CN111349664B - Method for producing long-chain dicarboxylic acid by using saturated fatty acid as substrate - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention relates to a method for producing long-chain dibasic acid by using saturated fatty acid as a substrate, which solves the technical problem that the fermentation process cannot be normally carried out due to serious bubbles in the existing production method of the long-chain dibasic acid, and comprises the following steps: (1) taking a glycerol freezing tube of the strain, inoculating the glycerol freezing tube into a shake flask filled with an YPD culture medium, and preparing a seed solution; inoculating the strain into a shake flask filled with a primary seed culture medium to prepare a primary seed solution; (2) inoculating the prepared first-stage seed liquid into a shake flask filled with a second-stage seed culture medium to prepare a second-stage seed liquid for later use; (3) adjusting pH, stirring speed, temperature and ventilation of the fermentation liquid, inoculating the obtained secondary seed liquid into a fermentation tank containing fermentation medium, supplementing glucose, adjusting pH of the fermentation liquid with sodium hydroxide, and adding laurinol solution of lauric acid to obtain long-chain dicarboxylic acid. The method can be widely applied to the field of preparation of long-chain dibasic acid.
Description
Technical Field
The invention relates to the field of biochemical engineering, in particular to a method for producing long-chain dibasic acid by using saturated fatty acid as a substrate.
Background
At present, the technical patent for producing long-chain dibasic acid at home and abroad mainly takes alkane and fatty acid derivatives as substrates to produce the long-chain dibasic acid by fermentation, and the alkane is obtained from petroleum, belongs to non-renewable resources and has larger price fluctuation; fatty acid derivatives, although renewable resources, are expensive. The long-chain saturated fatty acid is the best raw material for producing corresponding long-chain saturated dicarboxylic acid at present, not only has the characteristic of green and renewable, but also shortens the fermentation conversion time because one end of the long-chain saturated fatty acid is oxidized, and is the cheapest raw material. However, the current method of directly using fatty acid as raw material to convert into long-chain dibasic acid is not really used for production, on one hand, long-chain saturated fatty acid is solid and cannot be fed-batch, and on the other hand, the more important reason is that the fermentation cannot be completed because of serious foaming.
Chinese patent application No. 201110168672.X discloses a method for converting alkanes, fatty acids and fatty acid derivatives into corresponding dibasic acids, but the test is in a shake flask stage, and in the examples, no mention is made of a method for converting fatty acids into long-chain dibasic acids.
Chinese patent application No. 200410018255.7, discloses a method for converting oleic acid to DC 18. However, oleic acid is unsaturated fatty acid and is liquid at normal temperature, so that the problem of material supplement does not exist, and the product is unsaturated long-chain dibasic acid.
Application No. 201510176594.6 discloses a method for producing long-chain dibasic acid by fermentation using fatty acid derivatives as raw materials. The fatty acid derivative as the raw material has no problem of serious foaming and unsuccessful fermentation, but has much higher cost.
The melting point of lauric acid is 44 ℃, the melting point of lauryl alcohol is 24 ℃, lauric acid is solid at normal temperature, the lauryl alcohol is easy to crystallize at lower temperature, and the lauric acid and the lauryl alcohol are easy to generate soap-like foam in the process of adding flow, and all known antifoaming agents have no antifoaming effect.
Disclosure of Invention
The invention aims to solve the technical problem that the fermentation process cannot be normally carried out due to serious bubbles in the existing production method of long-chain dibasic acid, and provides the production method of long-chain dibasic acid which can eliminate bubbles in the fermentation process and has high conversion rate.
The strain used in the fermentation method is classified and named as Candida (Candida sp) TDTC018, and is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 10 months and 31 days, wherein the CGMCC is used for short, and the microbial research institute of China academy of sciences, No. 3 of Xilu No.1 of Beijing Korean district, Beijing; the preservation number is as follows: CGMCC No. 16659.
