A high-yield decarboxylic acid-producing strain and a fermentation method

Abstract

The application discloses a Candida viswanathii strain and a method for producing decanoic diacid by fermentation of the strain. The strain is Candida viswanathii, and the preservation number is CCTCC M 2021824. The application relates to a method for producing decanoic diacid by biological method of Candida viswanathii, which can maintain the content of fermentation substrate at a stable level in the fermentation process, and improve the content of fermentation product. The Candida viswanathii disclosed by the application can improve the production capacity of decanoic diacid, reduce the production cost, and is easy to be mass-produced in an industrialized production process.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain, particularly to a Candida viride strain that produces decacarboxylic acid, and a method for producing decacarboxylic acid by fermentation using this strain. Background Technology

[0002] Long-chain dicarboxylic acids are important raw materials for the synthesis of fragrances, nylon engineering plastics, hot melt adhesives, resins, cold-resistant plasticizers, pharmaceuticals, and pesticides. Their structural formula is HOOC(CH2). n COOH, where n is an integer greater than 7. Decadocic acid, also known as decacarbon long-chain dicarboxylic acid or sebacic acid, has the chemical formula HOOC(CH2)8COOH. As an important monomer raw material, decadocic acid is widely used in the production of polyamide engineering plastics, such as nylon 510, nylon 1010, and nylon 610. Decadocic acid can also be used to manufacture high-temperature lubricating oils, epoxy resin curing agents, synthetic greases, artificial fragrances, and cold-resistant plasticizers, making it one of the most widely used chemical raw materials.

[0003] Biological methods for preparing decacarboxylic acid (DAC) are characterized by simple production processes and environmental friendliness. Yu Zhihua of the Institute of Microbiology, Chinese Academy of Sciences (see *Acta Microbiologica Sinica*, 1989, Vol. 6: 0253-2654) obtained a high-yield strain through mutagenesis screening, achieving a DAC yield of over 71 g / L in a 16-liter fermenter. The DAC product was obtained through aqueous phase crystallization with a purity of over 99.6%. The Institute of Forestry and Soil Science, Chinese Academy of Sciences (see *Acta Microbiologica Sinica*, 1979, Vol. 1) screened *Candida lipolytica* to produce DAC with a yield of 30–40 g / L.

[0004] In the biological preparation of decacarbonyl dicarboxylic acid (DC10), decane or its analogues are often used as fermentation substrates. These substrates may undergo transformations during fermentation, including multi-step oxidation by the microbial cells to the target product, decacarbonyl dicarboxylic acid; consumption for cell growth maintenance; or volatilization into the external environment. The high volatility of fermentation substrates such as decane significantly limits the conversion rate of decacarbonyl dicarboxylic acid, and alkane costs also constitute the largest proportion of the total cost during fermentation. Therefore, improving the acid-producing performance of bacterial strains to reduce production costs is an important research direction. Summary of the Invention

[0005] The first objective of this invention is to provide a Candida vesicatoria strain to improve the acid concentration and conversion efficiency of decacarbonylate production by current Candida vesicatoria strains, thereby reducing production costs.

[0006] The present invention solves the above-mentioned technical problems and achieves the purpose of the present invention through the following technical solutions.

[0007] This invention provides a strain that produces high levels of decacarboxylic acid: a novel *Candida viswanathii* strain, CAes2121, with accession number CCTCC M 2021824, deposited on July 7, 2021, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China). In addition to possessing the superior production characteristics of its parent strain CAES2113, *Candida viswanathii* CAes2121 exhibits high enzyme activity in fermentation systems with pH values ​​above and below 7.0, and can also increase the decacarboxylic acid production rate, effectively reducing energy consumption and improving efficiency.

[0008] The strain provided in this invention is *Candida viswanathii*, which was developed from a single strain, CAES2113, through mutagenesis induced by a combination of LiCl and ARTP. The parent strain, CAES2113, was deposited on February 24, 2020, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC M 2020048 and classified as *Candida viswanathii*. Its biological and genetic characteristics are described in Chinese Invention Patent CN111748480A.

