CN111850059A - Method for improving fermentation conversion rate of fermentation substrate - Google Patents
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Abstract
The invention provides a method for improving fermentation conversion rate of a fermentation substrate. The method obviously reduces the content of the fermentation substrate in the fermentation tail gas and obviously improves the content of the fermentation product by controlling the concentration of the fermentation substrate in the fermentation production process. The method greatly reduces the difficulty of extraction and purification and wastewater treatment processes at the later stage of fermentation, simplifies the process and saves energy consumption; meanwhile, the quality of downstream products of fermentation products is improved.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation method for improving the fermentation conversion rate of a fermentation substrate of decane or analogues thereof, reducing impurities of a fermentation product, namely dodecanedioic acid and improving the yield of the dodecanedioic acid.
Background
The long chain dibasic acid (LCDA; also known as long chain dicarboxylic acid or long chain diacid) comprises the formula HOOC (CH)2)nA dibasic acid of COOH, wherein n is more than or equal to 7. Deca-dicarboxylic acid or deca-long chain dicarboxylic acid, sebacic acid, and has the chemical formula of HOOC (CH)2)8COOH. As an important monomer raw material, the monomer is widely used for producing polyamide engineering plastics, such as nylon 510, nylon 1010 and nylon 610. Can also be used for high-temperature resistant lubricating oil, epoxy resin curing agent, synthetic lubricating grease, artificial spice, cold-resistant plasticizer and the like, and is one of chemical raw materials with wide application.
The existing industrial production and application of the dodecanedioic acid is prepared by taking castor oil as a raw material through a chemical method. The main process flow is that castor oil is hydrolyzed to generate ricinoleic acid sodium soap, then ricinoleic acid is further prepared, ricinoleic acid is subjected to high-temperature pyrolysis by adding alkali and heating in the presence of phenol to generate deca-dibasic acid disodium salt, and then deca-dibasic acid is obtained by further heating, adding acid, decoloring and crystallizing. The method for preparing the dodecanedioic acid by adopting the castor oil catalytic cracking method has the advantages that the production process is complex, the reaction is carried out at the high temperature of 250-270 ℃, and phenol or o-cresol toxic reagents are used, so that the environment is polluted, and the development of the industry for producing the dodecanedioic acid by a chemical method is severely restricted.
The biological method for preparing the dodecanedioic acid has the characteristics of simple production process and environmental protection. The method comprises the steps of obtaining a high-yield strain from the residual shiitake of the microbiological institute of Chinese academy of sciences (see microbiological newspaper, 1989, 06: 0253-2654) through mutagenesis and screening, obtaining the yield of the dodecanedioic acid on a 16-liter fermentation tank to reach over 71g/L, obtaining the dodecanedioic acid product through aqueous phase crystallization, wherein the product purity is over 99.6 percent; the yield of the dodecanedioic acid produced by fermentation of the candida lipolytica screened by the institute of forestry and soil research (see the microbiological report of the Chinese academy of China, 1979, No. 01) is 30-40 g/L.
In the biological preparation of dodecanedioic acid, decane or an analogue thereof is often used as a fermentation substrate, and possible conversion pathways of such a fermentation substrate in the fermentation process include: the target product of the dodecanedioic acid is formed by the multi-step oxidation of the thalli, and the spent or volatilized bacteria are discharged out of a fermentation system along with fermentation tail gas. The volatile property of the fermentation substrate such as decane greatly restricts the conversion rate of the dodecanedioic acid, the metabolic products brought by the fermentation substrate such as decane in the fermentation process not only increase the difficulty of the process for extracting the dodecanedioic acid in the later period, but also bring great influence on the dodecanedioic acid product by residual impurities. Therefore, it is an important research direction to reduce the volatilization of fermentation substrates such as decane and the like by improving the fermentation process, improve the yield of dodecanedioic acid and reduce impurities.
Disclosure of Invention
The invention aims to provide a method for improving the fermentation conversion rate of a fermentation substrate, in particular to a method for improving the fermentation conversion rate when decane or analogues thereof are used as the fermentation substrate to prepare dodecanedioic acid.
