Wickerhamiella jfordiae and application thereof

Abstract

The present application relates to a strain of Candida viswanathii and its application, which is Candida viswanathii (Candida viswanathii) Candida viswanathii ) DLY-230301, which was preserved in the China General Microbiological Culture Collection Center on March 9, 2023, with the preservation number of CGMCC No. 26773. The Candida viswanathii bred in the present application can overcome the saponification problem in the fermentation process when producing long-chain dibasic acid by fermentation with fatty acid and its derivatives as the substrate, maintain the normal fermentation process, and broaden the selection of fermentation substrates.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to a strain of Candida virescens and its applications. Background Technology

[0002] Long-chain dicarboxylic acids (DCn) are aliphatic dicarboxylic acids (DCn) containing more than 10 carbon atoms in their carbon chain. These include both saturated and unsaturated dicarboxylic acids and are a class of fine chemical products with important and wide-ranging industrial applications. They are also crucial raw materials in the chemical industry for synthesizing high-grade fragrances, high-performance nylon engineering plastics, high-grade nylon hot melt adhesives, high-temperature dielectrics, high-grade paints and coatings, high-grade lubricants, cold-resistant plasticizers, resins, pharmaceuticals, and pesticides.

[0003] Fermentation for the production of long-chain dicarboxylic acids utilizes the unique oxidizing capabilities of microorganisms and the action of intracellular enzymes. Under ambient temperature and pressure, α- and ω-oxidation processes are used to oxidize the methyl groups at both ends of long-chain n-alkanes to carboxyl groups, generating various long-chain dicarboxylic acids of corresponding chain lengths. Many types of bacteria, molds, and actinomycetes can utilize petroleum hydrocarbons, among which *Candida* yeasts are high-yield microorganisms for the fermentation of alkane to produce long-chain dicarboxylic acids.

[0004] The core of fermentation production of long-chain dicarboxylic acids lies in the fermentation strain. The selection and breeding of fermentation strains play a crucial role in reducing process costs and improving product quality. Currently, *Candida tropicalis* is the main microorganism used to convert n-alkanes or fatty acids and their derivatives into corresponding long-chain dicarboxylic acids, and significant breakthroughs have been made in the fermentation processes developed around it. Although *Candida tropicalis* has achieved good technical results in the field of long-chain dicarboxylic acid preparation, this strain has certain shortcomings in substrate suitability, fermentation conditions, and conversion efficiency. Strain improvement is generally achieved through various mutagenesis methods and genetic engineering techniques, which can further improve the strain's suitability, stability, stress resistance, and ability to produce long-chain dicarboxylic acids, making it more suitable for industrial applications.

[0005] CN104862347A discloses a method for fermentation separation and coupling to produce long-chain dicarboxylic acids, including (1) fermenting *Candida* yeast. (1) The seed culture prepared by viswanathiiipe-1) is inoculated into a mixture of n-alkanes with pH 5.0-8.5 containing 5-40% (v / v) 10-18 carbon atoms and 95-60% (v / v) a liquid fermentation medium containing sugars as a carbon source; (2) The above mixture is fermented at 24-40℃ and aeration rate of 0.1-3.0 vvm for 42-194h. The centrifugal separation coupling device or plate and frame filter separation coupling device is started. The fermentation is carried out in the form of batch feeding fermentation, semi-continuous fermentation or continuous fermentation. The cells after separation coupling device are recycled back to the fermenter. The clear liquid after separation coupling device enters the extraction stage to prepare long-chain dicarboxylic acids. At the same time, liquid fermentation medium is added to the fermenter to continue the fermentation process; (3) During the above fermentation process, n-alkanes are added continuously or intermittently so that the concentration of n-alkanes in the fermentation liquid is always greater than or equal to 1% (v / v). When used to convert n-dodecane to DC12, the acid production reached 240 g / L, with an acid production rate greater than 1.5 g / h·L. CN110616158A discloses a method for synthesizing long-chain dodecanoic acid using Candida viswanathiiws biosynthesis, comprising the following steps: a seed culture stage, a fermentation culture stage, and a dodecanoic acid separation and recovery stage. The Candida viswanathiiws is 1201, accession number CCTCC No. 2019076. The conversion was carried out at pH 5.5–9.0 and 24–29°C for 48–52 hours, with n-alkane added daily starting from the 24th hour of fermentation to maintain a n-alkane concentration >20% (v / v) in the fermentation broth. In an industrial fermenter, when fermenting nC12 to produce DC12, the acid production reached 142 g / L after 52 hours of culture. At the end of 52 hours of fermentation, the average acid production rate was 2.73 g / h·L, with a conversion rate of 93.2%. DC12 has a purity of over 99%, with a monocarboxylic acid content of less than 0.01%. These strains all produce dicarboxylic acids through fermentation using petroleum-based n-alkanes as substrates. Under the same conditions, they also produce saponification reactions when using fatty acids and their derivatives as substrates.