[0009] The morphological characteristics of *Candida viswanathii* CAes2121 provided by this invention are: creamy white, with smooth, moist, glossy, round colonies with neat edges. Figure 1 and Figure 2 As shown.

[0010] A second aspect of the invention provides the application of the Candida albicans CAES2113, namely the application of the Candida albicans CAes2121 in the fermentation production of decacarbonyl dicarboxylic acids.

[0011] In some specific implementations, the substrates used in the fermentation production include decane, decacarbon fatty acids, decacarbon fatty acid derivatives, or mixtures thereof.

[0012] A third aspect of this invention is to provide a method for the biological production of long-chain decacarbondicarboxylic acids, the method comprising the following steps:

[0013] a) Seed culture and fermentation culture of Candida viswanathii CAes2121;

[0014] b) Extract decacarbon-2-carboxylic acid from the fermentation product obtained in step a).

[0015] In some specific implementations, the seed culture process includes strain activation, specifically by culturing the strain in YPD medium on a shaker for 1-2 days. The YPD medium comprises 2.0% (w / v) glucose, 1.0% (w / v) yeast extract, and 2.0% (w / v) peptone.

[0016] In some specific implementations, the temperature during seed culture and fermentation culture is 28℃~33℃, preferably 29~30℃.

[0017] In some specific implementations, the seed culture and fermentation culture used include a carbon source, a nitrogen source, inorganic salts, and / or nutrient factors. The carbon source includes one or more of sucrose, glucose, maltose, molasses, fructose, rhamnose, arabinose, and sorbitol, and the amount of carbon source added can be 1% to 10% (w / v); and / or, the nitrogen source includes one or more of yeast extract, corn steep liquor, urea, ammonia, ammonium sulfate, potassium nitrate, and ammonium nitrate, and the amount of nitrogen source added can be 0.1% to 3% (w / v); and / or, the inorganic salt includes one or more of potassium salt and sodium salt; preferably, the potassium salt includes one or more of potassium chloride, potassium nitrate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate, and the sodium salt includes one or more of sodium chloride, sodium nitrate, sodium dihydrogen phosphate, and disodium hydrogen phosphate, and the amount of inorganic salt added can be 0.1% to 1.5% (w / v); and / or, the nutritional factors include one or more of vitamin B1, vitamin B2, vitamin C, biotin, and amino acids, and the amount of nutritional factors added can be 0% to 1% (w / v).

[0018] In some specific embodiments, the seed culture medium includes: 1.0% to 4.0% sucrose or glucose, 0.3% to 1.5% corn steep liquor, 0.5% to 2.5% yeast extract, 0.01% to 0.3% urea, and 0.4% to 0.8% potassium dihydrogen phosphate.

[0019] In some specific embodiments, the fermentation culture medium includes: 1.0%–4.0% sucrose or glucose, 0–1.5% corn steep liquor, 0.5%–1.5% yeast extract, 0.4%–0.8% potassium dihydrogen phosphate, 0.1%–0.4% potassium nitrate, 0.15%–0.4% sodium chloride, 0.5%–0.8% ammonium sulfate, 0.05%–0.5% urea, and 0%–0.05% vitamin B2.

[0020] In some specific implementations, during the fermentation culture, the pH value of the fermentation system is controlled to be below 7.0 or above 7.0.

[0021] In some specific implementations, during the fermentation culture, the pH value of the fermentation system is controlled to be 4.0–6.8; specific pH values ​​can be: 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 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.

[0022] In some specific implementations, during the fermentation culture, the pH value of the fermentation system is controlled to be 7.0 to 8.5; specific pH value examples can be: 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5.

[0023] In some specific embodiments, during the fermentation culture, the substrate added to the fermentation medium includes one or more of n-alkanes, straight-chain saturated fatty acids, and straight-chain saturated fatty acid derivatives. Preferably, the substrate includes one or more of C10 n-alkanes, C10 straight-chain saturated fatty acids, C10 straight-chain saturated fatty acid esters, and C10 straight-chain saturated fatty acid salts.

