Production method of long-chain dicarboxylic acid

Abstract

The invention provides a production method of long-chain dicarboxylic acid, which comprises the following steps: carrying out fermentation culture on Candida vesiensis to obtain fermentation liquor, and extracting the long-chain dicarboxylic acid from the fermentation liquor, the Candida veronii is Candida veronii CATH2302, and the preservation number of the Candida veronii CATH2302 is CCTCC (China Center for Type Culture Collection) NO: M20232153, and the Candida veronii CATH2302 is CCTCC NO: M20232153. The fermentation substrate is selected from one or a combination of more of long-chain fatty acid, long-chain fatty acid ester or long-chain alkane with the carbon atom number of 9-18. According to the production method, the long-chain dibasic acid can be produced through fermentation under different acidic or alkaline fermentation conditions, the yield of the long-chain dibasic acid with the target chain length is remarkably increased, the bio-based content of the long-chain dibasic acid can also be increased, and the production method has the potential of industrial large-scale production.

Description

[0001] This case is a divisional application of the invention patent application with the application date of August 8, 2024, application number 202411081003.2, and invention name "A kind of Candida vesicerae and its application". Technical Field

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

[0003] Long-chain dibasic acids have a wide range of industrial uses. Long-chain dibasic acids can be used as raw materials to synthesize special polyamides, high-grade spices, high-grade hot melt adhesives, cold-resistant plasticizers, high-grade lubricants, high-grade rust inhibitors, high-grade paints and coatings, etc. At present, the methods for producing long-chain dibasic acids include catalytic oil method, chemical synthesis method and microbial fermentation method. Among them, the chemical synthesis method has a long route, requires high temperature and high pressure for the reaction, and has relatively strict requirements on the catalyst. Therefore, there are few varieties of long-chain dibasic acids at the scale of large-scale production, and there are only a few varieties such as twelve-carbon long-chain dibasic acids. The microbial fermentation method often uses petroleum-based alkanes as fermentation substrates, and obtains long-chain dibasic acids through microbial fermentation conversion. Its production process is at normal temperature and pressure, and large-scale industrial production can be achieved. Although new plant-based resources such as fatty acids or their derivatives can be selected as fermentation substrates when producing long-chain dibasic acids, the relevant engineering strains or processes generally have the problems of low dibasic acid yield and low amplification production efficiency, which are difficult to promote and apply in industry. Summary of the invention

[0004] The purpose of the present invention is to provide a method for producing long-chain dibasic acids, which uses Candida viswanathii CATH2302 to efficiently utilize fermentation substrates of fatty acids or their derivatives, and ferments and produces long-chain dibasic acids under different fermentation conditions of acidity or alkalinity, thereby significantly improving the yield of long-chain dibasic acids with target chain lengths, and can also increase the biobased content of long-chain dibasic acids. The production method has the potential for industrial scale-up production.

[0005] The general formula of the long-chain dibasic acid of the present invention is HOOC(CH2)nCOOH, wherein n is 6 to 18. Specifically, the long-chain dibasic acid is selected from any one of suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedicarboxylic acid, hexadecanedioic acid, heptadecanedioic acid, and octadecanedicarboxylic acid.

[0006] The invention provides a method for producing a long-chain dibasic acid, which comprises: fermenting and culturing Candida viswanathii to obtain a fermentation broth, and extracting the long-chain dibasic acid from the fermentation broth; the Candida viswanathii is Candida viswanathii CATH2302, and the preservation number is CCTCC NO: M20232153; and a fermentation substrate is selected from a combination of one or more of a long-chain fatty acid, a long-chain fatty acid ester or a long-chain alkane having 9 to 18 carbon atoms.

[0007] The Candida viswanathii CATH2302 described in the present invention was biologically deposited on November 8, 2023. The depository unit is the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), and is classified and named Candida viswanathii.

[0008] The Candida viswanathii CATH2302 of the present invention is obtained by mutagenesis screening, and is identified as Candida viswanathii by ITS (Internal Transcribed Spacer) sequence. The ITS sequence refers to GenBank No. MK394122.1, or to the sequence SEQ ID NO: 1 disclosed in the specification of Chinese patent application CN111748480B.