[0006] CN111748480A discloses a strain of *Candida viswanathii* CAES2113, with accession number CCTCC NO: M 2020048. The fermentation substrate includes any one or a combination of alkanes, straight-chain saturated fatty acids, straight-chain saturated fatty acid esters, and straight-chain saturated fatty acid salts, preferably including C9-C22 n-alkanes. It maintains high activity for extended periods under conditions with pH values ​​below or above 7.0 and exhibits high oxygen utilization, effectively reducing the amount of alkali used during fermentation and the amount of acid used in subsequent long-chain dicarboxylic acid extraction, simplifying the dicarboxylic acid extraction process and significantly reducing the amount of salt produced in the long-chain dicarboxylic acid production process. Although the fermentation substrate of this strain involves alkanes, straight-chain saturated fatty acids, fatty acid esters, and fatty acid salts, only alkanes are actually used. This is mainly because, under the same fermentation conditions, using fatty acids and their derivatives as substrates will result in a saponification reaction.

[0007] CN1292072C discloses a method for converting long-chain dicarboxylic acids using microbial fermentation, with the following pH values: 3.5–6.5 during the early fermentation stage and 7.0–8.5 during the later stages. During fermentation, especially in the acid-producing phase, the pH of the fermentation broth needs to be adjusted to above 7.0. This is primarily because many Candida species exhibit higher enzyme activity and catalytic efficiency in a slightly alkaline environment. An alkaline environment also allows long-chain dicarboxylic acids to exist in the form of dicarboxylate salts, promoting mass transfer during fermentation. Therefore, a large amount of alkali is typically added during fermentation to neutralize the continuously generated long-chain dicarboxylic acids and maintain a slightly alkaline environment, leading to saponification reactions when fatty acids and their derivatives are used as substrates. Furthermore, after fermentation, a large amount of acid is needed to reconstitute the salts of the long-chain dicarboxylic acids in the system, resulting in a large amount of high-concentration salt fermentation broth. The treatment of this high-salt wastewater severely hinders the development of the biological long-chain dicarboxylic acid industry.

[0008] Long-chain dicarboxylic acid fermentation is a typical four-phase system consisting of gas (oxygen), aqueous phase (fermentation broth), oil phase (alkane), and solid phase (cells). Regular pH adjustments, either neutral or alkaline, are required during fermentation. Currently, known long-chain dicarboxylic acid fermentation strains are typically evaluated using alkanes as substrates. However, due to differences in molecular characteristics and metabolic pathways between fatty acids / fatty acid esters and alkanes, saponification reactions will occur under the same fermentation conditions when fatty acids / fatty acid esters are used as substrates. As this reaction progresses, it not only affects the fermentation efficiency but can even lead to fermentation failure. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a strain of Candida virescens and its applications. The Candida virescens strain selected in this invention can overcome the saponification problem that occurs during fermentation when using fatty acids and their derivatives as substrates to produce long-chain dicarboxylic acids, maintaining a normal fermentation process and broadening the selection of fermentation substrates.

[0010] The first aspect of this invention provides a strain of Candida wanathii, namely Candida wanathii DLY-230301, which was deposited on March 9, 2023 at the China General Microbiological Culture Collection Center, with the accession number CGMCC No. 26773.

[0011] The main morphological characteristics of Candida viswanathii DLY-230301 provided by this invention are: white colony color, wrinkled colony surface, irregular edges, and oval individual strain under a microscope.

[0012] A second aspect of this invention provides the application of *Candida viswanathii* DLY-230301 in the production of long-chain dicarboxylic acids using at least one of fatty acids and their derivatives as fermentation substrates. Compared to the starting strain, this strain overcomes the saponification problem during fermentation while increasing the conversion efficiency by more than two times.

[0013] In the above applications, the fatty acids and their derivatives are at least one of C10-C18 fatty acids, fatty acid esters and fatty acid salts, preferably at least one of C10-C18 straight-chain saturated fatty acids and straight-chain saturated fatty acid esters.