[0024] In some specific embodiments, the substrate concentration is controlled at 0.5% to 3% during the fermentation culture. Preferably, the fermentation substrate concentration is controlled within any one of the following ranges: 0.5% to 1%, 1% to 1.5%, 1.5% to 2%, or 2% to 3%, or other concentration ranges formed by any combination of the endpoints of these ranges, such as a concentration range of 0.5% to 1.5% formed by the endpoints of 0.5% and 1.5%, or a concentration range of 1% to 2% formed by the endpoints of 1% and 2%.

[0025] In some specific implementations, during the fermentation process, the pressure is controlled at 0.05–0.15 MPa and the air flow rate is 0.1–0.7 vvm.

[0026] In some specific implementations, during the fermentation process, the pressure is controlled at 0.08–0.12 MPa and the air flow rate is 0.3–0.5 vvm.

[0027] In some specific implementations, during the fermentation culture, the dissolved oxygen is controlled at 5% or higher, preferably 10-50%, such as 20% or higher or 40% or higher.

[0028] In some specific implementations, during the fermentation culture, the concentration of live cells in the fermentation broth is controlled to be 8*10.7 cfu / mL ~1.5*10 9 cfu / mL, more preferably 2*10 8 cfu / mL ~9*10 8 The concentration of live cells can be controlled by adjusting the substrate flow rate or aeration rate. The inventors found that lower cell concentrations lead to longer fermentation cycles and lower acid production rates, while higher cell concentrations result in lower DC10 yields.

[0029] In some specific implementations, the step of extracting decacarboxylic acid can be referred to Chinese invention patents with publication numbers CN101985416B or CN103965035B.

[0030] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: a new Candida vesicatoria strain, CAes2121, was screened, which can maintain high activity for a long time in environments with pH values ​​below or above 7.0. When fermenting to produce decacarbonylic acid, it can control the number of live cells and maintain the content of fermentation substrate at a stable level during fermentation, thereby obtaining higher DC10 yield, faster acid production rate, improved efficiency and energy saving, and can be used for industrial-scale production.

[0031] Preservation Information

[0032] Strain: Candida viswanathii CAes2121

[0033] Date of preservation: July 7, 2021

[0034] Depository Institution: China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China)

[0035] Accession number: CCTCC M 2021824 Attached Figure Description

[0036] Figure 1 This is a colony morphology image of the first generation of Candida viswanathii (CAes2121) from Example 2 of the present invention.

[0037] Figure 2 This is a colony morphology image of the sixth generation of Candida viswanathii CAes2121, as described in Example 2 of this invention. Detailed Implementation

[0038] The preferred embodiments of the present invention will now be described in detail with reference to specific 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 essence; the scope of the present invention is not limited to the embodiments listed herein.

[0039] In this invention, the content of decacarboxylic acid and fermentation substrate in the fermentation broth can be detected using gas chromatography, liquid chromatography, liquid chromatography-mass spectrometry, or gas chromatography-mass spectrometry. Cell concentration is monitored using a Hamilton live cell concentration analyzer.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] The following example illustrates the detection of DC10 content in fermentation broth: The pH of the fermentation broth was adjusted to 3.0 using hydrochloric acid solution, and then 100 mL of diethyl ether was added to extract the dicarboxylic acid from the fermentation broth. The diethyl ether was then removed by evaporation to obtain dicarboxylic acid powder. The obtained dicarboxylic acid powder was 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.