In order to achieve the object, in a first aspect, the invention provides a method for improving fermentation conversion rate of a fermentation substrate, wherein the concentration of the fermentation substrate in the fermentation process is controlled to be 0.5% -3%, and the concentration of the fermentation substrate can be controlled by adjusting the addition rate of the fermentation substrate in the fermentation process. The concentration of the fermentation substrate is preferably controlled within any one of the ranges of 0.5% to 1%, 1% to 1.5%, 1.5% to 2%, and 2% to 3%.
Preferably, the fermentation substrate comprises decane, a ten carbon fatty acid derivative, or a mixture thereof; the product obtained by fermentation is dodecanedioic acid.
Preferably, the decane is of the formula CH3(CH2)8CH3The ten-carbon fatty acid is a ten-carbon straight-chain saturated fatty acid, and the ten-carbon fatty acid derivative includes a ten-carbon straight-chain saturated fatty acid ester, a ten-carbon straight-chain saturated fatty acid salt.
Preferably, the engineering bacteria for producing long-chain dicarboxylic acid used in the fermentation are selected from the group consisting of species in Corynebacterium (Corynebacterium), Geotrichum (Geotrichum), Candida (Candida), Pichia (Pichia), Rhodotorula (Rhodotorula), saccharomyces (saccharomyces), Yarrowia (Yarrowia); preferably a Candida species; more preferably Candida tropicalis (Candida tropicalis) or Candida sake (Candida desake).
In one embodiment of the invention, the pressure is controlled to be 0.05-0.15 Mpa and/or the air flow is controlled to be 0.1-0.7 vvm during the fermentation process.
In one embodiment of the invention, the fermentation temperature is controlled to be 28-33 ℃ during the fermentation process.
In a specific embodiment of the invention, the concentration of the fermentation substrate in the fermentation liquid is controlled to be 1% -2%, the pressure in the fermentation process is controlled to be 0.08-0.12 MPa, and the air flow is controlled to be 0.3-0.5 vvm.
In a specific embodiment of the invention, the strain used for fermentation is an engineering bacterium CATN145 for producing long-chain dicarboxylic acid, the engineering bacterium CATN145 is a Candida tropicalis (Candida tropicalis) bacterium, and the CAT N145 has been biologically deposited at 2011, 6 and 9 days, and the deposition unit is: china center for type culture Collection (Address: Wuhan, Wuhan university, China, zip code: 430072). The biological and genetic characteristics of the gene are described in Chinese patent application with publication number CN102839133A and publication date 2012-12-26.
In one embodiment of the present invention, the strain used for fermentation is an engineered strain producing long-chain dicarboxylic acid, Candida tropicalis 10468, which has been biologically deposited at 1/4 in 2017, and the deposition unit is: china center for type culture Collection (Address: Wuhan, Wuhan university, China), accession number: CCTCC NO: M2017164, and is classified and named as Candida tropicalis.
The invention also provides a method for improving fermentation conversion rate of fermentation substrate, which comprises the following steps:
(1) seed culture: inoculating the strain used for fermentation into a seed culture medium to culture until the seed is mature;
(2) fermentation culture: transferring the seed liquid obtained in the step (1) into a fermentation culture medium, and diluting the fermentation liquid by 30 times to obtain thallus optical density OD 620When the concentration reaches above 0.5 percent, adding a fermentation substrate, and controlling the concentration of the fermentation substrate to be kept at 0.5 to 3 percent.
In one embodiment of the invention, the seed culture medium comprises 1.0-3.0% of sucrose, 0.1-0.5% of urea, 0.1-1.0% of yeast extract, 0.2-1.0% of corn steep liquor and 0.4-1.2% of potassium dihydrogen phosphate.
In one embodiment of the invention, the fermentation medium comprises the following components: 2.0 to 5.0 percent of glucose, 0.1 to 1.0 percent of yeast extract, 0.2 to 1.0 percent of corn steep liquor, 0.1 to 1 percent of potassium nitrate, 0.1 to 1.0 percent of monopotassium phosphate and 0.1 to 0.5 percent of ammonium sulfate.
