CN111100884B - Method for preparing long-chain dicarboxylic acid by fermentation - Google Patents
Method for preparing long-chain dicarboxylic acid by fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 93
- 230000004151 fermentation Effects 0.000 title claims abstract description 93
- 238000000034 method Methods 0.000 title claims abstract description 39
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 title claims abstract description 17
- 150000001335 aliphatic alkanes Chemical class 0.000 claims abstract description 83
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 22
- 229930195729 fatty acid Natural products 0.000 claims abstract description 22
- 239000000194 fatty acid Substances 0.000 claims abstract description 22
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- 239000007788 liquid Substances 0.000 claims abstract description 18
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- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 12
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
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- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 7
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- 239000005642 Oleic acid Substances 0.000 claims description 6
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 6
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 6
- 150000003904 phospholipids Chemical class 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 125000004432 carbon atom Chemical group C* 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
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- 241000222178 Candida tropicalis Species 0.000 claims description 4
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 claims description 4
- 241000235648 Pichia Species 0.000 claims description 4
- 235000020778 linoleic acid Nutrition 0.000 claims description 4
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 claims description 4
- 238000009423 ventilation Methods 0.000 claims description 4
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 claims description 3
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 claims description 3
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 claims description 3
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 claims description 3
- 235000020661 alpha-linolenic acid Nutrition 0.000 claims description 3
- FRKBLBQTSTUKOV-UHFFFAOYSA-N diphosphatidyl glycerol Natural products OP(O)(=O)OCC(OP(O)(O)=O)COP(O)(O)=O FRKBLBQTSTUKOV-UHFFFAOYSA-N 0.000 claims description 3
- 229960004488 linolenic acid Drugs 0.000 claims description 3
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- WBHHMMIMDMUBKC-XLNAKTSKSA-N ricinelaidic acid Chemical compound CCCCCC[C@@H](O)C\C=C\CCCCCCCC(O)=O WBHHMMIMDMUBKC-XLNAKTSKSA-N 0.000 claims description 3
- 229960003656 ricinoleic acid Drugs 0.000 claims description 3
- FEUQNCSVHBHROZ-UHFFFAOYSA-N ricinoleic acid Natural products CCCCCCC(O[Si](C)(C)C)CC=CCCCCCCCC(=O)OC FEUQNCSVHBHROZ-UHFFFAOYSA-N 0.000 claims description 3
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- 241000223252 Rhodotorula Species 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 229960002969 oleic acid Drugs 0.000 claims description 2
- 230000001276 controlling effect Effects 0.000 claims 2
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- 238000006243 chemical reaction Methods 0.000 abstract description 19
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 34
- TVIDDXQYHWJXFK-UHFFFAOYSA-N dodecanedioic acid Chemical compound OC(=O)CCCCCCCCCCC(O)=O TVIDDXQYHWJXFK-UHFFFAOYSA-N 0.000 description 24
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- IIYFAKIEWZDVMP-UHFFFAOYSA-N tridecane Chemical compound CCCCCCCCCCCCC IIYFAKIEWZDVMP-UHFFFAOYSA-N 0.000 description 6
- 238000004945 emulsification Methods 0.000 description 4
- 239000003995 emulsifying agent Substances 0.000 description 4
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 4
- 239000003921 oil Substances 0.000 description 4
- HQHCYKULIHKCEB-UHFFFAOYSA-N tetradecanedioic acid Chemical compound OC(=O)CCCCCCCCCCCCC(O)=O HQHCYKULIHKCEB-UHFFFAOYSA-N 0.000 description 4
- 238000005273 aeration Methods 0.000 description 3
- 150000001991 dicarboxylic acids Chemical class 0.000 description 3
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- RSJKGSCJYJTIGS-UHFFFAOYSA-N undecane Chemical compound CCCCCCCCCCC RSJKGSCJYJTIGS-UHFFFAOYSA-N 0.000 description 2
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- 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
- C12P7/6409—Fatty acids
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- C—CHEMISTRY; METALLURGY
- 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
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to a method for preparing long-chain dicarboxylic acid by fermentation, which comprises the following steps of (1) culturing long-chain dicarboxylic acid zymophyte to obtain seed liquid; (2) adding fatty acid and water into alkane, and performing ultrasonic treatment to obtain micelle-like alkane; (3) adding the pretreated alkane and the seed liquid into a fermentation culture medium, and fermenting in a mode of interval pH value regulation until the fermentation is finished. The invention improves the alkane conversion rate and reduces the alkane loss by adding the alkane pretreatment procedure, thereby realizing the one-time addition of the alkane and simplifying the process.