[0043] The YPD culture medium formulations used in the following examples are as follows: 2% glucose, 1% yeast extract, and 2% peptone; the seed culture medium formulation is: 2% sucrose, 0.3% corn steep liquor, 0.5% yeast extract, 0.8% potassium dihydrogen phosphate, and 0.3% urea; the fermentation medium 1 formulation is: 4% sucrose, 1.5% yeast extract, 0.8% potassium dihydrogen phosphate, 0.4% potassium nitrate, 0.15% sodium chloride, and 0.05% urea; the fermentation medium 2 formulation is: 4% sucrose, 1.5% yeast extract, 0.8% potassium dihydrogen phosphate, 0.4% potassium nitrate, 0.15% sodium chloride, 0.5% ammonium sulfate, and 0.05% urea; the fermentation medium 3 formulation is: 4% sucrose, 1.5% yeast extract, 0.8% potassium dihydrogen phosphate, 0.4% potassium nitrate, 0.15% sodium chloride, 0.5% ammonium sulfate, 0.05% urea, and 0.01% vitamin B2. All the above culture media were sterilized at 121°C for 20 minutes before use.

[0044] Example 1: Obtaining Candida viswanathii strain CAes2121

[0045] Using *Candida viswanathii* strain CAES2113 (deposited on February 24, 2020, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), accession number CCTCC NO: M 2020048) as the starting strain, the glycerol tube of the starting strain was inoculated into a shake flask containing YPD medium, and 1 mL of sterile 0.5% LiCl solution was added to the medium. The culture was then incubated at 30℃ and 200 rpm for 20 h with shaking. Collect the cells by centrifugation of the well-grown culture medium and wash them 3-5 times with physiological saline. Resuspend the cells in 10% glycerol aqueous solution and induce mutagenesis using ARTP. The treatment conditions are: power 120W, gas flow rate 10SLM, distance 2mm, treatment time 100s. The mutagenized strain is cultured in YPD medium shake flasks for 24h at 29℃. After dilution, the treated bacterial solution is plated and cultured on YPD medium plates for 72h at 29℃. Single clones can be cultured. After multiple rounds of shake flask fermentation screening, a stable and high-yielding strain was obtained and named CAes2121, which is stored in glycerol tubes.

[0046] Example 2: Stability verification of Candida viswanathii strain CAes2121

[0047] The *Candida viswanathii* CAes2121 strain was verified through subculture experiments. After five subcultures, neither the colony morphology nor the ability to produce decacarboxylic acid showed significant changes. Colony morphology: The colonies were smooth, moist, milky white, glossy, round, and had neat edges. Figure 1 and Figure 2 It is evident that the Candida viswanathii CAes2121 of this invention exhibits good passage stability.

[0048] The production of decacarboxylic acid (DC10) using *Candida virescens* CAes2121 was verified by the following method: The first and sixth generation strains obtained above were used for shake-flask fermentation. One glycerol tube seed of *Candida virescens* CAes2121 (one from the first generation and one from the sixth generation) was inoculated into YPD medium and cultured for 24 h. Then, the culture was transferred to seed culture medium and cultured at 30℃ for 28 h. The OD of the seed culture was measured. 620 The concentration reached 0.8 (measured at a 30-fold dilution). The seed culture was inoculated into a shake flask containing fermentation medium 1, to which 3.5 mL of substrate n-decane was added. Fermentation was carried out at 30°C. After fermentation, the DC10 yields of the first and sixth generation strains were measured to be 117.88 g / L and 117.25 g / L, respectively.

[0049] Examples 3-5: Fermentation acid production of *Candida virescens* CAes2121 in shake flasks with different amounts of decacarbonane.

[0050] One glycerol tube seed of *Candida viswanathii* (CAes2121) obtained in Example 1 was inoculated into YPD medium and cultured for 24 h. Then, it was inoculated into seed culture medium and cultured at 29°C for 28 h. The OD of the seed culture was measured. 620 The concentration reached 0.82 (30 times diluted with water). 3.8 mL of seed culture was inoculated into a shake flask containing 15 mL of fermentation medium 1.

[0051] In Examples 3-5, the amount of n-decane added to fermentation medium 1 was 3.5 mL, 3.0 mL, and 2.0 mL, respectively. Fermentation was carried out at 29°C, and the fermentation was stopped when the n-alkane content in the fermentation broth was 0. The test results of the obtained fermentation broth are shown in Table 1 below.