The invention also provides a dodecanedioic acid prepared according to any one of the methods.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
by using the method, the concentration of the substrate in the fermentation liquid is controlled to be 0.5-3% and certain fermentation pressure, air flow and fermentation temperature are controlled during the production of the dodecanedioic acid by fermentation, so that the tail gas discharge amount of the fermentation substrate is greatly reduced, the conversion rate of the fermentation substrate is improved, and the yield of the dodecanedioic acid of the fermentation product is further improved. But also reduces the impurities generated in the fermentation process, greatly reduces the difficulty of extraction and purification and wastewater treatment processes in the later stage of fermentation, simplifies the process and saves energy consumption; meanwhile, the quality of downstream products of fermentation products is improved.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the invention, the method for testing the content of the dodecanedioic acid, the fermentation substrate and impurities in the fermentation liquid and the concentration of the fermentation substrate in the fermentation tail gas can adopt gas chromatography, liquid chromatography-mass spectrometry combined technology or gas chromatography-mass spectrometry combined technology.
The OD value in the present invention is a value measured when the optical density of the bacterial cells is diluted 30-fold in each of the following embodiments and examples, which are not particularly described.
The fermentation conversion rate of the fermentation substrate is weight conversion rate (w/w), namely the percentage of the mass of the product obtained by fermentation to the mass of the fermentation substrate.
In the process of producing the dodecanedioic acid by fermentation, a seed culture medium and a fermentation culture medium used when the strain producing the dodecanedioic acid is fermented and cultured at least comprise a carbon source and a nitrogen source, and optionally inorganic salt, a nutritional factor or a fermentation substrate. According to the common knowledge in the field of fermentation, the percentage of the added amount of the raw materials of the fermentation medium is the mass-to-volume ratio, namely w/v; % means g/100 mL.
Optionally, the carbon source includes, but is not limited to, one or more of glucose, sucrose and maltose; and/or the amount of the carbon source added is 1% to 10% (w/v), such as 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%.
Optionally, the nitrogen source includes, but is not limited to, one or more of yeast powder, yeast extract, corn steep liquor, ammonium sulfate, urea, and potassium nitrate; and/or the total amount of nitrogen sources added is 0.1% to 4% (w/v), such as 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.5%, 2.9%, 3.5%.
Optionally, the inorganic salts include, but are not limited to, one or more of potassium nitrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, magnesium sulfate, ferrous sulfate, calcium chloride, ferric chloride, copper sulfate; and/or the total amount of inorganic salts added is 0.1% to 1.5% (w/v), such as 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%.
In some preferred embodiments of the present invention, the seed medium may include sucrose 1.0-3.0%, urea 0.1-0.5%, yeast extract 0.1-1.0%, corn steep liquor 0.2-1.0%, and potassium dihydrogen phosphate 0.4-1.2%. Preferably, the Optical Density (OD) of the cells in the seed solution is measured620) When the dilution reaches more than 0.5 (30 times), inoculating the seed liquid into a fermentation culture medium for fermentation and transformation, wherein the inoculation amount can be 10-30% (v/v). Wherein the OD 620Can be 0.5 to 1.0, and can be about 0.8.
In some preferred embodiments of the invention, the amount of inoculation may be 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, 27%, 29%.
In some preferred embodiments of the invention, the fermentation medium may comprise the following components: 2.0 to 5.0 percent of glucose, 0.1 to 1.0 percent of yeast extract, 0.2 to 1.0 percent of corn steep liquor, 0.1 to 1 percent of potassium nitrate, 0.1 to 1.0 percent of monopotassium phosphate and 0.1 to 0.5 percent of ammonium sulfate (w/v).