Description
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to a method for preparing long-chain dicarboxylic acid by fermentation.
Background
Long chain dicarboxylic acids (Long chain dicarboxylic acids) refer to aliphatic dicarboxylic acids (DCn for short) with more than 10 carbon atoms in the carbon chain, including saturated and unsaturated dicarboxylic acids, and are fine chemical products with important and wide industrial application. And also important raw materials for synthesizing high-grade spices, high-performance nylon engineering plastics, high-grade nylon hot melt adhesives, high-temperature dielectrics, high-grade paints and coatings, high-grade lubricating oil, cold-resistant plasticizers, resins, medicines, pesticides and the like in the chemical industry.
The fermentation process of producing long chain binary acid is to oxidize the methyl at two ends of long chain n-alkane into carboxyl separately through alpha and omega oxidation at normal temperature and pressure to produce various long chain binary acids in corresponding chain length. There are many kinds of bacteria, molds and actinomycetes capable of utilizing petroleum hydrocarbons, among which yeast of the genus Candida (Candida) is a high-yield microorganism for producing dibasic acids by n-alkane fermentation. The long-chain diacid fermentation is a typical four-phase system of gas phase (oxygen), water phase (fermentation liquor), oil phase (alkane) and solid phase (thallus). The addition amount of the alkane substrate serving as the only carbon source at the later fermentation stage is usually 20-30%, and as the water solubility of the alkane is poor, more alkane in a ventilation fermentation system is lost due to the steam stripping effect, so that the long-chain dibasic acid fermentation mostly adopts batch fermentation, and the alkane is supplemented in the fermentation process. Chinese patent CN102115767A discloses a method for producing undecadicarboxylic acid by synchronously fermenting n-undecane with microorganism, which supplements n-undecane at 60, 90, and 120 hours to make the n-alkane concentration in the fermentation liquor always more than or equal to 5% (V/V).
In order to maintain the homogeneity of the four-phase system and to increase the solubility of alkanes in the fermentation system, it is also a common practice to add emulsifiers to the culture medium. Chinese patent CN102115768A discloses a method for producing hexadecanedioic acid by synchronously fermenting n-hexadecane with microorganisms, wherein 0.1-2 g/L of surfactant is added to enhance the emulsification effect of an oil phase. Emulsifiers are not generally required for microbial metabolism, and thus this way of adding emulsifiers can adversely affect normal microbial fermentation.
At present, in order to improve the dissolving effect of the alkane in the aqueous phase system, related researches are also carried out on the aspect of alkane pretreatment. Chinese patent CN103805643A discloses a method for producing long-chain dibasic acid, adding emulsified alkane in the fermentation process, improving the compatibility between water phase and oil phase in the fermentation system, and increasing the conversion rate of alkane in the fermentation system and the yield of dibasic acid products. The method adopts a microwave emulsification mode to pretreat the alkane, and although the emulsification effect of the alkane can be improved, the microwave radiation harm is large, and the practical application is difficult.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for preparing long-chain dicarboxylic acid by fermentation. The invention improves the alkane conversion rate and reduces the alkane loss by adding the alkane pretreatment procedure, thereby realizing the one-time addition of the alkane and simplifying the process.
The method for preparing the long-chain dicarboxylic acid by fermentation provided by the invention comprises the following steps:
(1) preparing a seed solution: culturing the long-chain dicarboxylic acid zymocyte to obtain seed liquid;
(2) alkane pretreatment: adding fatty acid and water into alkane, and performing ultrasonic treatment to obtain micelle-like alkane;
(3) and (3) fermenting long-chain dicarboxylic acid: adding the pretreated alkane and the seed liquid into a fermentation culture medium, and fermenting in a mode of interval pH value regulation until the fermentation is finished.