[0014] A third aspect of this invention provides the application of *Candida viswanathii* DLY-230301 in the production of long-chain dicarboxylic acids using alkanes as fermentation substrates. Compared to the starting strain, the concentration of long-chain dicarboxylic acids in the fermentation broth is increased by more than four times.

[0015] In the above applications, the alkane is a C10 to C18 alkane, preferably a C12 to C16 n-alkane.

[0016] In the application of this invention, the general chemical formula of the long-chain dicarboxylic acid is HOOC(CH2)nCOOH, where 10≤n≤18.

[0017] In the application of this invention, the fermentation process is specifically as follows: Candida viswanathii DLY-230301 is used as the fermentation strain, which is inoculated into a seed culture medium to obtain a seed liquid, which is then transferred to a fermentation culture medium for fermentation. Finally, the obtained fermentation liquid is extracted and purified to obtain a long-chain dicarboxylic acid product.

[0018] In this invention, the seed culture medium formula is as follows: carbon source 10–50 g / L, nitrogen source 0.5–5 g / L, inorganic salts 0.5–10 g / L, vitamin B1 0.01–0.1 g / L, and fermentation substrate 2%–10%. The carbon source is at least one of glucose, galactose, sucrose, maltose, trehalose, and mesotriose; the nitrogen source is at least one of yeast extract, corn steep liquor, urea, ammonium sulfate, and ammonia; and the inorganic salts are one or more of sodium chloride, potassium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, and ferric sulfate. Further optimization yielded the following seed culture medium formula: sucrose 10–50 g / L, corn steep liquor 0.5–5 g / L, yeast extract 0.5–5 g / L, urea 0.5–5 g / L, sodium chloride 0.5–10 g / L, potassium dihydrogen phosphate 0.5–10 g / L, magnesium sulfate 0.5–10 g / L, vitamin B1 0.03–0.1 g / L, and fermentation substrate 2%–5%.

[0019] In this invention, the seed culture preparation method is as follows: Candida viswanathii DLY-230301 is inoculated into the seed culture medium and cultured at a temperature of 25–40°C, preferably 28–35°C, and a rotation speed of 200–400 rpm until OD200 is reached. 620 It reaches 6.0 or above.

[0020] In this invention, the fermentation medium is formulated as follows: carbon source 10–50 g / L, nitrogen source 0.5–10 g / L, inorganic salts 0.5–10 g / L, vitamin B1 0.01–0.1 g / L, and fermentation substrate 10%–40%. The carbon source is at least one of glucose, galactose, sucrose, maltose, trehalose, and mesotriose; the nitrogen source is at least one of yeast extract, corn steep liquor, urea, ammonium sulfate, and ammonia; and the inorganic salts are one or more of sodium chloride, potassium chloride, sodium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, and ferric sulfate. Further optimization yielded the following fermentation medium formulation: sucrose 10–50 g / L, corn steep liquor 0.5–10 g / L, yeast extract 0.5–10 g / L, urea 0.5–10 g / L, ammonium sulfate 0.5–10 g / L, sodium acetate 0.5–10 g / L, sodium chloride 0.5–10 g / L, potassium dihydrogen phosphate 0.5–10 g / L, magnesium sulfate 0.5–10 g / L, vitamin B1 0.03–0.1 g / L, and fermentation substrate 10%–30%.

[0021] In the application of this invention, the fermentation substrate in the seed culture medium and fermentation culture medium is one or more of C10-C18 fatty acids, fatty acid esters, fatty acid salts, etc., more preferably one or more of C10-C18 straight-chain saturated fatty acids and straight-chain saturated fatty acid esters.

[0022] In this invention, the inoculation amount of the fermentation bacteria seed liquid is 5% to 20% of the fermentation medium volume. The fermentation conditions are: temperature 25 to 40°C, preferably 28 to 35°C; rotation speed 100 to 600 rpm, preferably 300 to 600 rpm; air flux 0.3 to 1.5 vvm, preferably 0.5 to 1.2 vvm; and fermentation time 72 to 144 hours.