The invention provides a method for producing long-chain dibasic acid by using saturated fatty acid as a substrate, which comprises the following steps: (1) preparing a first-level seed solution: taking a glycerol cryopreservation tube of the cryopreserved strain, inoculating the glycerol cryopreservation tube into a shake flask filled with an YPD culture medium, and performing shake culture for 12-24 hours to prepare a seed solution; inoculating the seed solution into a shake flask filled with a primary seed culture medium, and performing shake culture for 6-20 h to prepare a primary seed solution; (2) preparing a secondary seed solution: inoculating the primary seed solution prepared in the step (1) into a shake flask filled with a secondary seed culture medium, and performing shake culture for 8-24 hours to prepare a secondary seed solution for later use; (3) feeding and fermenting: adjusting the pH value of the fermentation liquor to 4-6, setting the stirring speed, temperature and ventilation quantity required by fermentation, inoculating the secondary seed liquid prepared in the step (2) into a fermentation tank filled with a fermentation culture medium, supplementing glucose, adjusting the pH value of the fermentation liquor by using sodium hydroxide, and then adding a lauryl alcohol solution of lauric acid in a flowing manner until the fermentation is finished to prepare the long-chain dicarboxylic acid.
Preferably, the saturated fatty acid is a straight-chain fatty acid having ten or more carbons, and the long-chain dibasic acid is a dibasic acid having twelve or more carbons.
Preferably, the saturated long-chain fatty acid is lauric acid, and the dibasic acid having twelve or more carbons is lauric acid.
Preferably, the component of the primary culture medium in the step (1) is 5-12 g/LKH2PO44-12 g/L of sucrose, 1-10 g/L of yeast extract, 1-4 g/L of urea and 0-40 ml/L of heavy wax oil; the component of the secondary culture medium in the step (2) is 5-12 g/LKH2PO44-12 g/L of sucrose, 1-10 g/L of yeast extract, 1-4 g/L of urea and 0-50 ml/L of heavy wax oil; the fermentation medium in the step (3) comprises 6-12 g/LKH2PO45-15 g/L sucrose, 1-10 g/L yeast extract, 1-5 g/L urea, 5-50 g/L corn steep liquor, 0-5 g/L NaCl, 4-14 g/L KNO31-5 g/L of Tween 60 and 0.1-5% of defoaming agent.
Preferably, in the step (3), the stirring speed is 300-750 rpm, and the temperature is 29-31 ℃.
Preferably, in the step (3), the pH value is adjusted by using sodium hydroxide, the pH value is adjusted to 6.8-7.2 within 12-18 h, the pH value is adjusted to 7.3-7.6 within 70-95 h, and the pH value is adjusted to 7.6-8.0 within 100-115 h.
Preferably, in the step (3), the lauric acid solution is prepared by dissolving lauric acid in lauric alcohol with a purity of 98% or more, and heating and maintaining the temperature to obtain a transparent solution.
Preferably, the molar ratio of lauryl alcohol to said lauric acid is 1: 1, heating and preserving heat at 50-55 ℃.
Preferably, in the step (3), the feeding fermentation process is completed in a 5L glass fermentation tank, and the feeding speed of the lauric acid lauryl alcohol solution is 2-7 ml/h.
Preferably, in the step (3), the fermentation period of the fed-batch fermentation is 116 h-186 h, and ethanol or n-dodecyl alkane can be fed as an antifoaming agent in the fermentation period.
According to the invention, a lauryl alcohol saturated solution of lauric acid is used as a substrate for fermentation conversion, a new strain Candida sp.CGMCC16659 is adopted to optimize the substrate state, the fed-batch speed and a pH value adjusting mode are controlled, ethanol or n-dodecyl alkane is added as a defoaming agent in the fermentation process, so that the fed-batch of lauric acid and lauryl alcohol is completed, the acid production concentration is higher than 120g/kg when 115 hours exist, and the conversion rate reaches 70%.
Drawings
FIG. 1 shows the feed rate of fed-batch fermentation in example 2 of the present invention.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as described in the claims.