In the invention, the long-chain dibasic acid zymocyte is a microorganism with complete alpha and omega-oxidation paths, such as at least one of candida, cryptococcus, endospore, hansenula, pichia, rhodotorula, torulopsis or hyphomycete, and the like, preferably candida tropicalis.
In the invention, the activated zymophyte is inoculated into a culture medium in the step (1), and the inoculation volume ratio is 3-10%, preferably 5-10%; the culture temperature is 25-37 ℃, preferably 28-32 ℃, and the culture time is 15-24 hours. The formula of the culture medium is as follows: 30-45 g/L of sucrose, 1.5-2 g/L of corn steep liquor, 1.5-1.8 g/L of yeast extract, 0.8-1.2 g/L of sodium chloride, 3.5-7.5 g/L of monopotassium phosphate, 1.2-1.8 g/L of magnesium sulfate, 1.2-4.8 g/L of urea, 1.5-2 g/L of ammonium sulfate and 1.5-1.8 g/L of sodium acetate.
In the invention, the alkane in the step (2) is alkane with 10-16 carbon atoms. The dosage of the alkane is 15 to 30 percent of the total volume of the fermentation liquor, preferably 20 to 25 percent.
In the present invention, the fatty acid in the step (2) is a fatty acid having 10 to 18 carbon atoms, and may be at least one of oleic acid, linoleic acid, ricinoleic acid, linolenic acid, and the like, and preferably oleic acid and linoleic acid. The addition amount of the fatty acid is 0.5-2% of the weight of the alkane. Furthermore, a certain amount of phospholipid, such as at least one of phosphatidyl glycerol, diphosphatidyl glycerol and the like, is added at the same time with the addition of the fatty acid, and the addition amount is 1-5 percent, preferably 2-5 percent of the mass of the fatty acid.
In the invention, the addition amount of the water in the step (2) is 1-2.5 times of the weight of the alkane.
In the invention, the ultrasonic treatment in the step (2) has the frequency of 25-40 kHz, the ultrasonic power density of 10-100W/L and the time of 10-30 minutes.
In the invention, the inoculation volume ratio of the fermentation seed liquid in the step (3) is 2-20%, preferably 10-20%. The fermentation temperature is 25-37 ℃, and preferably 28-32 ℃; the stirring speed is 120-500 rpm, preferably 200-400 rpm; the ventilation volume is 0.2-1.0 VVM, preferably 0.5-1.0 VVM; the fermentation time is 138-144 hours.
In the invention, the fermentation is carried out by adopting a mode of interval pH value regulation in the step (3), and the method specifically comprises the following steps: and (3) starting fermentation, controlling the pH value of a fermentation system to be 5-6, and controlling the pH value of the system to be 6.8-8.0 after 24 hours. Preferably, after 24 hours of fermentation, the pH value is increased once every 20-28 hours, and the pH value is increased by 0.1-0.3 every time until the fermentation is finished.
Compared with the prior art, the invention has the following advantages:
(1) according to the invention, through alkane pretreatment, namely adding fatty acid and water for ultrasonic treatment, alkane is favorably absorbed by fermenting microorganisms through pinocytosis, the rejection of the fermenting microorganisms is reduced, the alkane conversion rate and the fermenting effect are improved, the using amount of alkane in the fermentation period is reduced, and one-time addition of alkane substrate is realized on the process, so that the process steps are simplified.
(2) During the fermentation process, the zymophyte can autonomously secrete solubilizer and emulsifier, which generate pseudo-solubilization, so that the alkane exists in a micelle form and is taken up by endocytosis. The invention is easier to mediate endocytosis and improves the utilization rate of alkane through the synergistic action of the exogenous fatty acid and the phospholipid component.
(3) The existing conventional regulation and control mode is to add a certain amount of alkane while adjusting the pH gradient, and the production intensity in the fermentation period is inhibited because the alkane emulsification needs a process. By using alkane pretreatment in combination with pH gradient adjustment, alkanes can be rapidly utilized, thereby increasing production intensity.
(4) The fermentation process of the invention is beneficial to improving the homogeneity of a gas phase-water phase-oil phase-solid phase four-phase system, and on one hand, the loss of alkane along with tail gas under the steam stripping action is reduced; on the other hand, the mass transfer effect of the alkane can be improved, and the fermentation level is improved.