In some preferred embodiments of the present invention, the fermentation substrate, or feedstock referred to as a feedstock for fermentative conversion, used for the fermentative production of a ten-carbon diacid includes decane, a ten-carbon fatty acid derivative, or mixtures thereof. Preferably, the decane is of the formula CH3(CH2)8CH3The ten-carbon fatty acid is a ten-carbon straight-chain saturated fatty acid, and the ten-carbon fatty acid derivative includes a ten-carbon straight-chain saturated fatty acid ester, a ten-carbon straight-chain saturated fatty acid salt. Specifically, the ten-carbon fatty acid derivative comprises methyl ten-carbon straight-chain saturated fatty acid, butyl ten-carbon straight-chain saturated fatty acid, sodium ten-carbon straight-chain saturated fatty acid and potassium ten-carbon straight-chain saturated fatty acid. Preferably, the fermentation substrate is n-decane.
In some preferred embodiments of the present invention, the fermentation substrate may be added to the seed medium and fermentation medium first, or may be added during fermentation. Preferably, the Optical Density (OD) of the cells is determined during fermentation620) When the dilution reaches more than 0.5-0.9 (30 times), adding a fermentation substrate.
In a preferred embodiment of the invention, the concentration of the fermentation substrate is controlled during fermentation between 0.5% and 3%. Preferably, the fermentation substrate concentration is controlled within any one of the ranges 0.5% to 1%, 1% to 1.5%, 1.5% to 2%, or 2% to 3%, or any combination of the endpoints of these ranges, such as the 0.5% to 1.5% concentration range consisting of 0.5% and 1.5% endpoints, or the 1% to 2% concentration range consisting of 1% and 2% endpoints. The substrate concentration refers to the volume concentration (v/v) of the fermentation substrate in the fermentation liquid, can be controlled by adjusting the addition speed of the fermentation substrate in the fermentation process and is detected by gas chromatography-mass spectrometry.
The inventors have found that the concentration of fermentation substrate in the fermentation broth has a significant effect on the conversion of the synthesis of a ten-carbon diacid and the content of metabolic impurities, including a ten-carbon fatty acid (chemical formula CH) 3-(CH2)8-COOH) and/or a decahydroxy fatty acid (formula CH)2OH-(CH2)8-COOH). Through a large number of experiments and researches, the inventor unexpectedly discovers that when the concentration of a fermentation substrate in fermentation liquor is controlled to be 0.5% -3%, the requirement for thallus growth can be met, the volatilization amount of decane is reduced, the conversion rate of the fermentation substrate into dodecanedioic acid is greatly improved, and the content of metabolic impurities generated in the conversion process is reduced.
In a preferred embodiment of the invention, the fermentation substrate can be added in a batch or continuous feed.
In some preferred embodiments of the present invention, the fermentation temperature during the fermentation process is 28-33 ℃, and more preferably 29-30 ℃.
When the seeding tank or the fermentation tank is used for fermentation culture, the control pressure is the control tank pressure. In some preferred embodiments of the present invention, the pressure during fermentation is controlled to be 0.05 to 0.15MPa, more preferably 0.08 to 0.12 MPa.
If the air flow rate is controlled at a low level during fermentation, the amount of decane volatilized into the fermentation off-gas is reduced, but the cells are easily in an oxygen-limited state, and the growth of the cells is suppressed. In some preferred embodiments of the invention, the air flow rate during fermentation is controlled to be 0.1 to 0.7vvm, more preferably 0.3 to 0.5 vvm.
In some preferred embodiments of the invention, Dissolved Oxygen (DO) is controlled to be above 10%, preferably above 20% during the fermentative conversion process.
In some preferred embodiments of the present invention, the pH value is controlled to be 4.0 to 8.5, preferably 4.5 to 7.5 during the fermentation process. During the fermentation, the pH can be adjusted with 1N HCl or 1N NaOH.
In some preferred embodiments of the invention, the strain used for fermentation is Candida tropicalis (Candida tropicalis) or Candida sake (Candida sake).