[0052] Comparative Example 1

[0053] One glycerol tube containing the starting strain *Candida viswanathii* CAES2113 was taken and subjected to shake-flask fermentation using the same process as in Example 4. The OD of the seed culture before inoculation was measured. 620 The concentration reached 0.8 (diluted 30 times with water). Fermentation was carried out at 29°C. Fermentation was terminated when the concentration of fermentation substrate in the fermentation broth was 0. The test results of the obtained fermentation broth are shown in Table 1 below.

[0054] Table 1

[0055] Example 3 3.5 116.8 0.77 Example 4 3.0 96.0 0.68 Comparative Example 1 3.0 89.0 0.62 Example 5 2.0 69.0 0.51

[0056] As can be seen from Table 1, when performing shake-flask fermentation, the strain of this invention, compared with the starting strain, obtained a higher DC10 yield, a faster acid production rate, and more energy-efficient results under the same conditions.

[0057] Example 6: Fermentation of Candida albicans CAes2121 in a 10L fermenter to produce DC10

[0058] Candida albicans cells CAes2121 were inoculated into seed culture medium and cultured at 30℃. The seed culture cell optical density (OD) was measured... 620When the concentration of the seed culture reaches 0.5 (30-fold dilution), the seed culture is inoculated into fermentation medium 2 for fermentation transformation, with an inoculum size of 10% (v / v). The fermentation temperature is controlled at 30℃, the pH at 6.2, the air flow rate at 0.3 vvm, and the fermenter pressure at 0.08 MPa. A certain stirring speed is maintained, and dissolved oxygen is kept above 20% during fermentation. After the seed culture is inoculated into the fermenter, the cells begin to grow and reproduce. When the optical density (OD) of the cells in the fermentation broth reaches a certain level... 620 When the concentration reaches 0.5 (30-fold dilution), add the fermentation substrate n-decane. Control the decane concentration in the fermentation broth to be within the range of 0.5%–1% (v / v). During fermentation, control the flow rate of n-decane to maintain the concentration of live cells in the fermentation broth at 2*10⁻⁶. 8 cfu / mL ~9*10 8 The concentration of CFU / mL was 1300 g of n-decane, the fermentation substrate. Fermentation was stopped when the concentration of fermentation substrate in the fermentation broth reached 0. The test results of the obtained fermentation broth are shown in Table 2 below.

[0059] Comparative Example 2: Fermentation of Candida evie CAES2113 in a 10L fermenter to produce DC10

[0060] The fermentation method was the same as in Example 6, except for the strain: the starting strain *Candida viswanathii* CAES2113 was inoculated into seed culture medium with glycerol tubes, and the total amount of n-decane added as the fermentation substrate was 1300 g. Fermentation was stopped when the substrate content in the fermentation broth reached 0. The test results of the obtained fermentation broth are shown in Table 2 below.

[0061] Example 7: Fermentation of Candida albicans CAes2121 in a 10L fermenter to produce DC10

[0062] Candida albicans cells CAes2121 were inoculated into seed culture medium and cultured at 29℃. The seed culture cell optical density (OD2121) was determined as follows: 620 When the seed culture reaches 0.6 (30-fold dilution), it is inoculated into fermentation medium 2 for fermentation transformation at an inoculation rate of 10% (v / v). The fermentation temperature is controlled at 30℃, the pH at 7.2, the air flow rate at 0.3 vvm, and the fermenter pressure at 0.08 MPa. A certain stirring speed is maintained, and dissolved oxygen is kept above 20% during fermentation. After the seed culture is inoculated into the fermenter, the cells begin to grow and reproduce. When the optical density (OD) of the cells in the fermentation broth reaches a certain level... 620 When the concentration reaches 0.6 (30-fold dilution), add the fermentation substrate n-decane. Control the decane concentration in the fermentation broth to be within the range of 0.5%–1% (v / v). During fermentation, control the alkane flow rate to maintain the concentration of live cells in the fermentation broth at 2*10⁻⁶. 8 cfu / mL ~9*108 The concentration of CFU / mL was 1300 g of decane, the fermentation substrate. Fermentation was stopped when the concentration of fermentation substrate in the fermentation broth reached 0. The test results of the obtained fermentation broth are shown in Table 2 below.