Detailed Description
The method and effects of the present invention are further illustrated by the following examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments.
The experimental procedures in the following examples are, unless otherwise specified, conventional in the art. The test materials used in the following examples were purchased from biochemical reagent stores unless otherwise specified.
In the embodiment of the invention, candida tropicalis (Candida tropicalis) The mutant strain PF-UV-56 is used as a fermentation strain to carry out long-chain dicarboxylic acid fermentation by long alkane, and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC NO. 0356.
In the embodiment of the invention, fermentation is carried out by adopting a mode of interval pH value regulation, and the pH control values within the range of 0-144 h are respectively subjected to interval regulation from the beginning of fermentation.
The calculation formula of the alkane conversion rate T is as follows:
. Wherein M is1、M2、M3Respectively the initial added mass of alkane, the residual mass in the fermentation liquor, the mass of alkane recovered from tail gas, and g.
The calculation formula of the production strength K of the long-chain dibasic acid is as follows:
. Wherein C is the fermentation concentration of the long-chain dicarboxylic acid in the fermentation liquor, g/L; h is total fermentation time H.
Example 1
The formula of the culture medium is as follows: 30g/L of sucrose, 2g/L of corn steep liquor, 1.5g/L of yeast extract, 1.2g/L of sodium chloride, 3.5g/L of monopotassium phosphate, 1.8g/L of magnesium sulfate, 4g/L of urea, 1.5g/L of ammonium sulfate and 1.8g/L of sodium acetate.
(1) Preparing a seed solution: inoculating 50mL of zymocyte liquid into a 3L shake flask containing 450mL of seed culture medium, culturing at 32 ℃ and 120rpm of shaking table for 48 hours to obtain zymocyte seed liquid.
(2) Alkane pretreatment: 750g of dodecane (with the volume of about 1L) is taken, 750g of water and 15g of linoleic acid are added, and ultrasonic treatment is carried out for 10 minutes, wherein the ultrasonic frequency is 40kHz, and the power density is 50W/L.
(3) And (3) fermenting long-chain dicarboxylic acid: adding 500mL of fermentation seed liquid into 2.75L of fermentation culture medium, and then adding 1.75L of dodecane pretreated by the step (2). The fermentation parameters were as follows: the fermentation temperature was 32 ℃, the number of stirring revolutions was 400rpm, and the aeration was set at 0.8 vvm. The pH values of 0-24 h, 24-48 h, 48-72 h, 72-96 h, 96-120 h and 120-144 h are respectively controlled to be 5, 7, 7.2, 7.4, 7.6 and 7.8.
The concentration of the dodecanedioic acid in the final fermentation system is 156.6g/L, the conversion rate of the dodecane is 82.1%, the alkane is recovered from tail gas by 40.2g, and the production intensity is 1.08 g/(L.h).
Example 2
The formula of the culture medium is as follows: 35g/L of sucrose, 1.5g/L of corn steep liquor, 1.8g/L of yeast extract, 0.8g/L of sodium chloride, 7.5g/L of monopotassium phosphate, 1.2/L of magnesium sulfate, 4.8g/L of urea, 2g/L of ammonium sulfate and 1.5g/L of sodium acetate.
(1) Preparing a seed solution: inoculating 50mL of zymocyte liquid into a 3L shake flask containing 950mL of seed culture medium, culturing at 28 ℃ and 120rpm of shaking table for 48 hours to obtain zymocyte seed liquid.
(2) Alkane pretreatment: adding 1875g of tridecane (about 2.5L in volume), adding 3750g of water and 37.5g of oleic acid, and performing ultrasonic treatment for 20 minutes at an ultrasonic frequency of 30kHz and a power density of 20W/L.
(3) And (3) fermenting long-chain dicarboxylic acid: adding 1L of the fermentation seed liquid into 2.75L of the fermentation culture medium, and then adding 6.25L of the dodecane pretreated in the step (2). The fermentation parameters were as follows: the fermentation temperature was 28 ℃, the stirring speed was 300rpm, and the aeration was set at 0.5 vvm; the pH values of 0-24 h, 24-44 h, 44-64 h, 64-84 h, 84-104 h, 104-124 h and 124-144 h are respectively controlled to be 6, 7, 7.2, 7.3, 7.4, 7.6 and 7.9.