[0023] In this invention, an alkaline pH adjuster is used to control the pH of the fermentation system to 7.2-7.5. The alkaline pH adjuster is at least one of sodium hydroxide, potassium hydroxide, ammonia, etc., with a mass concentration of 5%-40%. More preferably, the pH is controlled in stages during fermentation. Specifically, from 0 to 24 hours, the pH is controlled to be 4.0-7.0; after 24 hours, the pH is controlled to be 7.0-7.5. That is, starting from the 24th hour, the pH is gradually increased according to the fermentation progress, with the final fermentation pH not exceeding 7.5.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) This invention uses wild Candida viswanathii CICC No. 33310 as the starting strain and obtains the target strain Candida viswanathii DLY-230301 through ultraviolet mutagenesis and single carbon source directional screening. This strain can overcome the saponification problem during fermentation and maintain a normal fermentation process when using fatty acids and their derivatives as fermentation substrates to produce long-chain dicarboxylic acids, thus broadening the selection of fermentation substrates. Compared with the starting strain, the conversion efficiency is increased by more than 2 times.

[0026] (2) The *Candida viswanathii* DLY-230301 strain selected in this invention also exhibits excellent fermentation performance when using alkanes as substrates to produce long-chain dicarboxylic acids. The concentration of long-chain dicarboxylic acids in the terminated fermentation broth can reach over 150 g / L. Compared with the starting strain, the concentration of long-chain dicarboxylic acids in the fermentation broth is increased by more than 4 times.

[0027] Instructions for the Preservation of Biological Materials

[0028] The *Candida viswanathii* DLY-230301 provided by this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 26773; deposit date: March 9, 2023; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0029] The strains of the present invention and their applications are further described in detail below through examples. These examples are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of the present invention is not limited to the following examples. In the present invention, % of the fermentation substrate refers to a mass percentage.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0031] In this embodiment of the invention, the concentration of long-chain dicarboxylic acids in the solution was determined by titration with a sodium hydroxide standard solution, and the purity of the long-chain dicarboxylic acids was analyzed by gas chromatography. The gas chromatography column stationary phase was bonded cross-linked polyethylene glycol-20M; the column temperature was initially 150℃, held for 2 min, then increased to 200℃ at a rate of 6℃ / min, and held for 30 min; the carrier gas N2 flow rate was 1.4 mL / min; the hydrogen flow rate was 40 mL / min; the air flow rate was 400 mL / min; and the injection volume was 1 μL.

[0032] Example 1

[0033] Starting with wild-type *Candida virescens* CICC No. 33310, the target strain *Candida virescens* DLY-230301 was obtained through ultraviolet mutagenesis, single-carbon source directional screening, and chlorpromazine hydrochloride screening. The specific breeding process is as follows:

[0034] (1) The starting strain, *Candida virescens* CICC No. 33310, was cultured in seed culture medium to the logarithmic growth phase. The cells were collected by centrifugation, washed twice with physiological saline, dispersed by glass beads, and filtered through a sterile filter membrane to obtain a single-cell suspension. 5 mL of the single-cell suspension was placed in a petri dish and irradiated for 30 seconds at a distance of 15 cm from a 30 W UV lamp. The suspension was then added to seed culture medium containing 5% methyl lauryl and cultured in the dark for 48 hours. The suspension was then diluted 10... -5 The mixture was spread onto YPD agar plates and cultured in the dark for 48 hours to obtain the mutant strain.

[0035] (2) The mutant strain was spread on YPD plate medium containing 10 mg / L chlorpromazine hydrochloride and cultured for 48 h. Three strains were obtained on the medium. The fermentation performance of these strains was compared and the target strain was finally screened out as Candida viswanathii DLY-230301.

[0036] The YPD plate culture medium formula is as follows: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, and agar powder 20g / L.

[0037] Example 2

[0038] The seed culture medium formula is as follows: sucrose 30 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; methyl laurate 2%.

[0039] The fermentation medium formula is as follows: sucrose 20 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; ammonium sulfate 1.0 g / L; sodium acetate 2.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; methyl laurate 25%.

[0040] The preserved *Candida viswanathii* strain DLY-230301 was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker at 30°C and 200 rpm. The pH was not controlled during the culture. The culture was continued until the OD value was reached.620 The pH was 7.6, yielding the seed culture. The seed culture was inoculated into a 5L fermenter containing fermentation medium at an inoculation rate of 10%. The temperature was controlled at 32℃, the rotation speed at 500 rpm, and the air flow rate at 1.0 vvm. The pH of the fermentation system was adjusted to 7.2-7.5 using a 30% sodium hydroxide solution.