In a preferred embodiment of the present invention, the method for increasing the production of dodecanedioic acid may comprise the steps of: when the fermentation culture strain produces the dodecanedioic acid, the concentration of a fermentation substrate in the fermentation liquid is controlled to be 0.5-3%, the pressure in the fermentation process is controlled to be 0.05-0.15 MPa, and the air flow is controlled to be 0.1-0.7 vvm.
In a preferred embodiment of the present invention, the method for increasing the production of dodecanedioic acid may comprise the steps of: when the fermentation culture strain produces the dodecanedioic acid, the concentration of a fermentation substrate in the fermentation liquid is controlled to be 0.5% -2%, the pressure in the fermentation process is controlled to be 0.08-0.12 MPa, and the air flow is controlled to be 0.3-0.5 vvm.
In a preferred embodiment of the present invention, the method for increasing the production of dodecanedioic acid may comprise the steps of:
(1) Seed culture: inoculating the strain Candida for fermentation into a seed culture medium or a seed tank containing the seed culture medium, and culturing until the seeds are mature.
(2) Fermentation culture: transferring the seed liquid obtained in the step (1) into a fermentation medium or a fermentation tank containing the fermentation medium, and determining the Optical Density (OD) of thallus of the fermentation liquid620) When the concentration reaches more than 0.5 (diluted by 30 times), adding a fermentation substrate, and controlling the concentration of the fermentation substrate to be kept between 0.5 and 3 percent.
Preferably, (1) seed culture: inoculating the strain used for fermentation into a seed tank, controlling the culture temperature to be 28-33 ℃, the tank pressure to be 0.05-0.15 MPa, the air flow to be 0.3-0.7 vvm, and OD after the seed liquid is diluted by 30 times620At a temperature of 0.5 or more, inoculating the seed solution into a fermentation tank containing a fermentation medium in an amount of 10-30% (v/v, relative to the initial volume of fermentation).
Preferably, (2) fermentation culture: transferring the seed liquid obtained in the step (1) into a fermentation medium or a fermentation tankOptical Density (OD) of fermentation broth620) When the concentration of the fermentation substrate in the fermentation liquid reaches 0.5 (diluted by 30 times) or more, the fermentation substrate is added, the concentration of the fermentation substrate in the fermentation liquid is controlled to be 0.5-3% by controlling the adding rate of the fermentation substrate, and the total adding amount of the fermentation substrate is preferably 25-60% (v/v, relative to the initial volume of fermentation), and is preferably 35-40%. In the fermentation process, the culture temperature is controlled to be 28-33 ℃, the pH value in the fermentation process is controlled to be 5.0-8.5, the air flow is controlled to be 0.3-0.7 vvm, the tank pressure is controlled to be 0.05-0.15 MPa, a certain stirring speed is kept, and the dissolved oxygen is controlled to be more than 10%.
Preferably, (2) during fermentation culture, fermentation is continued after the fermentation substrate is stopped being supplemented until the fermentation substrate in the fermentation liquid is detected to be 0, namely the fermentation is finished, and the fermentation is stopped.
In general, the dodecanedioic acid fermentation broth obtained after the fermentation is finished contains water, bacteria and other impurities, and the dodecanedioic acid needs to be separated and purified from the fermentation broth, and the separation and purification can be specifically carried out by referring to a refining method or a process disclosed in publication No. CN101985416B or CN 103965035B.
The technical solution of the present invention is further specifically described below by way of examples.
The culture medium, the culture fermentation method and the long-chain dicarboxylic acid detection method are as follows:
1. seed culture medium, formula (w/v) is: 2.0 percent of sucrose, 0.5 percent of yeast powder (the total nitrogen content is 12 percent by weight), 0.4 percent of corn steep liquor (the total nitrogen content is 2.5 percent by weight), 0.8 percent of monopotassium phosphate and 0.3 percent of urea.
2. The fermentation medium has the following formula (w/v): 3.0 percent of glucose, 0.8 percent of yeast extract (total nitrogen content is 7 percent by weight), 0.6 percent of corn steep liquor (total nitrogen content is 2.5 percent by weight), 0.5 percent of potassium nitrate, 0.5 percent of monopotassium phosphate and 0.3 percent of ammonium sulfate.
