Method for producing octadecane dibasic acid by fermentation of Candida vesica

Abstract

The present invention belongs to the field of biochemical engineering technology, and specifically relates to a method for producing octadecadicarboxylic acid by fermenting Candida visweiss. The method for producing octadecadicarboxylic acid by fermenting Candida visweiss described in the present invention comprises the following steps: (1) transferring the Candida visweiss seed liquid into a fermentation tank with a prepared culture medium for culturing; (2) adding substrate in stages or continuously during the fermentation process; (3) collecting the fermentation liquid after the fermentation is completed, extracting octadecenoic acid, and then hydrogenating it to obtain octadecadicarboxylic acid; the Candida visweiss has a preservation number of CCTCC NO: M 20222052, which is preserved in the China Center for Type Culture Collection, with the preservation address at Wuhan University, the preservation date being December 23, 2022, and the Latin name is Candida viswanathii The method of the present invention has high conversion efficiency, high product purity and high yield.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for producing octadecandicacid by fermenting Candida visweiss. Background Art

[0002] Long-chain dibasic acids are the main intermediates of polyamide engineering plastics. They can also be used in hot melt adhesives, high-grade lubricants, high-grade rust inhibitors, high-grade powder coatings and other application fields. Long-chain dibasic acids can be used to make engineering plastics with excellent performance, including modified nylon, etc. The diesters synthesized with octanol can be used as high-grade lubricants with high temperature resistance, and can also be used as base oils for low-temperature lubricating esters. In recent years, with the development and growth of synthetic biology, the use of microbial fermentation conversion is a low-cost, simple and easy-to-operate approach to replace conventional chemical synthesis. At present, most of the 10-16 carbon chain dibasic acids on the market are produced by fermentation, but there is a lack of fermentation production of dibasic acids with more than 17 carbons, especially normal alkanes as raw materials. As the carbon chain lengthens, its physical and chemical properties change, and the difficulty of using fermentation strains increases greatly. At the same time, due to the low content of natural resources such as petroleum and coal in nature, the difficulty and cost of purification also increase significantly.

[0003] Chinese invention patent CN101698859B discloses a method for producing n-hexadecane dicarboxylic acid, and Chinese invention patent CN110564784B discloses a method for producing tetradecane dicarboxylic acid by fermentation of Candida visvensis. Both methods use microbial fermentation conversion to produce long-chain dicarboxylic acids, and the substrate raw materials are all corresponding normal alkanes. The strict requirements on the raw materials limit the production of long-chain dicarboxylic acids. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for producing octadecane dibasic acid by fermentation of Candida vesichella, which overcomes the limitation that the substrate raw material must use the corresponding normal alkane when producing long-chain dibasic acid by microbial fermentation conversion method. The substrate source is more extensive, avoiding dependence on a single raw material. The method of the present invention has high conversion efficiency, high product purity and high yield.

[0005] The technical solution of the present invention is as follows:

[0006] The method for producing octadecane dibasic acid by fermentation of Candida visweiss of the present invention comprises the following steps:

[0007] (1) Transferring the Candida vesica seed solution into a fermentation tank with prepared culture medium for culturing;

[0008] (2) Adding substrate in stages or continuously during fermentation;

[0009] (3) After the fermentation is completed, the fermentation liquid is collected, octadecenoic acid is extracted, and then hydrogenated to obtain octadecadienoic acid;

[0010] The Candida vesica has a deposit number of CCTCC NO: M 20222052, and is deposited in the China Center for Type Culture Collection, Wuhan University, on December 23, 2022. Its Latin name is Candida viswanathii .

[0011] The substrate includes one or more of linoleic acid, elaidic acid, oleic acid and their ester derivatives and fatty acid salts.

[0012] The purity of the substrate is ≥90%.

[0013] The first addition time of the staged addition of substrate is 20-30 hours of cultivation in the fermenter, and the subsequent addition is carried out in several times.

[0014] The continuous addition of substrate begins from 20 hours of cultivation in the fermenter, and the mass concentration of the substrate is controlled to be 2-6% during the fermentation process.