[0009] Specifically, the mutagenesis screening method comprises the following steps: connecting the starting strain Candida viswanathii CAES2113 (preservation number: CCTCC NO: M 2020048, disclosed in Chinese invention patent CN111748480B) to a glycerol tube in a shake flask containing YPD medium, adding 0.02% to 0.05% of a chemical mutagenic agent (e.g., 5-fluorouracil), and culturing at 29 to 31°C and 200 to 250 rpm for 24 hours. The culture solution with good growth is centrifuged to collect the bacteria and washed with physiological saline for 3 to 5 times, and diluted to a bacterial concentration of 10 6cfu / ml, and then use ultraviolet mutagenesis, operating conditions: ultraviolet wavelength 260mm, ultraviolet lamp power 20W, irradiation distance 20cm, irradiation time 30s~1min, while irradiating and stirring the bacterial solution, with a lethality of 80% as the standard. After ultraviolet mutagenesis treatment, it is placed in a light-proof ice bath for 1~2h, gradient dilution, and the bacterial solution with appropriate dilution is applied to the solid plate, and cultured in the dark until a single colony is produced. Pick a monoclonal clone with good growth to a well plate containing a primary screening medium, wherein the primary screening medium contains 1%~3% long-chain fatty acid esters or long-chain fatty acids, and ferment at a natural pH value of 29~31°C, and the fermentation cycle is 70~80 hours. After the fermentation is completed, the initial screening is performed, and the strains with relatively high yields of the target chain length long-chain dibasic acid are selected, and then these strains are screened again by well plate and shake flask fermentation, and finally a stable high-yield strain is obtained, which is stored in a glycerol tube and identified as belonging to Candida visnatus using the ITS (Internal Transcribed Spacer) sequence.

[0010] In some embodiments, the long-chain fatty acid is selected from a combination of one or more of nonadecanoic acid, decanoic acid, undecanoic acid, lauric acid (also known as dodecanoic acid), tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid (also known as hexadecanoic acid), heptadecanoic acid and octadecanoic acid.

[0011] In some embodiments, the long-chain fatty acid ester is selected from a combination of one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester or fatty acid butyl ester having 9 to 18 carbon atoms.

[0012] In some embodiments, the long-chain fatty acid ester is selected from a combination of one or more of nonadecanoate, decadecanoate, undecanoate, laurate, tridecanoate, myristate, pentadecanoate, palmitate, heptadecanoate or octadecanoate.

[0013] In some embodiments, the long-chain fatty acid ester is selected from a combination of one or more of methyl laurate, ethyl laurate, butyl laurate, laurin, methyl myristate, ethyl myristate, butyl myristate, methyl palmitate, ethyl palmitate or butyl palmitate.

[0014] In some embodiments, the long-chain alkane is selected from a combination of one or more of nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, and octadecane, and is more preferably a long-chain normal alkane. The long-chain alkane may be a petroleum-based alkane or coal-derived alkane derived from petroleum or coal, or a bio-based alkane obtained by processing vegetable oils and fats.

[0015] When the fermentation substrate used is derived from plant oils and fats, such as coconut oil or palm oil, the corresponding long-chain dibasic acid obtained by fermentation conversion will have a corresponding bio-based content. For example, lauric acid can be obtained by hydrolysis of coconut oil or high-temperature and high-pressure decomposition, and methyl laurate can be prepared by esterification of lauric acid and methanol. The dodecanedicarboxylic acid produced by fermentation using such lauric acid or methyl laurate as a fermentation substrate will have a corresponding bio-based content.

[0016] In some embodiments, the long chain alkane is a petroleum-based n-alkane or a bio-based n-alkane.