3. The strains used for the fermentation:
candida tropicalis (Candida tropicalis) strain CATN145, biopreserved 6/9.2011, the depository: china center for type culture Collection (Address: Wuhan university, Wuhan, China, zip code: 430072), preservation number: CCTCC M2011192.
Candida tropicalis (Candida tropicalis) strain 10468, biopreserved 4/1 in 2017, depository: china center for type culture Collection (Address: Wuhan, Wuhan university, China), accession number: CCTCC M2017164.
4. Method for detecting yield of dodecanedioic acid by Gas Chromatography (GC) technology
(1) And (3) detecting the content of fermentation liquor products and impurities: the fermentation liquor is pretreated by conventional gas chromatography, and is detected by gas chromatography (internal standard method), wherein the chromatographic conditions are as follows:
a chromatographic column: supelco SPB-5030 m 0.53mm 0.5 μm (cat 54983).
Gas chromatograph (Shimadzu, GC-2014).
The method comprises the following steps: the initial temperature is 100 ℃, the temperature is raised to 230 ℃ at the speed of 15 ℃/min, and the temperature is kept for 2 min. The carrier gas is hydrogen, the injection port temperature is 280 ℃, the FID temperature is 280 ℃, and the injection amount is 4 mu L.
And calculating the yield of the dibasic acid according to the peak area of the dibasic acid product and the peak area ratio of the internal standard with known concentration, and calculating the impurity content according to the peak area of the dibasic acid product and the peak area of the impurity.
(2) The decane detection method in the fermentation tail gas detection comprises the following steps: collecting the gas sample in a glass syringe, detecting by gas chromatography,
a chromatographic column: GDX-50230 m 0.53mm 25 μm (cat 0510032).
Gas chromatograph (Shimadzu, GC-2014).
The method comprises the following steps: the sample inlet is 100 ℃, the column temperature is 80 ℃, the detector is 250 ℃, and the sample loading is 1.0mL by using carrier gas nitrogen.
The concentration was calculated from the peak area.
(3) And (3) detecting the purity and impurity content of the solid product: the solid sample is pretreated by the conventional gas chromatography, and is detected by the gas chromatography (normalization method),
chromatographic conditions are as follows: a chromatographic column: supelco SPB-5030 m 0.53mm 0.5 μm (cat 54983).
Gas chromatograph (Shimadzu, GC-2014).
The method comprises the following steps: the initial temperature is 100 ℃, the temperature is raised to 230 ℃ at the speed of 15 ℃/min, and the temperature is kept for 2 min. The carrier gas is hydrogen, the injection port temperature is 280 ℃, the FID temperature is 280 ℃, and the injection amount is 4 mu L.
And calculating the purity and the impurity content of the product according to the peak area of the dibasic acid product and the peak area of the impurity.
EXAMPLE 1 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation method comprises the following steps: inoculating Candida tropicalis CATN145 thallus to a seed culture medium, culturing at 30 deg.C, and measuring the Optical Density (OD) of the thallus in the seed solution620) When the concentration reaches 0.5 (diluted by 30 times), the seed solution is inoculated into a fermentation culture medium for fermentation and transformation, and the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 30 ℃, controlling the pH value to be 7.2 in the fermentation process, controlling the air flow to be 0.3vvm, controlling the pressure of the fermentation tank to be 0.08MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is maintained to be more than 20% in the fermentation process. After the seed liquid is inoculated into a fermentation tank, the thalli begin to grow and reproduce, and when the Optical Density (OD) of the thalli in the fermentation liquid is 620) When the concentration reached 0.5 (diluted 30 times), n-decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 0.5-1% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 170 h.
And (3) detection results: the average content of decane monitored in real time in the fermentation tail gas is 2000mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 184g/L, the conversion rate of the decadic acid is 85.2%, the content of impurity decadic fatty acid is 2.50%, and the content of impurity decadic hydroxyl fatty acid is 2.00%.
