Fermentation method for producing long carbon chain dibasic acid

Abstract

The present invention belongs to the field of biochemical engineering technology, and specifically relates to a fermentation method for producing long-chain dibasic acids. The method of the present invention comprises the following steps: (1) transferring the seed liquid of Candida visweiss into a fermentation tank with a culture medium prepared for culturing; (2) adding substrate and glycerol during the fermentation tank culturing process; (3) collecting the fermentation liquid after the fermentation is completed, and extracting the long-chain dibasic acids; the deposit number of the Candida visweiss is CCTCC NO: M 20222052. The present invention uses Candida visweiss as the fermentation strain, and adds glycerol during the fermentation process, which can be used as a carbon source to provide energy for yeast cells, and does not affect the expression of enzymes in the long-chain dibasic acid catalytic pathway. At the same time, it also reduces the stress of dibasic acid salts on yeast cells, improves the vitality of yeast cells, effectively improves the conversion rate, increases the acid production, and reduces the degradation of dibasic acids, shortening the fermentation cycle.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a fermentation method for producing long carbon chain dibasic acids. Background Art

[0002] As an important fine chemical raw material, long-chain dibasic acids can be used to synthesize a series of high-value-added special chemicals such as high-performance nylon engineering plastics, special nylon for machinery, fragrances for daily chemical products, advanced paints and coatings, polyamide hot melt adhesives and cold-resistant plasticizers for clothing, resins, high-temperature electrolytes, advanced lubricants, pesticides and pharmaceuticals.

[0003] Long-chain dibasic acids cannot be obtained directly from nature. They can be prepared by vegetable oil cracking or chemical synthesis, but the preparation process is complex, the reaction conditions are harsh, and there are many by-products. At room temperature and pressure, the biological fermentation method uses alkanes with different carbon chain lengths as substrates and generates dibasic acids with different carbon chain lengths through a series of enzyme-catalyzed reactions in cells. Compared with the chemical synthesis method, the biological fermentation method of long-chain dibasic acids has the advantages of strong reaction specificity, mild reaction conditions, and green environmental protection.

[0004] During the fermentation process of long-chain dibasic acids, alkanes serve as both fermentation substrates and carbon sources for yeast. Part of the dibasic acids produced by fermentation are degraded by yeast to provide energy for the life activities of the cells, resulting in a decrease in the production of dibasic acids; part of them exist outside the cells in the form of salts. As the fermentation proceeds, dibasic acid salts gradually accumulate, causing osmotic stress to the yeast, gradually reducing the cell activity of the yeast and eventually causing the yeast to die, resulting in a decrease in the density of yeast in the fermentation tank and a decrease in the fermentation efficiency of dibasic acids.

[0005] In order to improve the conversion efficiency of alkanes, Qiu Yongjun et al. (2004) supplemented the carbon source for Candida by adding glucose or sucrose twice during the fermentation process. However, in the fermentation process of dibasic acids, the α and ω oxidation pathways catalyze the conversion of alkanes to dibasic acids. The enzymes involved in this pathway are induced by alkanes, and sugar substances will inhibit the expression of enzymes in this pathway.

[0006] Chinese invention patent CN103805642B discloses a fermentation method for producing long-chain dibasic acids, and reports that using glycerol as a defoaming agent improves fermentation efficiency. However, through multiple experiments, it was found that glycerol is not stable as a defoaming agent, and even increased foaming occurs. Therefore, the method of producing long-chain dibasic acids by biological fermentation still needs to be continuously improved to improve conversion efficiency. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a fermentation method for producing long-chain dibasic acids, using Candida visweiss as the fermentation strain, and adding glycerol during the fermentation process, which can not only provide energy for yeast cells as a carbon source, but also does not affect the expression of enzymes in the long-chain dibasic acid catalytic pathway, and at the same time reduces the stress of dibasic acid salts on yeast cells, improves the activity of yeast cells, and thus increases the acid production.