[0017] In some embodiments, the fermentation substrate is selected from one or more of the following combinations: a combination of long-chain fatty acids and long-chain fatty acid esters; a combination of long-chain fatty acids and long-chain alkanes; a combination of long-chain fatty acid esters and long-chain alkanes; a combination of long-chain fatty acid salts and long-chain alkanes; or a combination of long-chain fatty acids, long-chain fatty acid esters and long-chain alkanes. In the above combination consisting of two or three different types of fermentation substrates, the mass ratio of different types of fermentation substrates is not particularly limited. For example, when the fermentation substrate is a combination of long-chain fatty acids and long-chain fatty acid esters, the mass ratio of long-chain fatty acids and long-chain fatty acid esters is not particularly limited, for example, it can be 1:99 to 99:1.

[0018] In some embodiments, the fermentation substrate is a combination of long-chain fatty acids and long-chain fatty acid esters in a mass ratio of 1:99 to 99:1; a combination of long-chain fatty acids and long-chain alkanes in a mass ratio of 1:99 to 99:1; a combination of long-chain fatty acid esters and long-chain alkanes in a mass ratio of 1:99 to 99:1; a combination of long-chain fatty acid salts and long-chain alkanes in a mass ratio of 1:99 to 99:1; or a combination of long-chain fatty acids, long-chain fatty acid esters and long-chain alkanes in any mass ratio.

[0019] In some specific embodiments, when fermentation is used to produce dodecane dibasic acid, the fermentation substrate is selected from any one of dodecane, lauric acid, lauric acid ester, a combination of lauric acid and dodecane, a combination of lauric acid ester and dodecane, and a combination of lauric acid and lauric acid ester, wherein the lauric acid ester is methyl laurate or ethyl laurate.

[0020] In some specific embodiments, when fermentation produces tetradecanedioic acid, the fermentation substrate is selected from any one of tetradecane, myristic acid, myristic acid ester, a combination of myristic acid and tetradecane, a combination of myristic acid ester and tetradecane, and a combination of myristic acid and myristic acid ester, wherein myristic acid ester is methyl myristate or ethyl myristate.

[0021] In some specific embodiments, when fermenting to produce hexadecane dibasic acid, the fermentation substrate is selected from any one of hexadecane, palmitic acid, palmitate, a combination of palmitic acid and hexadecane, a combination of palmitate and hexadecane, and a combination of palmitic acid and palmitate, wherein palmitate is methyl palmitate or ethyl palmitate.

[0022] In some embodiments, the temperature of the fermentation culture is 28°C to 35°C, for example, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C.

[0023] In some embodiments, the air volume of the fermentation culture is 0.2 to 0.8 vvm, for example, 0.2 vvm, 0.3 vvm, 0.4 vvm, 0.5 vvm, 0.6 vvm, 0.7 vvm or 0.8 vvm.

[0024] In some embodiments, the pressure of the fermentation culture is 0.05-0.2 MPa, for example, 0.05 MPa, 0.08 MPa, 0.11 MPa, 0.12 MPa, 0.15 MPa, 0.2 MPa.

[0025] In some embodiments, the dissolved oxygen in the fermentation culture is 10-90%, such as 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80% or 90%, preferably 20-80%.

[0026] In some embodiments, the fermentation culture is acidic; preferably, the pH value during the fermentation culture is 4.5-6.9, and further 4.5-6.5, such as 4.5, 4.8, 5.0, 5.5, 5.8, 6.0, 6.4 or 6.5. Specifically, it can be adjusted by adding sodium hydroxide or dilute sulfuric acid.

[0027] In some embodiments, the fermentation culture is alkaline; preferably, the pH value during the fermentation culture is 7.0 to 7.5, such as 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5. Specifically, it can be adjusted by adding sodium hydroxide or dilute sulfuric acid.

[0028] In some embodiments, during fermentation culture, when the optical density OD620 of the strain diluted 30 times reaches 0.5-1.0, preferably 0.7-1.0, such as 0.7, 0.8, 0.9 or 1.0, fermentation substrate is added to start fermentation conversion.

[0029] In some embodiments, the fermentation substrate is added in a one-time addition, batch addition or continuous flow addition.