EXAMPLE 2 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation method comprises the following steps: inoculating Candida tropicalis CATN145 thallus to a seed culture medium, culturing at 30 deg.C, and measuring the Optical Density (OD) of the thallus in the seed solution620) When the concentration reaches 0.5 (diluted by 30 times), the seed solution is inoculated into a fermentation culture medium for fermentation and transformation, and the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 30 ℃, controlling the pH value to be 7.2 in the fermentation process, controlling the air flow to be 0.3vvm, controlling the pressure of the fermentation tank to be 0.08MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is maintained to be more than 20% in the fermentation process. Inoculating seed liquid for fermentation After the fermentation, the thallus starts to grow and breed, and when the Optical Density (OD) of the thallus in the fermentation liquor620) When the concentration reached 0.5 (diluted 30 times), n-decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 1.0-1.5% (v/v), and the total addition amount of decane as a fermentation substrate is 1300 g. When the fermentation substrate in the fermentation liquid is detected to be 0, the fermentation is stopped, and the fermentation time is 165 h.
And (3) detection results: the average decane content monitored in real time in the fermentation tail gas is 2500mg/m3. After the fermentation is finished, the yield of the decarburized dibasic acid in the fermentation liquor is 182g/L, the conversion rate of the decarburized dibasic acid is 84.0%, the content of impurity decarburized fatty acid is 1.00%, and the content of impurity decarburized hydroxy fatty acid is 0.32%.
EXAMPLE 3 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation method comprises the following steps: inoculating Candida tropicalis CATN145 thallus to a seed culture medium, culturing at 30 deg.C, and measuring the Optical Density (OD) of the thallus in the seed solution620) When the concentration reaches 0.5 (diluted by 30 times), the seed solution is inoculated into a fermentation culture medium for fermentation and transformation, and the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 30 ℃, controlling the pH value to be 7.2 in the fermentation process, controlling the air flow to be 0.3vvm, controlling the pressure of the fermentation tank to be 0.08MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is maintained to be more than 20% in the fermentation process. After the seed liquid is inoculated into a fermentation tank, the thalli begin to grow and reproduce, and when the Optical Density (OD) of the thalli in the fermentation liquid is 620) When the concentration reached 0.5 (diluted 30 times), n-decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 1.5-2% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 168 h.
And (3) detection results: the average content of decane monitored in real time in the fermentation tail gas is 2700mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 180g/L, the conversion rate of the decadic acid is 83.5%, the content of impurity decadic fatty acid is 1.1%, and the content of impurity decahydroxyl fatty acid is 0.78%.
EXAMPLE 4 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation method comprises the following steps: inoculating Candida tropicalis CATN145 thallus to a seed culture medium, culturing at 30 deg.C, and measuring the Optical Density (OD) of the thallus in the seed solution620) When the concentration reaches 0.5 (diluted by 30 times), the seed solution is inoculated into a fermentation culture medium for fermentation and transformation, and the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 30 ℃, controlling the pH value to be 7.2 in the fermentation process, controlling the air flow to be 0.3vvm, controlling the pressure of the fermentation tank to be 0.08MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is maintained to be more than 20% in the fermentation process. After the seed liquid is inoculated into a fermentation tank, the thalli begin to grow and reproduce, and when the Optical Density (OD) of the thalli in the fermentation liquid is 620) When the concentration reached 0.5 (diluted 30 times), n-decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 2-3% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. When the fermentation substrate in the fermentation liquid is detected to be 0, the fermentation is stopped, and the fermentation time is 155 h.
And (3) detection results: the average content of decane monitored in real time in the fermentation tail gas is 3200mg/m3. After the fermentation is finished, the yield of the decarburized dibasic acid in the fermentation liquor is 162.5g/L, the conversion rate of the decarburized dibasic acid is 75.0%, the content of impurity decarburized fatty acid is 1.3%, and the content of impurity decarburized hydroxy fatty acid is 1.0%.