[0015] In step (3), the preparation method of octadecenedioic acid is as follows: octadecenoic acid is used as a raw material, a fixed bed equipped with a composite catalyst is used in the device, a hydrogenation reaction is carried out under ultrasonic conditions, the hydrogenation reaction product is subjected to reduced pressure distillation, and a 230-260°C / 20mmHg fraction is collected to obtain octadecenedioic acid.

[0016] The hydrogenation reaction conditions are as follows: ultrasonic energy density is 0.2-0.6kW / L, hydrogenation reaction pressure is 13-16MPa, hydrogenation reaction temperature is 180-240°C, raw material octadecenoic acid space velocity is 0.2-1m³ / (m³ catalyst·h), and hydrogen circulation volume is 2100-11000m³ / (m³ catalyst·h).

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention overcomes the limitation that the substrate raw material must use the corresponding normal alkane when producing long-chain dibasic acid by microbial fermentation conversion method. The substrate source is more extensive, avoiding the dependence on a single raw material; it also avoids the limitation on the production process caused by the physical property that n-octadecane will become solid at a temperature below 28°C, and the raw substrate is liquid at room temperature; the process design of multiple staged substrate addition or flow addition of substrate is adopted during the fermentation process, and the process flexibility is stronger; the hydrogenation process is relatively mature, the conversion efficiency is high, the product purity is high, the yield is high, and the cost is low.

[0019] 2. The fermentation strain of the present invention, Candida visweiss, has a preservation number of CCTCC NO: M 20222052. The yeast strain is inoculated into the culture medium and fermented for 150 hours. The molar conversion rate of the substrate is 60-68%, and the conversion rate of the hydrogenation process is 94-99%. DETAILED DESCRIPTION

[0020] To make the technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified, all raw materials used in the embodiments are commercially available.

[0021] Example 1

[0022] 1. Preparation of Master Seed Slant and Working Seed Slant

[0023] Take 0.5 mL of the frozen tube of Candida vesica CCTCC NO: M 20222052 and inoculate it into a blank YEPD slant, spread it evenly with a sterile bamboo stick, put it into an incubator, control the temperature at 30℃, and culture it for 3 days. After the slant grows well, it becomes the main seed slant; pick a ring of main seeds on the clean bench and inoculate it into a blank YEPD slant, spread it evenly with a sterile bamboo stick, put it into an incubator, control the temperature at 30℃, and culture it for 2 days. After the slant grows well, it becomes the working seed slant.

[0024] The YEPD medium contains 10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, and the rest is water, which is sterilized at 121°C for 30 min before use. Solid YEPD slant medium is prepared by adding 2% agar powder to the above ingredients, and then divided into stoppered glass test tubes. After sterilization at 121°C for 30 min, the stoppered end is placed horizontally at an angle of 15° and cooled and solidified before use.

[0025] 2. Preparation of seeds for malt wort

[0026] On the clean bench, scrape the two working seed slants into a 3L glass shake bottle filled with 400mL sterile malt juice, seal it with 10 layers of sterile gauze, put it into a shaker, control the temperature at 30℃, the shaker speed at 230r / min, and culture for 24h.

[0027] The malt juice is liquid malt juice with a sugar content of 20%, which is sterilized at 121°C for 30 minutes before use.

[0028] 3. Preparation of seeds for primary seed tank

[0029] After the wort seed culture was completed, samples were taken from the clean bench and the OD value was >9 and the pH value was <4.5. When there was no bacteria under the 100x oil immersion microscope and the seeds grew normally, the wort was inoculated with a 0.3m 3 1m of seed culture medium 3 The inoculation process of the first-level seed tank is carried out according to the aseptic operation process. After the inoculation is completed, the culture temperature is controlled at 30°C, the stirring speed is 260r / min, and the ventilation volume is 18m 3 / h, apparent pressure 0.1MPa, and seed culture in the first-level seed tank is completed after 24 hours of cultivation.

[0030] The seed culture medium consists of 15 g / L glucose, 6 g / L KH2PO4, 3 g / L yeast extract, 0.2 g / L corn flour, 3 g / L urea, and the remaining ingredients are water. It is sterilized at 121°C for 30 minutes before use.