[0030] Preferably, during fermentation culture, the concentration of the fermentation substrate is controlled below 8% (v / v), preferably 0.01-5% (v / v), such as 0.5%, 1%, or 2%.

[0031] In some embodiments, when the fermentation substrate is solid, it is first heated to dissolve it and then added into the fermentation liquid; wherein the temperature of heating the fermentation substrate can be 45-65°C, for example, 45°C, 50°C, 55°C.

[0032] In some embodiments, the fermentation time is 130 to 180 hours.

[0033] In some embodiments, the culture medium for fermentation culture contains at least a carbon source, a nitrogen source and / or an inorganic salt.

[0034] In some preferred embodiments, the carbon source is selected from a combination of one or more of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, more preferably selected from a combination of one or more of glucose, lactose, and sucrose.

[0035] In some preferred embodiments, the nitrogen source is selected from a combination of one or more of yeast extract, peptone, corn steep liquor, urea, ammonium salt and nitrate.

[0036] In some preferred embodiments, the inorganic salt is selected from a combination of one or more of sulfates, hydrochlorides, nitrates and phosphates, more preferably selected from a combination of one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, calcium chloride and potassium nitrate.

[0037] In some preferred embodiments, the culture medium includes a seed culture medium and a fermentation culture medium, both of which contain the carbon source, the nitrogen source and the inorganic salt. Candida visweiss is first cultured in the seed culture medium and then inoculated into the fermentation culture medium for culture and fermentation conversion.

[0038] In some specific embodiments, the seed culture medium contains at least 1% to 3.5% sucrose, 0.1% to 1.5% corn steep liquor, 0.1% to 1.0% yeast extract, 0.2% to 2.0% potassium dihydrogen phosphate, 0.1% to 0.8% urea, and 0.01% to 0.10% (w / v) defoaming agent.

[0039] In some specific embodiments, the fermentation medium contains at least 1% to 5% glucose, 0.1% to 1.0% corn steep liquor, 0.1% to 0.6% yeast extract, 0.1% to 0.8% potassium nitrate, 0.1% to 0.8% potassium dihydrogen phosphate, 0.05% to 0.35% ammonium sulfate, 0.02% to 0.30% urea, and 0.05% to 0.40% sodium chloride (w / v).

[0040] Preferably, the defoamer includes a polyether defoamer, a silicone defoamer, a silicone ether composite defoamer, a mineral oil defoamer or a nonionic surfactant, and examples thereof include commercially available polyether defoamers, Dow DF-103 defoamer, Sigma Aldrich's defoamer 204, polyethylene glycol monostearate or dimethyl silicone oil.

[0041] In some embodiments, during fermentation culture, the inoculation amount of Candida visweiss is 5-50% (v / v) of the fermentation medium, that is, the volume ratio between the seed liquid and the fermentation medium is (5-50):100.

[0042] In some embodiments, the extraction includes adjusting the pH value of the fermentation broth to 2-5, and performing solid-liquid separation to obtain a crude long-chain dibasic acid. Specifically, dilute sulfuric acid or dilute hydrochloric acid can be used to adjust the pH value.

[0043] In some embodiments, the solid-liquid separation may be performed by centrifugation or filtration to separate the solid from the liquid.

[0044] In some embodiments, the present invention provides a fermentation broth containing Candida viswanathii CATH2302 and long-chain dibasic acid, wherein the deposit number of Candida viswanathii CATH2302 is CCTCC NO: M 20232153, and the biobased content of the long-chain dibasic acid is 1-100%.

[0045] The long-chain dibasic acid product prepared by the method of the present invention has a biobased content of 1 to 100%, and further 5 to 100%. The biobased content can be measured by a method for measuring the radioactive C14 content, such as the standard ASTM-D6866 method of the American Society for Testing and Materials.