The concentration of the tridecane dibasic acid in the final fermentation system is 144.2g/L, the conversion rate of the tridecane is 79.2 percent, 86.5g of alkane is recovered from tail gas, and the production intensity is 1.01 g/(L.h).
Example 3
The formula of the seed culture medium is as follows: 30g/L of sucrose, 2g/L of corn steep liquor, 1.5g/L of yeast extract, 1.2g/L of sodium chloride, 3.5g/L of monopotassium phosphate, 1.8/L of magnesium sulfate and 4g/L of urea.
The formula of the fermentation medium is as follows: 30g/L of sucrose, 2g/L of corn steep liquor, 1.5g/L of yeast extract, 1.2g/L of sodium chloride, 3.5g/L of monopotassium phosphate, 1.8g/L of magnesium sulfate, 4g/L of urea, 1.5g/L of ammonium sulfate and 1.8g/L of sodium acetate.
(1) Preparing a fermentation seed solution: inoculating 50mL of zymocyte liquid into a 3L shake flask containing 450mL of seed culture medium, culturing at 30 ℃ and 120rpm of shaking table for 48 hours to obtain zymocyte seed liquid.
(2) Alkane pretreatment: 750g of dodecane (volume is about 1L) is taken, 1500g of water and 7.5g of oleic acid are added, and ultrasonic treatment is carried out for 20 minutes, wherein the ultrasonic frequency is 25kHz, and the power density is 50W/L.
(3) And (3) fermenting long-chain dicarboxylic acid: adding 500mL of fermentation seed liquid into 2L of fermentation culture medium, and then adding 2.5L of dodecane pretreated by the step (2). The fermentation parameters were as follows: the fermentation temperature was 30 ℃, the stirring speed was 400rpm, and the aeration was set at 0.8 vvm; the pH values of 0-24 h, 24-48 h, 48-72 h, 72-96 h, 96-120 h and 120-144 h are respectively controlled to be 5, 7.0, 7.3, 7.6, 7.7 and 8.0.
The concentration of the dodecanedioic acid in the final fermentation system is 150.2g/L, the conversion rate of the dodecane is 80.1%, 41.5g of alkane is recovered from tail gas, and the production intensity is 1.04 g/(L.h).
Example 4
The difference from example 1 is that: 0.75g of phosphatidylglycerol was added simultaneously with the addition of the fatty acid. The concentration of the dodecanedioic acid in the final fermentation system is 162.6g/L, the conversion rate of the dodecane is 85.5%, the alkane is recovered from tail gas by 30.7g, and the production intensity is 1.13 g/(L.h).
Example 5
The difference from example 3 is that: while adding the fatty acid, 0.75g of diphosphatidyl glycerol was added. The concentration of the dodecanedioic acid in the final fermentation system is 156.2g/L, the conversion rate of the dodecane is 82.2%, the alkane is recovered from tail gas by 36.9g, and the production intensity is 1.08 g/(L.h).
Example 6
The difference from example 1 is that: the fatty acid is ricinoleic acid. The concentration of the dodecanedioic acid in the final fermentation system is 148.2g/L, the conversion rate of the dodecane is 79.6%, 43.5g of alkane is recovered from tail gas, and the production intensity is 1.03 g/(L.h).
Example 7
The difference from example 1 is that: the fatty acid is linolenic acid. The concentration of dodecanedioic acid in the final fermentation system is 153.2g/L, the conversion rate of dodecane is 81.9%, the tail gas is recovered by 42.1g of alkane, and the production intensity is 1.06 g/(L.h).
Example 8
The difference from example 1 is that: tetradecane is used for fermentation to prepare tetradecanedioic acid. The concentration of the tetradecanedioic acid in the final fermentation system is 101.6g/L, the conversion rate of the hexadecane is 50.2%, 113.2g of alkane is recovered from tail gas, and the production intensity is 0.71 g/(L.h).
Comparative example 1
The difference from example 1 is that: and (2) performing alkane pretreatment in an ultrasonic mode without adding linoleic acid. The concentration of dodecanedioic acid in the final fermentation system is 122.3g/L, the conversion rate of dodecane is 65.6%, 178g of alkane is recovered from tail gas, and the production intensity is 0.84 g/(L.h).