[0008] The technical solution of the present invention is as follows:

[0009] The fermentation method for producing long carbon chain dibasic acid of the present invention comprises the following steps:

[0010] (1) Transferring the Candida vesica seed solution into a fermentation tank with prepared culture medium for culturing;

[0011] (2) Substrate and glycerol are added during the fermentation process, and the addition of substrate and glycerol includes the first addition and subsequent addition;

[0012] (3) After fermentation, the fermentation liquid is collected and the long-chain dibasic acid is extracted;

[0013] 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 .

[0014] Preferably, the total volume of glycerol added accounts for 1%-4% of the initial culture medium volume of the fermenter.

[0015] Preferably, the first glycerol addition is 18-25 hours after the start of fermentation tank culture, when the residual sugar concentration is ≤5 g / L; the volume of the first glycerol addition accounts for 20%-30% of the total glycerol addition volume.

[0016] Preferably, the first addition of substrate is 0.5-1 h after the first addition of glycerol, and each subsequent addition of substrate is spaced 6-8 h apart.

[0017] Preferably, the subsequent addition of glycerol is 0.5-1 h before the addition of the substrate, and the glycerol is continued to be added after the last addition of the substrate, with a time interval of 6-8 h until all the glycerol is added.

[0018] Preferably, the substrate includes one or more of n-decane, n-undecane, n-dodecane, n-tridecane, n-hexadecane, and n-octadecane.

[0019] The long carbon chain dibasic acid is of the chemical formula HOOC(CH 2 ) nThe dibasic acid of COOH, wherein n≥8, the long carbon chain dibasic acid includes one or more of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, hexadecanedioic acid, and octadecane dibasic acid.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention uses Candida visnatus as the fermentation strain, and adds glycerol during the fermentation process, which can be used as a carbon source to provide energy for yeast cells, and does not affect the expression of enzymes in the long-chain dibasic acid catalytic pathway, while also reducing the stress of dibasic acid salts on yeast cells, improving the vitality of yeast cells, effectively improving the conversion rate, and increasing the acid production. At the same time, the degradation of dibasic acids is reduced, and the fermentation cycle is shortened.

[0022] 2. Conventional nutritional supplements during the fermentation process, such as yeast powder, corn flour, etc., are organic components with color and complex ingredients, which will cause substantial interference and impact on subsequent extraction and sewage treatment. The method of using glycerol as a nutritional supplement in the present invention can effectively avoid this problem. DETAILED DESCRIPTION

[0023] The present invention is described in detail by the following examples. Unless otherwise specified, all raw materials used in the examples are commercially available.

[0024] Example 1

[0025] The fermentation method for producing long carbon chain dibasic acid of the present invention comprises the following steps:

[0026] 1. Preparation of seed slants

[0027] 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.

[0028] 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.

[0029] 2. Preparation of seeds for malt wort

[0030] On the clean bench, scrape the sludge from the two working seed slopes into a 3L glass shake bottle filled with 400mL of 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.

[0031] The malt juice is liquid malt juice with a sugar content of 15%, which is sterilized at 121°C for 30 minutes before use.

[0032] 3. Preparing seeds for seed tanks

[0033] After the wort seed culture is completed, samples are taken on the clean bench to test the OD 620 When the value is greater than 9, the pH value is less than 4.5, and there is no foreign bacteria under the 100x oil immersion microscope, and the seeds grow normally, the seeds are inoculated with 0.3m 3 0.5m 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.

[0034] After the first-level seed tank is cultivated, samples are taken to test OD 620 When the value is greater than 12, the pH value is less than 3.5, and there is no foreign bacteria under the 100x oil immersion microscope and the seeds grow normally, the pressure difference method is used to transfer them into a 3m 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.

[0035] The components of the seed culture medium are: glucose 15g / L, KH 2 PO 4 6g / L, yeast extract 3g / L, corn flour 0.2g / L, urea 3g / L.