[0046] The technical solution provided by the present invention has the following beneficial effects:

[0047] 1. The Candida viswanathii CATH2302 of the present invention can not only utilize traditional alkanes as fermentation substrates, but also can efficiently utilize fatty acid derivatives, such as long-chain fatty acids, long-chain fatty acid esters, etc. as fermentation substrates, and can especially utilize long-chain fatty acids, long-chain fatty acid esters and compositions thereof with long-chain alkanes as fermentation substrates, and the yield of target long-chain dibasic acids is significantly increased, which is significantly improved compared with existing strains, and has the potential for industrial scale-up production.

[0048] 2. In the production method of Candida viswanathii CATH2302 and long-chain dibasic acids of the present invention, fermentation conversion can be carried out not only under alkaline conditions, but also under acidic conditions (for example, pH value below 6.5). In particular, when the fermentation conditions are acidic, the amount of alkali solution used in the fermentation process can be effectively reduced, the salt content in the fermentation liquid, fermentation treatment liquid and wastewater can be greatly reduced, and the subsequent process of extracting long-chain dibasic acids can be simplified.

[0049] 3. Some fermentation substrates of fatty acids and their derivatives come from the seeds of oil-rich tropical crops such as palm trees and coconut trees. This new type of renewable resource can replace fossil raw materials such as petroleum alkanes, which is of great significance to the sustainable development of industrial processes.

[0050] Strain deposit information:

[0051] Candida viswanathii CATH2302, the deposit number is CCTCC NO: M20232153, the deposit date is November 8, 2023, the depository is the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), and the classification name is Candida viswanathii. DETAILED DESCRIPTION

[0052] The present invention is further described below by specific examples, which are only used as an illustration of the present invention and do not limit the scope of the present invention. Unless otherwise specified, the raw materials and materials used in the following examples are all commercially available, and the methods and conditions used are all known methods and conventional conditions in the art. Raw materials that need to be sterilized are sterilized at 121°C for 20 minutes.

[0053] Unless otherwise specified, fermentation substrates are all commercially available, wherein bio-based alkanes are alkanes obtained by processing vegetable oils and fats as raw materials, petroleum-based alkanes are alkanes derived from petroleum; lauric acid, methyl laurate, and methyl myristic acid are derived from natural coconut oil.

[0054] Materials and methods:

[0055] 1. Strains

[0056] Starting strain: Candida viswanathii CAES2113 (deposit number: CCTCC NO: M 2020048, disclosed in Chinese invention patent CN111748480B), hereinafter referred to as CAES2113.

[0057] The strain of the present invention: Candida viswanathii CATH2302 (deposit number: CCTCCNO: M 20232153), hereinafter referred to as CATH2302.

[0058] 2. Culture medium

[0059] YPD medium (w / v): 2% peptone, 2% glucose and 1% yeast extract (OXOID, No. LP0021), and 1.5% agar powder should also be added to the solid culture medium.

[0060] Seed culture medium (w / v): sucrose 2.5%, yeast extract 0.4%, corn steep liquor 0.7%, potassium dihydrogen phosphate 0.7%, urea 0.4%, commercial defoamer 204 0.04%.

[0061] Fermentation medium (w / v): 3.5% glucose, 0.6% corn steep liquor, 0.3% yeast extract, 0.5% potassium nitrate, 0.3% potassium dihydrogen phosphate, 0.15% urea, 0.1% ammonium sulfate and 0.15% sodium chloride.

[0062] 3. Product detection method: High performance gas chromatography was used to detect the long-chain dibasic acid in the fermentation broth, and the product concentration was calculated based on the ratio of the peak area of ​​the product to the peak area of ​​the internal standard of known concentration.

[0063] 4. Acquisition of Candida visweiss CATH2302

[0064] Mutagenesis screening: The starting strain Candida viswanathii CAES2113 glycerol tube was connected to a shake flask containing YPD medium, and 0.02% chemical mutagenic agent 5-fluorouracil was added. The culture was shaken at 30°C and 250 rpm for 24 hours. The culture medium with good growth was centrifuged to collect the bacteria and washed 3 to 5 times with physiological saline, and diluted to a bacterial concentration of 10 6cfu / ml, and then use ultraviolet mutagenesis, operating conditions: ultraviolet wavelength 260mm, ultraviolet lamp power 20W, irradiation distance 20cm, irradiation time 1min, stirring the bacterial solution while irradiating, with a lethality of 80% as the standard. After ultraviolet mutagenesis treatment, place it in a dark ice bath for 2h, gradient dilution, and apply the bacterial solution of appropriate dilution to the solid plate, and culture it in the dark until a single colony is produced.