[0063] Comparative Example 3: Fermentation of Candida evie CAES2113 in a 10L fermenter to produce DC10

[0064] The fermentation method was the same as in Example 7, except for the strain: Candida viswanathii (CAES2113) glycerol tubes were inoculated into seed culture medium, and the total amount of n-decane, the fermentation substrate, was 1300 g. Fermentation was stopped when the substrate content in the fermentation broth reached 0. The test results of the obtained fermentation broth are shown in Table 2 below.

[0065] Example 8: Fermentation of Candida albicans CAes2121 in a 10L fermenter to produce DC10

[0066] The fermentation method is the same as in Example 6, except that the tank pressure is controlled at 0.11 MPa and the dissolved oxygen is maintained above 40% during fermentation. The concentration of live cells in the fermentation broth is maintained at 8*10⁻⁶ by controlling the flow rate of n-decane during fermentation. 7 cfu / mL ~2*10 8 The concentration of CFU / mL was 1300 g of n-decane, the fermentation substrate. Fermentation was stopped when the concentration of fermentation substrate in the fermentation broth reached 0. The test results of the obtained fermentation broth are shown in Table 2 below.

[0067] Example 9: Fermentation of Candida albicans CAes2121 in a 10L fermenter to produce DC10

[0068] The fermentation method was the same as in Example 7, except that the dissolved oxygen level was below 10% during fermentation. The concentration of live cells in the fermentation broth was maintained at 9*10⁻⁶ by controlling the alkane flow rate during fermentation. 8 cfu / mL ~1.1*10 9 The concentration of CFU / mL was 1300 g of n-decane, the fermentation substrate. Fermentation was stopped when the concentration of fermentation substrate in the fermentation broth reached 0. The test results of the obtained fermentation broth are shown in Table 2 below.

[0069] Example 10: Fermentation of Candida albicans CAes2121 in a 10L fermenter to produce DC10

[0070] The fermentation method is the same as in Example 6, except that the seed liquid is inoculated into fermentation medium 3 for fermentation. During the fermentation process, the dissolved oxygen is controlled to be above 10%. When the fermentation substrate in the fermentation liquid is detected to be 0, the fermentation is stopped. The detection results of the obtained fermentation liquid are shown in Table 2 below.

[0071] Comparative Example 4: Fermentation of Candida albicans CAes2121 in a 10L fermenter to produce DC10

[0072] The fermentation method is the same as in Example 6, except that the dissolved oxygen is controlled above 40% during fermentation, and the concentration of live cells in the fermentation broth is maintained at 2*10⁻⁶ by controlling the alkane flow rate. 7 cfu / mL ~8*10 7 When the concentration of CFU / mL in the fermentation broth was 0, fermentation was stopped. The results of the fermentation broth analysis are shown in Table 2 below.

[0073] Comparative Example 5: Fermentation of Candida albicans CAes2121 in a 10L fermenter to produce DC10

[0074] The fermentation method is the same as in Example 6, except that: dissolved oxygen is controlled above 10% during fermentation, and the concentration of live cells in the fermentation broth is maintained at 2*10⁻⁶ by controlling the alkane flow rate. 9 cfu / mL ~5*10 9 When the concentration of CFU / mL in the fermentation broth was 0, fermentation was stopped. The results of the fermentation broth analysis are shown in Table 2 below.

[0075] Table 2

[0076]

[0077] As can be seen from Table 2, when using a fermenter for fermentation, the strain of this invention, compared with the starting strain, yields a higher DC10 yield, a faster acid production rate, and saves more energy under the same conditions.