[0036] 4. Fermentation culture

[0037] After the seed culture in the secondary seed tank is completed, samples are taken and the OD 620 When the value is greater than 12, the pH value is less than 3.5, and there is no foreign bacteria under the 100x oil immersion microscope and the seeds grow normally, the pressure difference method is used to transfer them into a 13m 3 25m3 of fermentation medium (containing 200L n-dodecane) 3 After the transfer, the fermentation conditions were controlled as follows: temperature 30°C, stirring speed 180r / min, ventilation volume 480m3 / h, pressure 0.1MPa, pH 6.4, constant control when pH drops to 4.5 as the seeds grow. When the fermentation reaches 10h and the residual sugar is less than 5g / L, glucose is added.

[0038] The fermentation medium components are as follows: carbon source, glucose 60 g / L, added twice in 50% initial carbon source and 50% feeding carbon source; nitrogen source, urea 12 g / L; inorganic salt, potassium dihydrogen phosphate 10 g / L.

[0039] Glycerol was first added when the fermentation reached 25h and the residual sugar concentration was ≤5g / L. 700L of n-dodecane was added 0.5h later, and the pH was adjusted to 7.2 for constant control. 600L of n-dodecane was added 6h later, and 400L of n-dodecane was added every 6h thereafter. The remaining n-dodecane was added for the last time, with a total addition of 4000L. When the fermentation reached 60h, the pH was adjusted to 7.6 for constant control. The addition time of glycerol was 0.5h before each addition of n-alkanes; after the last addition of n-alkanes, glycerol was added every 6h until all the glycerol was added.

[0040] Among them, the addition of glycerol: the total volume of glycerol added accounts for 4% of the initial culture medium volume of the fermentation tank; the volume of the first added glycerol accounts for 20% of the total volume of glycerol added.

[0041] 5. Fermentation broth acid production detection

[0042] 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 hydrochloric acid solution, 100 mL of ether was added to extract the dibasic acid in the fermentation broth with a separatory funnel, and the ether was evaporated and removed with a water bath to obtain a crude dibasic acid powder; the obtained dibasic acid powder was dissolved in ethanol and titrated with 0.1 mol / L NaOH solution, and the dibasic acid content in the fermentation broth was calculated according to the formula, and the final acid production result was converted according to the lower tank volume of 20.5 m³.

[0043] The fermentation cycle was 116h, and the cell density in the lower tank was OD 620 =1.003 (the measurement result of the fermentation broth diluted 30 times), calculated acid production: 159.83 / L.

[0044] Example 2

[0045] The fermentation process was similar to that of Example 1, except that the fermentation substrate was n-decane;

[0046] During the fermentation and cultivation process, glycerol was first added when the fermentation reached 24 hours and the residual sugar concentration was ≤5g / L. 700L of n-alkanes were added 0.5 hours later, and the pH was adjusted to 7.2 for constant control. 600L of n-alkanes were added 7 hours later, and 400L of n-alkanes were added every 7 hours thereafter. The remaining n-alkanes were added for the last time, and the total addition amount was 4000L. When the fermentation reached 60 hours, the pH was adjusted to 7.6 for constant control. The addition time of glycerol was 1 hour before each addition of n-alkanes; after the last addition of n-alkanes, glycerol was added every 7 hours until all the glycerol was added.

[0047] Among them, the addition of glycerol: the total volume of glycerol added accounts for 2.3% of the initial culture medium volume of the fermenter; the volume of the first added glycerol accounts for 25% of the total volume of glycerol added.

[0048] The fermentation cycle was 132h, and the cell density in the lower tank was OD 620 =0.745, calculated acid production: 124.47 g / L.

[0049] Example 3

[0050] The fermentation process was similar to that of Example 1, except that the fermentation substrate was n-undecane;

[0051] During the fermentation process, glycerol was added for the first time when the fermentation reached 22 hours and the residual sugar concentration was ≤5g / L. After 1 hour, 700L of n-alkanes were added, and the pH was adjusted to 7.2 for constant control. After 8 hours, 600L of n-alkanes were added. After that, 400L of n-alkanes were added every 8 hours. The remaining n-alkanes were added for the last time, and the total addition amount was 4000L. When the fermentation reached 60 hours, the pH was adjusted to 7.6 for constant control. The glycerol addition time was 1 hour before each addition of n-alkanes; after the last addition of n-alkanes, glycerol was added every 8 hours until all the glycerol was added.