[0041] Fermentation was completed after 144 hours. Samples were taken for analysis, and the concentration of long-chain dicarboxylic acids in the fermentation broth was 152.8 g / L, with a purity of 98.5% for monocarboxylic acids. Simultaneously, a parallel experiment was conducted using *Candida viride* CICC No. 33310 as the fermentation starter. After 144 hours of fermentation, the concentration of long-chain dicarboxylic acids in the fermentation broth was 50.6 g / L, with a purity of 98.4% for monocarboxylic acids.

[0042] Example 3

[0043] The seed culture medium formula is as follows: sucrose 30 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; methyl palmitate 2%.

[0044] The fermentation medium formula is as follows: sucrose 20 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; ammonium sulfate 1.0 g / L; sodium acetate 2.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; methyl palmitate 10%.

[0045] The preserved *Candida viswanathii* strain DLY-230301 was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker at 30°C and 200 rpm. The pH was not controlled during the culture process. The culture was continued until the OD value was reached. 620 The pH was 7.7, yielding the seed culture. The seed culture was inoculated into a 5L fermenter containing fermentation medium at an inoculation rate of 10%. The temperature was controlled at 32℃, the rotation speed at 500 rpm, and the air flow rate at 1.0 vvm. The pH of the fermentation system was adjusted to 7.2-7.5 using a 30% sodium hydroxide solution.

[0046] After 96 hours of fermentation, samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 30.3 g / L, and the purity of monocarboxylic acids was 98.6%. Parallel experiments were conducted using *Candida viride* CICC No. 33310 as the fermentation starter. During this parallel experiment, significant saponification occurred, leading to the termination of the fermentation process.

[0047] Example 4

[0048] The seed culture medium formula is as follows: sucrose 30 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; decanoic acid 2%.

[0049] The fermentation medium formula is as follows: sucrose 20 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; ammonium sulfate 1.0 g / L; sodium acetate 2.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; decanoic acid 15%.

[0050] The preserved *Candida viswanathii* strain DLY-230301 was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker at 30°C and 200 rpm. The pH was not controlled during the culture process. The culture was continued until the OD value was reached. 620 The pH was set to 7.5 to obtain the seed culture. The seed culture was inoculated into a 5L fermenter containing fermentation medium at an inoculation rate of 10%. The temperature was controlled at 32℃, the rotation speed at 500rpm, and the air flow rate at 1.0vvm. The pH of the fermentation system was adjusted to 7.2-7.5 using a 30% sodium hydroxide solution.

[0051] After 144 hours of fermentation, samples were taken and analyzed. The concentration of long-chain dicarboxylic acids in the fermentation broth was 59.7 g / L, and the purity of monoacids was 99.1%. Simultaneously, a parallel experiment was conducted using *Candida viride* CICC No. 33310 as the fermentation strain. After 144 hours of fermentation, samples were taken and analyzed. The concentration of long-chain dicarboxylic acids in the fermentation broth was 9.6 g / L, and the purity of monoacids was 99.0%.

[0052] Example 5

[0053] The seed culture medium formula is as follows: sucrose 30 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; methyl stearate 2%.

[0054] The fermentation medium formula is as follows: sucrose 20 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; ammonium sulfate 1.0 g / L; sodium acetate 2.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; methyl stearate 10%.

[0055] The preserved *Candida viswanathii* strain DLY-230301 was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker at 30°C and 200 rpm. The pH was not controlled during the culture process. The culture was continued until the OD value was reached. 620 The pH was 8.0 to obtain the seed culture. The seed culture was inoculated into a 5L fermenter containing fermentation medium at an inoculation rate of 10%. The temperature was controlled at 32℃, the rotation speed at 500rpm, and the air flow rate at 1.0vvm. The pH of the fermentation system was adjusted to 7.2-7.5 using a 30% sodium hydroxide solution.

[0056] After 96 hours of fermentation, samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 23.5 g / L, and the purity of monocarboxylic acids was 98.5%. Parallel experiments were conducted using *Candida viride* CICC No. 33310 as the fermentation starter. Severe saponification occurred during this process, leading to the termination of fermentation.

[0057] Example 6

[0058] The seed culture medium formula is as follows: sucrose 30 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; and 12-carbon n-alkanes 2%.

[0059] The fermentation medium formula is as follows: sucrose 20 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; ammonium sulfate 1.0 g / L; sodium acetate 2.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; and 25% n-dodecane.