Example 1 fermentation with ethanol as antifoam agent
(1) Preparing a first-level seed solution: inoculating frozen glycerol cryopreservation tube (seed tube) of CGMCC16659 strain into 500ml shake flask containing 50ml YPD culture medium (yeast extract 5g/L, peptone 10g/L, glucose 10g/L), and shake culturing at 30 deg.C and 250rpm for 12 hr to obtain seed solution; inoculating the seed solution into a medium (KH) containing first-class seed culture medium2PO 45 g/L, sucrose 4g/L, yeast extract 1g/L, urea 1g/L, heavy wax oil 20 ml/L; sterilizing at 115 ℃ for 30min), and shake culturing at 30 ℃ and 250rpm for 20h to obtain a first-stage seed solution;
(2) preparing a secondary seed solution: inoculating 10ml of the shaken primary seeds into a medium containing 40ml of secondary seeds (KH)2PO 412 g/L, sucrose 12g/L, yeast extract 10g/L, urea 4g/L, and heavy wax oil 50 ml/L; sterilized at 115 ℃ for 30min), and shake-cultured at 30 ℃ and 250rpm for 16h in a 500ml shake flask; preparing a secondary seed solution for later use;
(3) feeding and fermenting: inoculating 200ml of the second-stage seed liquid into 1.5L fermentation medium (KH)2PO49g/L, 10g/L of cane sugar, 5g/L of yeast extract, 3g/L of urea, 27g/L of corn steep liquor, 3g/L of NaCl, and KNO39g/L, tween 603g/L and 2.5 percent of defoaming agent; 115 ℃ sterilization for 15min), adjusting the pH value of the fermentation liquor to 4.00 before inoculation, adjusting the pH value of the fermentation liquor to 29 ℃, rotating speed to 750rpm, ventilation capacity to 0.4vvm, beginning to supplement glucose after 6h, speed to 2g/h, stopping adding glucose after 14h, then slowly adjusting the pH value of the fermentation liquor to 7.20 by NaOH within 3h, beginning to slowly add lauryl alcohol solution of lauric acid preheated to 50 ℃ when 18h (the molar ratio is 1: 1) the feeding rate was 2ml/L, and the specific fermentation parameters are shown in Table 1.
TABLE 1 fermentation data during fed-batch fermentation in example 1
NaOH needs to be added very slowly in the fermentation process to increase the pH value, if excessive foam occurs in the process, the lauric acid solution is stopped to be fed, and 10-15 ml of 95% ethanol is quickly fed to ensure that the foam is gradually reduced until the fermentation can be continued. The pH value is adjusted to 7.30 within 70h, the pH value is adjusted to 7.60 within 95h, the pH value is adjusted to 8.0 within 115h, the lauric acid lauryl alcohol solution is stopped to be supplemented within 24h before the fermentation is finished, the total fermentation time is 186h, the total mass of the mixed solution is 583g, the acid production concentration is 124g/kg, and the conversion rate is 61.6%.
Example 2 fermentation with dodecyl alkane as antifoam
(1) Preparing a first-level seed solution: inoculating frozen glycerol cryopreservation tube (seed tube) of CGMCC16659 strain into 500ml shake flask containing 50ml YPD culture medium (yeast extract 5g/L, peptone 10g/L, glucose 10g/L), and shake culturing at 30 deg.C and 250rpm for 12 hr to obtain seed solution; inoculating seed liquid into first-class seed culture medium (KH)2PO49g/L, 8g/L of sucrose, 5g/L of yeast extract, 2g/L of urea and 20ml/L of heavy wax oil; sterilizing at 115 ℃ for 30min), and shake culturing at 30 ℃ and 250rpm for 20h to obtain a first-stage seed solution;
(2) preparing a secondary seed solution: inoculating 10ml of the shaken first-class seeds into a medium containing 40ml of second-class seeds (KH)2PO45g/L, 4g/L of sucrose, 1g/L of yeast, 1g/L of urine and 50ml/L of heavy wax oil; sterilizing at 115 ℃ for 30min), and performing shake culture at 30 ℃ and 250rpm for 8-24 h to prepare a secondary seed solution for later use;
(3) feeding and fermenting: inoculating 200ml of the second-stage seed liquid into 1.5L fermentation medium (KH)2PO412g/L, 15g/L of cane sugar, 10g/L of yeast extract, 5g/L of urea, 50g/L of corn steep liquor, 5g/L of NaCl, and KNO314g/L, tween 605g/L and 5% of defoaming agent; 115 ℃ sterilization for 15min), adjusting the pH value of the fermentation liquor to 6.00 before inoculation, adding glucose at 31 ℃, 300rpm, the ventilation rate of 0.4vvm, beginning to supplement glucose after 6h, the speed of 2g/h, stopping adding glucose after 14h, then slowly adjusting the pH value of the fermentation liquor to 7.20 by NaOH within 3h, and slowly adding lauryl alcohol solution of lauric acid preheated to 55 ℃ when 18h (the molar ratio is 1: 1) the feed rate was 7ml/L, as shown in FIG. 1.