[0031] 4. Preparation of seeds for secondary seed tank

[0032] After the first-level seed tank is cultivated, samples are taken to detect OD value>12, pH<3.5, and 100 times oil immersion microscope is used to check for bacteria-free and normal seed growth. 3 5m of seed culture medium 3 The seed transfer process of the secondary seed tank is carried out according to the aseptic operation process. After the transfer is completed, the culture temperature is controlled at 30℃, the stirring speed is 200r / min, and the ventilation volume is 180m 3 / h, apparent pressure 0.1MPa, and seed culture in the secondary seed tank is completed after 12h of culture.

[0033] 5. Fermentation to produce acid

[0034] After the seed culture in the secondary seed tank is completed, samples are taken to detect OD value>12, pH<3.5, and 100 times oil immersion microscope to check for bacteria and normal seed growth. Then, the pressure difference method is used to transfer the seeds into a 13m 3 25m of fermentation medium 3 In the fermentation tank, the transfer process is carried out according to the aseptic operation process. After the transfer is completed, the culture temperature is controlled at 30°C, the stirring speed is 180r / min, and the ventilation volume is 480m 3 / h, apparent pressure 0.1MPa, culture for 12h, when residual sugar <5g / L, start to add glucose, when fermentation to 20h, residual sugar concentration ≤10g / L, add 600L of substrate linoleic acid for the first time, substrate purity ≥90%, enter the conversion stage.

[0035] The components of the fermentation medium are as follows: carbon source: glucose 60 g / L, nitrogen source: urea 12 g / L, inorganic salt: potassium dihydrogen phosphate 10 g / L, trace elements: zinc sulfate, manganese sulfate, copper sulfate mixture 0.008 g / L.

[0036] 6.Conversion process control

[0037] During the fermentation conversion period, the dissolved oxygen was controlled to >20% by controlling the ventilation volume and rotation speed, and the pH of the fermentation liquid was controlled by adding alkali solution. Fermentation liquid samples were taken for testing every 12 hours to ensure that there were no abnormalities in the seed, pH, OD, and residual hydrocarbon values.

[0038] Fermentation liquid pH control: When the seeds are transferred to the fermentation tank, the pH is adjusted to 6.4. As the seeds grow, the pH drops to 4.5 and is kept constant. After the first addition of linoleic acid, the pH is adjusted to 7.2 and kept constant. After fermentation for 60 hours, the pH is adjusted to 7.6 and kept constant.

[0039] After the first addition of substrate, subsequent additions were made at a rate of 400 L linoleic acid every 8 h, with the last addition being 300 L. When the total amount reached 4000 L, the addition was stopped.

[0040] 7. Fermentation broth acid production detection

[0041] The acid production of the fermentation broth was detected according to the determination method disclosed in Chinese patent CN1048754C, that is, the pH of the fermentation broth sample was adjusted to 3 with a hydrochloric acid solution, 100 mL of ether was added to a separatory funnel to extract the dibasic acid in the fermentation broth, and the ether was evaporated and removed in a water bath to obtain a crude dibasic acid powder; the powder was dissolved in ethanol and titrated with a 0.1 mol / L NaOH solution, and the dibasic acid content in the fermentation broth was calculated according to the formula.

[0042] 8. Extraction of octadecenoic acid

[0043] The fermentation liquid was filtered through a ceramic membrane, the obtained clear liquid was decolorized and filtered with activated carbon, and then dilute sulfuric acid was added for acidification and crystallization. The crystallization end point was controlled at 2.8-3.2. After plate and frame filtration, water washing and drying, octadecenoic acid solid was obtained, and the molar conversion rate was 63.2%.

[0044] 9. Hydrogenation reaction

[0045] After preheating octadecenoic acid to 106°C, it is injected into the reaction tower with an air velocity of 0.2m³ / (m³ catalyst·h). Hydrogen is compressed into the reaction tower with a hydrogen circulation compressor, and the hydrogen circulation volume is 11000m³ / (m³ catalyst·h). A fixed bed composite catalyst is used, and the reaction temperature is 180°C at a pressure of 13MPa and an ultrasonic energy density of 0.2kW / L for hydrogenation reaction. After hydrogenation, the reaction product is sent to a separator for preliminary separation of the crude octadecanoic acid product and hydrogen. The separated hydrogen is returned to the reaction tower for recycling, and the crude octadecanoic acid product enters the distillation device for vacuum distillation treatment. The fraction of 230-260°C / 20mmHg is collected, which is the octadecanoic acid product. After testing, the product purity is 99.1% and the yield is 98.3%.