[0078] Example 11 Preparation of decacarbonylic acid products

[0079] The fermentation broth obtained in Example 6 was acidified by adjusting the pH value to 3.2 with sulfuric acid and filtered to obtain solids. The solids were then dissolved in acetic acid, and activated carbon was added at no more than 5% of the volume of the clear liquid. The mixture was decolorized at 85°C for 70 min, filtered to obtain clear liquid, cooled to 65°C and kept at that temperature for 1 h, then cooled to 35°C to crystallize. The high-purity decacarbonyl dicarboxylic acid product was obtained by centrifugation.

[0080] As can be seen from the comparative examples above, the *Candida viswanathii* CAes2121 used in this invention has a better ability to produce decacarbonyl dicarboxylic acid than its parent *Candida viswanathii* CAes2121. In Example 6, the decacarbonyl dicarboxylic acid content reached 185 g / L, and its acid production rate reached 1.23 g / h·L. The method of this invention achieves better results in ensuring fermentation efficiency and acid production by controlling fermentation process parameters such as the concentration of cells in the fermentation broth, the concentration of the fermentation substrate, pressure, and air flow rate. The *Candida viswanathii* CAes2121 of this invention can be used for the industrial production of decacarbonyl dicarboxylic acid using biological methods.

[0081] 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 strain that produces high levels of decacarbonylic acid, characterized in that, The strain described is *Candida viswanathii* CAes2121, with accession number CCTCC M 2021824, and the depositary institution is the China Center for Type Culture Collection.

2. An inoculum containing the Candida albicans strain CAes2121 as described in claim 1.

3. The application of Candida virescens as described in claim 1 in the fermentation production of decacarboxylic acid.

4. The application as described in claim 3, characterized in that, The substrates used in the fermentation process include decane, deca-carboxylic acids, deca-carboxylic acid derivatives, or mixtures thereof.

5. A method for producing long-chain decacarbondicarboxylic acid by biological means, the method comprising the following steps: a) Cultivating and fermenting the seed culture of Candida viswanathii CAes2121 as described in claim 1; b) Extract decacarbonyl dicarboxylic acid from the fermentation product obtained in step a).

6. The method as described in claim 5, characterized in that, The temperature during seed culture and fermentation culture is 28℃~33℃.

7. The method as described in claim 5, characterized in that, The seed culture and fermentation culture are conducted using a seed culture medium and a fermentation culture medium that include a carbon source, a nitrogen source, inorganic salts and / or nutrient factors, and the temperature during the seed culture and fermentation culture is 29℃~30℃.

8. The method as described in claim 7, characterized in that, The carbon source includes one or more of sucrose, glucose, maltose, molasses, fructose, rhamnose, arabinose, and sorbitol, and the amount of the carbon source added is 1% to 10% (w / v); and / or, The nitrogen source includes one or more of yeast extract, corn steep liquor, urea, ammonia, ammonium sulfate, potassium nitrate, and ammonium nitrate, and the amount of nitrogen source added is 0.1%~3% (w / v); and / or, The inorganic salt includes one or more of potassium and sodium salts; the amount of the inorganic salt added is 0.1% to 1.5% (w / v); and / or, The nutritional factors include one or more of vitamin B1, vitamin B2, vitamin C, biotin, and amino acids, and the amount of the nutritional factors added is 0~1% (w / v).

9. The method as described in claim 5, characterized in that, During the fermentation process, the pH of the fermentation system is controlled to be either below 7.0 or above 7.

0.

10. The method as described in claim 5, characterized in that, During the fermentation culture, the substrate concentration is controlled at 0.5% to 3%.

11. The method as described in claim 5, characterized in that, During the fermentation culture, the concentration of live cells in the fermentation broth is controlled at 8*10. 7 cfu / mL ~1.5*10 9 cfu / mL.

12. The method as described in claim 5, characterized in that, During the fermentation culture, the concentration of live cells in the fermentation broth is controlled at 2*10⁻⁶. 8 cfu / mL ~9*10 8 cfu / mL.

13. The method as described in claim 7, characterized in that, The potassium salt includes one or more of potassium chloride, potassium nitrate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate, and the sodium salt includes one or more of sodium chloride, sodium nitrate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

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