[0060] The preserved *Candida viswanathii* strain DLY-230301 was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker at 30°C and 200 rpm. The pH was not controlled during the culture process. The culture was continued until the OD value was reached. 620 The pH was 7.8, yielding the seed culture. The seed culture was inoculated into a 5L fermenter containing fermentation medium at an inoculation rate of 10%. The temperature was controlled at 32℃, the rotation speed at 500 rpm, and the air flow rate at 1.0 vvm. The pH of the fermentation system was adjusted to 7.2-7.5 using a 30% sodium hydroxide solution.

[0061] After 144 hours of fermentation, samples were taken and analyzed. The concentration of long-chain dicarboxylic acids in the fermentation broth was 163.6 g / L, and the purity of monoacids was 98.7%. Simultaneously, a parallel experiment was conducted using *Candida viride* CICC No. 33310 as the fermentation starter. After 144 hours of fermentation, samples were taken and analyzed. The concentration of long-chain dicarboxylic acids in the fermentation broth was 42.2 g / L, and the purity of monoacids was 98.6%.

[0062] Example 7

[0063] The seed culture medium formula is as follows: sucrose 30 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; methyl laurate 2%.

[0064] The fermentation medium formula is as follows: sucrose 20 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; ammonium sulfate 1.0 g / L; sodium acetate 2.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; methyl laurate 25%.

[0065] The preserved *Candida viswanathii* strain DLY-230301 was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker at 30°C and 200 rpm. The pH was not controlled during the culture process. The culture was continued until the OD value was reached. 620 The seed culture was obtained at a pH of 7.5. The seed culture was inoculated into a 5L fermenter containing fermentation medium at an inoculation rate of 10%. The temperature was controlled at 32℃, the rotation speed at 500 rpm, and the air flux at 1.0 vvm. During fermentation, a 30% sodium hydroxide solution was used for pH gradient control: from 0-24h, the system pH was controlled at 5.0; from 24h onwards, the pH was gradually increased according to the fermentation progress, with the final fermentation pH reaching 7.5 until fermentation was completed.

[0066] After 120 hours of fermentation, samples were taken and analyzed. The concentration of long-chain dicarboxylic acids in the fermentation broth was 157.2 g / L, and the purity of monocarboxylic acids was 98.5%. Simultaneously, a parallel experiment was conducted using *Candida viride* CICC No. 33310 as the fermentation starter. After 120 hours of fermentation, samples were taken and analyzed. The concentration of long-chain dicarboxylic acids in the fermentation broth was 50.2 g / L, and the purity of monocarboxylic acids was 98.7%.

[0067] Comparative Example 1

[0068] Similar to Example 2, except that the fermentation strain used was the Candida tropicalis mutant PF-UV-56, with accession number CGMCC No. 0356. Severe saponification occurred during fermentation, leading to its termination.

[0069] Comparative Example 2

[0070] Similar to Example 6, except that the fermentation strain used was the Candida tropicalis mutant PF-UV-56, with accession number CGMCC No. 0356. After 144 hours of fermentation, samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 91.21 g / L, and the purity of monocarboxylic acids was 18.7%.

Claims

1. A strain of Candida virescens, characterized by: It is Candida virescens ( Candida viswanathii DLY-230301 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 9, 2023, with accession number CGMCC No. 26773.

2. The Candida albicans according to claim 1, characterized in that: The main morphological characteristics are: the colony color is white, the colony surface is wrinkled, the edges are irregular, and the individual strains are oval under a microscope.

3. A Candida viviparous yeast strain according to any one of claims 1-2 ( Candida viswanathii Application of DLY-230301 in the fermentation production of long-chain dicarboxylic acids using at least one of fatty acids and their derivatives as fermentation substrates.

4. The application according to claim 3, characterized in that: The fatty acids and their derivatives are at least one of C10-C18 fatty acids, fatty acid esters and fatty acid salts, preferably at least one of C10-C18 straight-chain saturated fatty acids and straight-chain saturated fatty acid esters.

5. A Candida virescens yeast as described in any one of claims 1-2 ( Candida viswanathii Application of DLY-230301 in the production of long-chain dicarboxylic acids using alkanes as fermentation substrates.

6. The application according to claim 5, characterized in that: The alkane is a C10 to C18 alkane, preferably a C12 to C16 n-alkane.