[0065] Pick a monoclone with good growth to a well plate containing a primary screening medium (a fermentation medium containing 3% fermentation substrate methyl laurate), and ferment at a natural pH value of 29°C for 70 hours. After the fermentation is completed, the strain with relatively high yield of dodecanedioic acid is selected, and then these strains are rescreened through the well plate, and the rescreened strains are rescreened by 500ml shake flask fermentation, and the 500ml shake flask fermentation is fermented at a natural pH value of 29°C for 110 hours. Finally, a stable high-yield strain is obtained and stored in a glycerol tube. Using ITS (Internal Transcribed Spacer) sequence identification, the purified single colony ITS sequence obtained in the above steps is subjected to DNA sequencing, and compared with the ITS sequence of the known Candida viswanathii (see GenBank No. MK394122.1), it is identified as Candida viswanathii, and thus it is named Candida viswanathii CATH2302.

[0066] 5. Stability verification of Candida viswanathii CATH2302 strain

[0067] The continuous passage experiment verified that the stability of the morphology, growth and production performance of the CATH2302 strain was good. After five passages, the colony morphology did not change significantly. The surface of the CATH2302 colony was smooth and moist, milky white, shiny, round, and had neat edges. The results showed that Candida viswanathii CATH2302 had good passage stability.

[0068] Examples 1 to 6 Production of long-chain dibasic acids by fermentation of Candida viswanathii CATH2302 in a fermenter

[0069] (1) Activation culture: 100 mL of 10° Baume wort (pH 5.4) was inoculated into a glycerol tube seed of Candida visweiss, and the activation culture was carried out in a shake flask at a temperature of 29°C and a rotation speed of 220 rpm. When the OD620 value of the bacteria reached 0.7 (diluted 30 times), the culture was terminated to obtain the shake flask seeds.

[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 amount of 1.6% (v / v). The culture was carried out at a temperature of 29° C., an air volume of 0.33 vvm, a pressure of 0.12 MPa, and a dissolved oxygen content of 15%. The culture was terminated when the OD620 value of the seed solution reached 0.8 (diluted 30 times) to obtain the seed solution.

[0071] (3) Fermentation culture: The seed liquid obtained in step (2) is inoculated into a fermentation tank filled with fermentation medium, with an inoculation amount of 15% (v / v), and fermentation culture is carried out under the conditions of temperature 29°C, air volume 0.31vvm, pressure 0.12MPa, and dissolved oxygen 45%. When the OD620 value of the fermentation liquid reaches 0.8 (diluted 30 times), the fermentation substrate is added to the fermentation tank in batches for fermentation conversion, and the concentration of the fermentation substrate is controlled to be 1% (v / v). After fermentation for 150 hours, the addition of the fermentation substrate is stopped. When the fermentation substrate detection is 0, the fermentation is stopped, and the total fermentation time is calculated. When the fermentation substrate is solid, it is heated to 45-65°C and dissolved before being added to the fermentation tank.

[0072] The fermentation strains, fermentation substrates, pH values ​​during fermentation culture, the amount of fermentation substrate added, the acid production, and the total fermentation time of Examples 1 to 6 are shown in Table 1 below, wherein DC12 represents dodecanedioic acid, DC14 represents tetradecandioic acid, and the acid production represents the concentration of the target long-chain dicarboxylic acid detected in the fermentation broth.

[0073] Comparative Example 1

[0074] The method for preparing long-chain dibasic acid is the same as that in Example 1, except that the strain used is the starting strain Candida viswanathii CAES2113. The results are shown in Table 1 below.

[0075] Comparative Example 2

[0076] The method for preparing long-chain dibasic acid is the same as that in Example 5, except that the strain used is the starting strain Candida viswanathii CAES2113. The results are shown in Table 1 below.