[0052] Among them, the addition of glycerol: the total volume of glycerol added accounts for 2.88% of the initial culture medium volume of the fermenter; the volume of the first added glycerol accounts for 20% of the total volume of glycerol added.

[0053] The fermentation cycle was 135h, and the cell density in the lower tank was OD 620 =0.755, calculated acid production: 129.64 g / L.

[0054] Example 4

[0055] The fermentation process was similar to that of Example 1, except that the fermentation substrate was n-tridecane;

[0056] During the fermentation and cultivation process, glycerol was added for the first time when the fermentation reached 18h ​​and the residual sugar concentration was ≤5g / L. After 1h, 700L of n-alkanes were added, and the pH was adjusted to 7.2 for constant control. After 8h, 600L of n-alkanes were added, and then 400L of n-alkanes were added every 8h. The remaining n-alkanes were added for the last time, and the total addition amount was 4000L. When the fermentation reached 60h, the pH was adjusted to 7.6 for constant control. The addition time of glycerol was 1h before each addition of n-alkanes; after the last addition of n-alkanes, glycerol was added every 8h until all the glycerol was added.

[0057] Among them, the addition of glycerol: the total volume of glycerol added accounts for 1% of the initial culture medium volume of the fermentation tank; the volume of the first added glycerol accounts for 30% of the total volume of glycerol added.

[0058] The fermentation cycle was 132h, and the cell density in the lower tank was OD 620 =0.845, calculated acid production: 150.50 g / L.

[0059] Example 5

[0060] The fermentation process was similar to that of Example 1, except that the fermentation substrate was n-hexadecane;

[0061] During the fermentation and cultivation process, glycerol was first added when the fermentation reached 20h and the residual sugar concentration was ≤5g / L. 700L of n-alkanes were added 0.5h later, and the pH was adjusted to 7.2 for constant control. 600L of n-alkanes were added 8h later, and 400L of n-alkanes were added every 8h thereafter. The remaining n-alkanes were added for the last time, and the total addition amount was 4000L. When the fermentation reached 60h, the pH was adjusted to 7.6 for constant control. The addition time of glycerol was 0.5h before each addition of n-alkanes; after the last addition of n-alkanes, glycerol was added every 8h until all the glycerol was added.

[0062] Among them, the addition of glycerol: the total volume of glycerol added accounted for 2.69% of the initial culture medium volume of the fermenter; the volume of the first added glycerol accounted for 25% of the total volume of glycerol added.

[0063] The fermentation cycle is 120h, and the cell density in the lower tank is OD 620 =0.953, calculated acid production: 159.37 g / L.

[0064] Example 6

[0065] The fermentation process was similar to that of Example 1, except that the fermentation substrate was n-octadecane;

[0066] During the fermentation process, glycerol was added for the first time when the fermentation reached 25 hours and the residual sugar concentration was ≤5g / L. After 1 hour, 700L of n-alkanes were added, and the pH was adjusted to 7.2 for constant control. After 8 hours, 600L of n-alkanes were added, and then 400L of n-alkanes were added every 8 hours. The remaining n-alkanes were added for the last time, and the total addition amount was 4000L. When the fermentation reached 60 hours, the pH was adjusted to 7.6 for constant control. The addition time of glycerol was 1 hour before each addition of n-alkanes; after the last addition of n-alkanes, glycerol was added every 8 hours until all the glycerol was added.

[0067] Among them, the addition of glycerol: the total volume of glycerol added accounted for 2.69% of the initial culture medium volume of the fermenter; the volume of the first added glycerol accounted for 25% of the total volume of glycerol added.