NaOH is added very slowly in the fermentation process to increase the pH, during the process, if excessive foam is generated, the lauric acid solution is stopped to be fed, the ventilation volume is reduced to 0.3vvm, and sterile n-dodecane is slowly fed to gradually reduce the foam until the fermentation can be continued. The pH value is adjusted to 7.40 within 80 hours and 7.60 within 100 hours, the material feeding is stopped in about 115 hours because of large foam, the aeration is reduced until the conversion of the bottom materials is completed after 178 hours, the total mass of the mixed solution is 569g, the acid production concentration is 121g/kg, and the conversion rate is 60.3%.
Claims (7)
1. A method for producing long-chain dibasic acid by using saturated fatty acid as a substrate is characterized by comprising the following steps: (1) preparing a first-level seed solution: taking a glycerol cryopreservation tube of the cryopreserved strain, inoculating the glycerol cryopreservation tube into a shake flask filled with an YPD culture medium, and performing shake culture for 12-24 hours to prepare a seed solution; inoculating the seed solution into a shake flask filled with a primary seed culture medium, and performing shake culture for 6-20 h to prepare a primary seed solution, wherein the strain is Candida (Candida sp) TDTC018 with the preservation number of CGMCC 16659; (2) preparing a secondary seed solution: inoculating the primary seed liquid prepared in the step (1) into a shake flask filled with a secondary seed culture medium, and performing shake culture for 8-24 h to prepare a secondary seed liquid for later use; (3) feeding and fermenting: adjusting the pH value of fermentation liquor to 4-6, setting the stirring speed and temperature required by fermentation, inoculating the secondary seed liquid prepared in the step (2) into a fermentation tank filled with a fermentation culture medium, supplementing glucose, adjusting the pH value of the fermentation liquor by using sodium hydroxide, and then adding a lauryl alcohol solution of lauric acid in a flowing manner until the fermentation is finished to prepare long-chain dibasic acid; the lauryl alcohol solution of the lauric acid is a solution of the lauric acid dissolved in the lauryl alcohol; the molar ratio of the lauryl alcohol to the lauric acid is 1: 1, the heating and heat preservation temperature is 50-55 ℃; and ethanol or n-dodecyl alkane is added as a defoaming agent in the fermentation period, the saturated fatty acid is lauric acid, and the long-chain dibasic acid is lauric acid.
2. The method for producing long-chain dicarboxylic acid by using saturated fatty acid as a substrate according to claim 1, wherein the primary medium in step (1) comprises 5-12 g/LKH2PO44-12 g/L of sucrose, 1-10 g/L of yeast extract, 1-4 g/L of urea and 0-40 ml/L of heavy wax oil; the secondary culture medium in the step (2) comprises 5-12 g/LKH2PO44-12 g/L of sucrose, 1-10 g/L of yeast extract, 1-4 g/L of urea and 0-50 ml/L of heavy wax oil; the fermentation medium in the step (3) comprises 6-12 g/LKH2PO4，5-15 g/L sucrose, 1-10 g/L yeast extract, 1-5 g/L urea, 5-50 g/L corn steep liquor, 0-5 g/L NaCl, 4-14 g/L KNO31-5 g/L of Tween 60 and 0.1-5% of defoaming agent.
3. The method for producing the long-chain dicarboxylic acid by using the saturated fatty acid as the substrate according to claim 1, wherein the stirring speed in the step (3) is 300-750 rpm, and the temperature is 29-31 ℃.
4. The method for producing the long-chain dicarboxylic acid by using the saturated fatty acid as the substrate according to claim 1, wherein in the step (3), the pH value is adjusted by using sodium hydroxide, the pH value is adjusted to 6.8-7.2 within 12-18 h, the pH value is adjusted to 7.3-7.6 within 70-95 h, and the pH value is adjusted to 7.6-8.0 within 100-120 h.
5. The method for producing long-chain dibasic acid using saturated fatty acid as substrate according to claim 1, wherein in the step (3), the lauric acid solution is prepared by dissolving lauric acid in lauric alcohol with a purity of 98% or more, and heating and maintaining the temperature to obtain a transparent solution.
6. The method for producing long-chain dicarboxylic acid by using saturated fatty acid as a substrate according to claim 1, wherein the feeding fermentation process in step (3) is performed in a 5L glass fermentation tank, and the feeding rate of the lauric acid in lauryl alcohol solution is 2-7 ml/h.
7. The method for producing long-chain dicarboxylic acid by using saturated fatty acid as a substrate according to claim 1, wherein the fermentation period of the fed-batch fermentation in step (3) is 116-186 h.
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