[0077] Table 1

[0078]

[0079] As can be seen from Table 1, Example 1 and Comparative Example 1 used a combination of lauric acid and petroleum-based n-dodecane as substrates, the difference being that the Candida visweiss strains were different, and Example 1 showed that the DC12 yield of the strain CATH2302 of the present invention was significantly increased, and the fermentation time was shortened. Similarly, compared with Comparative Example 2, Example 5 showed that the DC14 yield of the strain of the present invention was also significantly increased, and the fermentation time was shortened. Examples 2-4 show that the strain of the present invention can also utilize a single alkane, a single fatty acid ester, or a composition of a fatty acid ester and a fatty acid to ferment and produce the target DC12, and the acid production is all above 150 g / L, and Example 3 even reaches more than 190 g / L. Example 6 shows that the strain of the present invention is also suitable for fermenting and producing long-chain dibasic acids under high pH conditions.

[0080] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for producing a long-chain dibasic acid, comprising: The invention relates to a method for obtaining a fermentation broth by fermenting and culturing Candida viswanathii, and extracting a long-chain dibasic acid from the fermentation broth, wherein the Candida viswanathii is Candida viswanathii CATH2302, and the deposit number is CCTCC NO: M 20232153; and the fermentation substrate is selected from a combination of one or more of long-chain fatty acids, long-chain fatty acid esters or long-chain alkanes having 9 to 18 carbon atoms.

2. The production method according to claim 1, characterized in that The long-chain fatty acid is selected from one or more combinations of nonadecanoic acid, deadecanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid and octadecanoic acid.

3. The production method according to claim 1, characterized in that The long-chain fatty acid ester is selected from a combination of one or more of nonadecanoate, decadecanoate, undecanoate, laurate, tridecanoate, myristate, pentadecanoate, palmitate, heptadecanoate or octadecanoate; or, the long-chain fatty acid ester is selected from a combination of one or more of methyl laurate, ethyl laurate, butyl laurate, laurin, methyl myristate, ethyl myristate, butyl myristate, methyl palmitate, ethyl palmitate or butyl palmitate.

4. The production method according to claim 3, characterized in that: The long-chain fatty acid ester is selected from one or more combinations of methyl laurate, ethyl laurate, butyl laurate, laurin, methyl myristate, ethyl myristate, butyl myristate, methyl palmitate, ethyl palmitate or butyl palmitate.

5. The production method according to claim 1, characterized in that: The long-chain alkane is selected from one or more combinations of nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane and octadecane.

6. The production method according to claim 5, characterized in that The long-chain alkane is a petroleum-based normal alkane or a bio-based normal alkane.

7. The production method according to claim 1, characterized in that: The fermentation substrate is a combination of long-chain fatty acids and long-chain fatty acid esters in a mass ratio of 1:99 to 99:1; a combination of long-chain fatty acids and long-chain alkanes in a mass ratio of 1:99 to 99:1; a combination of long-chain fatty acid esters and long-chain alkanes in a mass ratio of 1:99 to 99:1; a combination of long-chain fatty acid salts and long-chain alkanes in a mass ratio of 1:99 to 99:1; or a combination of long-chain fatty acids, long-chain fatty acid esters and long-chain alkanes in any mass ratio.

8. The production method according to claim 1, characterized in that: The fermentation culture temperature is 28°C to 35°C, and / or the dissolved oxygen is 10 to 90%, and / or the air volume is 0.2 to 0.8 vvm, and / or the pressure is 0.05 to 0.2 MPa, and / or the fermentation culture is under acidic or alkaline conditions.

9. The production method according to claim 1, characterized in that: During fermentation culture, the concentration of the fermentation substrate is controlled to be below 8%, and further controlled to be 0.01 to 5%.

10. The production method according to claim 1, characterized in that: The extraction comprises adjusting the pH value of the fermentation liquid to 2-5, and separating the solid from the liquid to obtain a crude long-chain dibasic acid.

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