Comparative example 2
The difference from example 1 is that: and (3) adding no water in the step (2), and performing alkane pretreatment in an ultrasonic mode. The concentration of the dodecanedioic acid in the final fermentation system is 120.1g/L, the conversion rate of the dodecane is 64.1%, 182g of alkane is recovered from tail gas, and the production intensity is 0.83 g/(L.h).
Comparative example 3
The difference from example 1 is that: and (3) adding linoleic acid, and then simply stirring and mixing, and performing alkane pretreatment in an ultrasonic mode. The concentration of the dodecanedioic acid in the final fermentation system is 120.9g/L, the conversion rate of the dodecane is 63.9%, the alkane is recovered from tail gas by 186g, and the production intensity is 0.83 g/(L.h).
Comparative example 4
The difference from example 1 is that: the fermentation process does not adopt a mode of interval pH value regulation, and the pH value of the whole fermentation process is 7.0. The concentration of the dodecanedioic acid in the final fermentation system is 125.2g/L, the conversion rate of the dodecane is 66.7%, 175g of alkane is recovered from tail gas, and the production intensity is 0.87 g/(L.h).
Comparative example 5
The difference from example 1 is that: the alkane pretreatment process was performed in the manner disclosed in chinese patent CN103805643A, and the treated alkane was added in batches. The concentration of the dodecanedioic acid in the final fermentation system is 136.2g/L, the conversion rate of the dodecane is 75.5%, the tail gas recovers 90g of alkane, and the production intensity is 0.94 g/(L.h).
Comparative example 6
The difference from example 1 is that: the alkane pretreatment process is carried out according to the method disclosed by Chinese patent CN103805643A, and the treated alkane is added at one time in the early stage of fermentation. The concentration of the dodecanedioic acid in the final fermentation system is 123.1g/L, the conversion rate of the dodecane is 64.2%, 169g of alkane is recovered from tail gas, and the production intensity is 0.85 g/(L.h).
Claims (19)
1. A method for preparing long-chain dicarboxylic acid by fermentation is characterized by comprising the following steps:
(1) preparing a seed solution: culturing the long-chain dicarboxylic acid zymocyte to obtain seed liquid;
(2) alkane pretreatment: adding fatty acid and water into alkane, and performing ultrasonic treatment to obtain micelle-like alkane; the fatty acid is a fatty acid with 10-18 carbon atoms, and the alkane is an alkane with 10-16 carbon atoms;
(3) and (3) fermenting long-chain dicarboxylic acid: adding the pretreated alkane and the seed liquid into a fermentation culture medium, and fermenting in a mode of interval pH value regulation until the fermentation is finished; the mode of regulating the pH value in an interval mode is as follows: and (3) starting fermentation, controlling the pH value of a fermentation system to be 5-6, and controlling the pH value of the system to be 6.8-8.0 after 24 hours.
2. The method of claim 1, wherein: the long-chain binary acid zymocyte is at least one of candida, cryptococcus, endospore mold, hansenula, pichia, rhodotorula, torulopsis or hyphomycete with complete alpha and omega-oxidation ways.
3. The method of claim 2, wherein: the long-chain dibasic acid zymophyte is candida tropicalis.
4. The method of claim 1, wherein: inoculating the activated zymophyte into a seed culture medium, wherein the inoculation volume ratio is 3-10%, the culture temperature is 25-37 ℃, and the culture time is 15-24 hours.
5. The method of claim 4, wherein: the inoculation volume ratio is 5-10%, and the culture temperature is 28-32 ℃.
6. The method according to claim 1 or 4, characterized in that: the formula of the seed culture medium for preparing the seed solution in the step (1) is as follows: 30-45 g/L of sucrose, 1.5-2 g/L of corn steep liquor, 1.5-1.8 g/L of yeast extract, 0.8-1.2 g/L of sodium chloride, 3.5-7.5 g/L of monopotassium phosphate, 1.2-1.8 g/L of magnesium sulfate and 1.2-4.8 g/L of urea.
7. The method of claim 1, wherein: the dosage of the alkane in the step (2) is 15 to 30 percent of the total volume of the fermentation liquor.
