, The extraction process includes: adjusting the pH of the fermentation broth to 2-5, performing solid-liquid separation, and obtaining medium- and long-chain dicarboxylic acids.

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