[0046] Example 2

[0047] Seed culture was transferred to a fermenter according to Reference Example 1, and the fermentation substrate was elaidic acid. After 20 hours of fermentation, the substrate was added by flow addition. The substrate content was detected every 2 hours during the flow addition process, and the substrate mass concentration was controlled to be 4%. When the total amount added reached 4000L, the addition was stopped. Extraction and hydrogenation reaction were performed according to Example 1 to obtain an octadecane dibasic acid product. After testing, the molar conversion rate of the extracted substrate was 64.7%, the product purity was 99.5%, and the yield was 98.9%.

[0048] Example 3

[0049] Reference Example 2, except that the fermentation substrate is oleic acid, and after 20 hours of fermentation, the substrate is fed in a manner, the substrate content is detected every 2 hours during the feeding process, the substrate mass concentration is controlled to be 2%, and the addition is stopped when the total amount added reaches 4000 L. After testing, the molar conversion rate of the extracted substrate is 62.8%, the product purity is 99.2%, and the yield is 98.3%.

[0050] Example 4

[0051] Reference Example 2, except that the fermentation substrate is sodium oleate, and after 20 hours of fermentation, the substrate is fed in a manner, the substrate content is detected every 2 hours during the feeding process, the substrate mass concentration is controlled to be 6%, and the addition is stopped when the total amount added reaches 4000 L. After testing, the molar conversion rate of the extracted substrate is 61.9%, the product purity is 99.3%, and the yield is 98.5%.

[0052] Example 5

[0053] Reference Example 1, except that the fermentation substrate is methyl linoleate, and when the substrate is first added and the fermenter is cultured for 30 hours, the hydrogenation reaction conditions are: ultrasonic energy density is 0.6kW / L, hydrogenation reaction pressure is 16MPa, hydrogenation reaction temperature is 240°C, raw material octadecenoic acid space velocity is 1m³ / (m³ catalyst·h), hydrogen circulation volume is 2100m³ / (m³ catalyst·h). After testing, the molar conversion rate of the extracted substrate is 60.7%, the product purity is 98.9%, and the yield is 98.1%.

Claims

1. A method for producing octadecane dibasic acid by fermentation of Candida vesichella, characterized in that: The following steps are involved: (1) Transferring the Candida vesica seed solution into a fermentation tank with prepared culture medium for culturing; (2) Adding substrate in stages or continuously during fermentation; (3) After the fermentation is completed, the fermentation liquid is collected, octadecenoic acid is extracted, and then hydrogenated to obtain octadecadienoic acid; The Candida vesica has a deposit number of CCTCC NO: M 20222052, and is deposited in the China Center for Type Culture Collection, Wuhan University, on December 23, 2022. Its Latin name is Candida viswanathii; The substrate includes one or more of linoleic acid, elaidic acid, oleic acid, sodium oleate and methyl linoleate.

2. The method according to claim 1, characterized in that: The purity of the substrate is ≥90%.

3. The method according to claim 1, characterized in that: The first addition time of the staged addition of substrate is 20-30 hours of fermentation tank cultivation.

4. The method according to claim 1, characterized in that: The continuous addition of substrate begins from 20 hours of cultivation in the fermenter, and the mass concentration of the substrate is controlled to be 2%-6% during the fermentation process.

5. The method according to claim 1, characterized in that: In step (3), the preparation method of octadecenedioic acid is as follows: octadecenoic acid is used as a raw material, a fixed bed equipped with a composite catalyst is used in the device, a hydrogenation reaction is carried out under ultrasonic conditions, the hydrogenation reaction product is subjected to reduced pressure distillation, and a 230-260°C / 20mmHg fraction is collected to obtain octadecenedioic acid.