7. The application according to any one of claims 3-6, characterized in that: The general formula of the long-chain dicarboxylic acid is HOOC(CH2)nCOOH, where 10≤n≤18.

8. The application according to claim 3 or 5, characterized in that: The fermentation process specifically involves: using Candida albicans ( Candida viswanathii DLY-230301 is a fermentation bacterium. It is inoculated into a seed culture medium to obtain a seed liquid, which is then transferred to a fermentation culture medium for fermentation. Finally, the fermentation liquid is extracted and purified to obtain a long-chain dicarboxylic acid product.

9. The application according to claim 8, characterized in that: The seed culture medium formula is as follows: carbon source 10-50 g / L, nitrogen source 0.5-5 g / L, inorganic salt 0.5-10 g / L, vitamin B1 0.01-0.1 g / L, and fermentation substrate 2%-10%; wherein the carbon source is at least one of glucose, galactose, sucrose, maltose, trehalose, and mesotriose; the nitrogen source is at least one of yeast extract, corn steep liquor, urea, ammonium sulfate, and ammonia water; and the inorganic salt is one or more of sodium chloride, potassium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, and ferric sulfate.

10. The application according to claim 9, characterized in that: The seed culture medium formula is as follows: sucrose 10-50 g / L, corn steep liquor 0.5-5 g / L, yeast extract 0.5-5 g / L, urea 0.5-5 g / L, sodium chloride 0.5-10 g / L, potassium dihydrogen phosphate 0.5-10 g / L, magnesium sulfate 0.5-10 g / L, vitamin B1 0.03-0.1 g / L, and fermentation substrate 2%-5%.

11. The application according to claim 8, 9 or 10, characterized in that: The method for preparing the seed culture is as follows: *Candida virescens* (…) Candida viswanathii DLY-230301 was inoculated into seed culture medium and cultured at a temperature of 25–40℃, preferably 28–35℃, and a rotation speed of 200–400 rpm until OD. 620 It reaches 6.0 or above.

12. The application according to claim 8, characterized in that: The fermentation medium is formulated as follows: carbon source 10-50 g / L, nitrogen source 0.5-10 g / L, inorganic salts 0.5-10 g / L, vitamin B1 0.01-0.1 g / L, and fermentation substrate 10%-40%; wherein the carbon source is at least one of glucose, galactose, sucrose, maltose, trehalose, and mesotriose; the nitrogen source is at least one of yeast extract, corn steep liquor, urea, ammonium sulfate, and ammonia water; and the inorganic salts include one or more of sodium chloride, potassium chloride, sodium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, and ferric sulfate.

13. The application according to claim 12, characterized in that: The fermentation medium formula is as follows: sucrose 10-50 g / L, corn steep liquor 0.5-10 g / L, yeast extract 0.5-10 g / L, urea 0.5-10 g / L, ammonium sulfate 0.5-10 g / L, sodium acetate 0.5-10 g / L, sodium chloride 0.5-10 g / L, potassium dihydrogen phosphate 0.5-10 g / L, magnesium sulfate 0.5-10 g / L, vitamin B1 0.03-0.1 g / L, and fermentation substrate 10%-30%.

14. The application according to claim 9, 10, 12 or 13, characterized in that: The fermentation substrate is one or more of C10-C18 fatty acids, fatty acid esters, and fatty acid salts, more preferably one or more of C10-C18 straight-chain saturated fatty acids and straight-chain saturated fatty acid esters.

15. The application according to claim 8, 9, 10, 12 or 13, characterized in that: The inoculation volume of the seed culture is 5% to 20% of the fermentation medium volume.

16. The application according to claim 8, characterized in that: The fermentation conditions are as follows: temperature 25-40℃, preferably 28-35℃; rotation speed 100-600rpm, preferably 300-600rpm; air flux 0.3-1.5vvm, preferably 0.5-1.2vvm; and fermentation time 72-144h.

17. The application according to claim 8 or 16, characterized in that: The pH of the fermentation system is adjusted to 7.2-7.5 using an alkaline pH adjuster; the alkaline pH adjuster is at least one of sodium hydroxide, potassium hydroxide, and ammonia water, with a mass concentration of 5% to 40%.

18. The application according to claim 8, characterized in that: During the fermentation process, the pH was controlled in stages. Specifically, from 0 to 24 hours, the pH of the system was controlled at 4.0 to 7.0; after 24 hours, the pH was controlled at 7.0 to 7.5, and the final fermentation pH did not exceed 7.5.