[0068] The fermentation cycle was 129 hours, and the cell density in the lower tank was OD 620 =0.790, calculated acid production: 163g / L.

[0069] Comparative Example 1

[0070] Reference Example 1, except that no glycerol was added;

[0071] The fermentation cycle is 140h, and the cell density in the lower tank is OD 620 =0.822, calculated acid production: 149.45g / L.

[0072] From the comparison between Example 1 and Comparative Example 1, it can be seen that due to the addition of glycerol, the fermentation period was shortened by 24 hours and the calculated acid production was increased by 6.94%.

[0073] Comparative Example 2

[0074] Reference Example 2, except that no glycerol was added;

[0075] The fermentation cycle is 148h, and the cell density in the lower tank is OD 620 =0.602, calculated acid production: 115.22g / L.

[0076] From the comparison between Example 2 and Comparative Example 2, it can be seen that due to the addition of glycerol, the fermentation period was shortened by 16 h, and the calculated acid production was increased by 8.03%.

[0077] Comparative Example 3

[0078] Reference Example 3, except that no glycerol was added;

[0079] The fermentation cycle was 143h, and the cell density in the lower tank was OD 620 =0.652, calculated acid production: 120.51 g / L.

[0080] From the comparison between Example 3 and Comparative Example 3, it can be seen that due to the addition of glycerol, the fermentation period is shortened by 8 hours and the calculated acid production is increased by 7.58%.

[0081] Comparative Example 4

[0082] Reference Example 4, except that no glycerol was added;

[0083] The fermentation cycle was 151h, and the cell density in the lower tank was OD 620 =0.740, calculated acid production: 139.55g / L.

[0084] From the comparison between Example 4 and Comparative Example 4, it can be seen that due to the addition of glycerol, the fermentation period was shortened by 19 hours and the calculated acid production was increased by 7.85%.

[0085] Comparative Example 5

[0086] Reference Example 5, except that no glycerol was added;

[0087] The fermentation cycle was 139h, and the cell density in the lower tank was OD 620 =0.806, calculated acid production: 146.62g / L.

[0088] From the comparison between Example 5 and Comparative Example 5, it can be seen that due to the addition of glycerol, the fermentation period was shortened by 19 hours and the calculated acid production was increased by 8.7%.

[0089] Comparative Example 6

[0090] Reference Example 6, except that no glycerol was added;

[0091] The fermentation cycle was 139h, and the cell density in the lower tank was OD 620 =0.721, calculated acid production: 143.9 / L.

[0092] From the comparison between Example 6 and Comparative Example 6, it can be seen that due to the addition of glycerol, the fermentation period was shortened by 10 h, and the calculated acid production was increased by 13.27%.

Claims

1. A fermentation method for producing long carbon chain dibasic acids, 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) Substrate and glycerol are added during the fermentation process, and the addition of substrate and glycerol includes the first addition and subsequent addition; (3) After fermentation, the fermentation liquid is collected and the long-chain dibasic acid is extracted; The Candida viswanathii has a deposit number of CCTCC NO: M 20222052, and is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University. The deposit date is December 23, 2022, and the Latin name is Candida viswanathii; The total volume of glycerol added is 1%-4% of the initial culture medium volume of the fermenter; The substrate includes one or more of n-decane, n-undecane, n-dodecane, n-tridecane, n-hexadecane and n-octadecane.

2. The method according to claim 1, characterized in that: The first time to add glycerol is 18-25h after the start of fermentation tank culture, when the residual sugar concentration is ≤5g / L; the volume of the first added glycerol accounts for 20%-30% of the total volume of glycerol added.

3. The method according to claim 2, characterized in that: The first substrate addition time is 0.5-1h after the first addition of glycerol, and the interval between each subsequent substrate addition is 6-8h.

4. The method according to claim 3, characterized in that: The time for subsequent glycerol addition is 0.5-1h before the addition of substrate. After the last substrate addition, glycerol is added again at intervals of 6-8h until all the glycerol is added.