8. The method of claim 7, wherein: the dosage of the alkane in the step (2) is 20 to 25 percent of the total volume of the fermentation liquor.
9. The method of claim 1, wherein: the addition amount of the fatty acid in the step (2) is 0.5-2% of the weight of the alkane.
10. The method according to claim 1 or 9, characterized in that: the fatty acid in the step (2) is at least one of oleic acid, linoleic acid, ricinoleic acid and linolenic acid.
11. The method of claim 10, wherein: the fatty acid in the step (2) is oleic acid or linoleic acid.
12. The method of claim 1, wherein: and (2) adding the fatty acid and the phospholipid at the same time, wherein the addition amount of the phospholipid is 1-5% of the mass of the fatty acid.
13. The method of claim 12, wherein: the phospholipid is at least one of phosphatidyl glycerol and diphosphatidyl glycerol, and the addition amount of the phospholipid is 2-5% of the mass of the fatty acid.
14. The method of claim 1, wherein: the addition amount of the water in the step (2) is 1-2.5 times of the weight of the alkane.
15. The method of claim 1, wherein: the ultrasonic treatment in the step (2) has the frequency of 25-40 kHz, the ultrasonic power density of 10-100W/L and the time of 10-30 minutes.
16. The method of claim 1, wherein: the inoculation volume ratio of the fermentation seed liquid in the step (3) is 2-20%, the fermentation temperature is 25-37 ℃, the stirring speed is 120-500 rpm, the ventilation volume is 0.2-1.0 VVM, and the fermentation time is 138-144 hours.
17. The method of claim 16, wherein: the inoculation volume ratio of the fermentation seed liquid in the step (3) is 10-20%, the fermentation temperature is 28-32 ℃, the stirring speed is 200-400 rpm, and the ventilation volume is 0.5-1.0 VVM.
18. The method of claim 1, wherein: the fermentation medium in the step (3) comprises the following components: 30-45 g/L of sucrose, 1.5-2 g/L of corn steep liquor, 1.5-1.8 g/L of yeast extract, 0.8-1.2 g/L of sodium chloride, 3.5-7.5 g/L of monopotassium phosphate, 1.2-1.8 g/L of magnesium sulfate, 1.2-4.8 g/L of urea, 1.5-2 g/L of ammonium sulfate and 1.5-1.8 g/L of sodium acetate.
19. The method of claim 1, wherein: and (3) adopting a mode of interval pH value regulation, increasing the pH value once every 20-28 hours after fermenting for 24 hours, and increasing the pH value by 0.1-0.3 every time until the fermentation is finished.
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| CN1071951A (en) * | 1991-10-29 | 1993-05-12 | 中国石油化工总公司抚顺石油化工研究院 | The method of asynchronous microbiological fermentative production long-chain alpha, omega-dibasic acid |
| CN103805642A (en) * | 2012-11-07 | 2014-05-21 | 中国石油化工股份有限公司 | Fermentation method for production of long-chain dicarboxylic acids |
| CN103805643A (en) * | 2012-11-07 | 2014-05-21 | 中国石油化工股份有限公司 | Production method for long-chain dicarboxylic acids |
| CN103898173A (en) * | 2014-01-02 | 2014-07-02 | 中国科学院等离子体物理研究所 | Method for preparing fatty acid colloid solution applied to crypthecodinium cohnii for producing docosahexaenoic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1071951A (en) * | 1991-10-29 | 1993-05-12 | 中国石油化工总公司抚顺石油化工研究院 | The method of asynchronous microbiological fermentative production long-chain alpha, omega-dibasic acid |
| CN103805642A (en) * | 2012-11-07 | 2014-05-21 | 中国石油化工股份有限公司 | Fermentation method for production of long-chain dicarboxylic acids |
| CN103805643A (en) * | 2012-11-07 | 2014-05-21 | 中国石油化工股份有限公司 | Production method for long-chain dicarboxylic acids |
| CN103898173A (en) * | 2014-01-02 | 2014-07-02 | 中国科学院等离子体物理研究所 | Method for preparing fatty acid colloid solution applied to crypthecodinium cohnii for producing docosahexaenoic acid |
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