Comparative example 1 production of dodecanedioic acid by fermentation of strain CATN145
The fermentation process was the same as in example 5, except that: the addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 4-6% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 150 hours.
And (3) detection results: the average content of decane in the fermentation tail gas is 4600mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 144g/L, the conversion rate of the decadic acid is 66.4%, the content of impurity decadic fatty acid is 3.7%, and the content of impurity decahydroxyl fatty acid is 2.5%.
Comparative example 2 production of dodecanedioic acid by fermentation of strain CATN145
The fermentation process was the same as in example 5, except that: the addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 8-10% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. When the fermentation substrate in the fermentation liquid is detected to be 0, the fermentation is stopped, and the fermentation time is 145 h.
And (3) detection results: the average content of decane in the fermentation tail gas is 6200mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 132g/L, the conversion rate of the decadic acid is 60.9%, the content of impurity decadic fatty acid is 3.9%, and the content of impurity decadic hydroxy fatty acid is 2.2%.
EXAMPLE 5 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation method comprises the following steps: inoculating Candida tropicalis CATN145 thallus to a seed culture medium, culturing at 27 deg.C, and measuring the Optical Density (OD) of the thallus in the seed solution620) When the concentration reaches 0.5 (diluted by 30 times), the seed solution is inoculated into a fermentation culture medium for fermentation and transformation, and the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 30 ℃, controlling the pH value to be 7.2 in the fermentation process, controlling the air flow to be 0.3vvm, controlling the pressure of the fermentation tank to be 0.08MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is maintained to be more than 20% in the fermentation process. After the seed liquid is inoculated into a fermentation tank, the thalli begin to grow and reproduce, and when the Optical Density (OD) of the thalli in the fermentation liquid is 620) When the concentration reached 0.5 (diluted 30 times), n-decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 1-2% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 220 h.
And (3) detection results: the average content of decane monitored in real time in the fermentation tail gas is 1000mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 141g/L, the conversion rate of the decadic acid is 65.1 percent, the content of impurity decadic fatty acid is 3.30 percent, and the content of impurity decahydroxyl fatty acid is 2.00 percent.
EXAMPLE 6 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation process was the same as in example 5, except that: the fermentation temperature is controlled to be 29 ℃ in the fermentation process, and the total addition amount of fermentation substrate decane is 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 160 h.
And (3) detection results: the average content of decane monitored in real time in the fermentation tail gas is 2250mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 185g/L, the conversion rate of the decadic acid is 85.6 percent, the content of impurity decadic fatty acid is 0.87 percent, and the content of impurity decahydroxyl fatty acid is 0.32 percent.
EXAMPLE 7 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation process was the same as in example 5, except that: the fermentation temperature is controlled to be 31 ℃ in the fermentation process, and the total addition amount of fermentation substrate decane is 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 140 h.
And (3) detection results: the average content of decane monitored in real time in the fermentation tail gas is 2800mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 169g/L, the conversion rate of the decadic acid is 78%, the content of impurity decadic fatty acid is 1.67%, and the content of impurity decahydroxyl fatty acid is 1.22%.
EXAMPLE 8 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation process was the same as in example 5, except that: the fermentation temperature is controlled to be 33 ℃ in the fermentation process, and the total addition amount of fermentation substrate decane is 1300 g. When the fermentation substrate in the fermentation liquid is detected to be 0, the fermentation is stopped, and the fermentation time is 185 h.
And (3) detection results: the average content of decane monitored in real time in fermentation tail gas is 3500mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 162g/L, the conversion rate of the decadic acid is 75%, the content of impurity decadic fatty acid is 4.53%, and the content of impurity decahydroxyl fatty acid is 3.68%.
EXAMPLE 9 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation method comprises the following steps: inoculating Candida tropicalis CATN145 thallus to a seed culture medium, culturing at 30 deg.C, and measuring the Optical Density (OD) of the thallus in the seed solution620) When the concentration reaches 0.5 (diluted by 30 times)Inoculating the seed liquid into a fermentation culture medium for fermentation and transformation, wherein the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 29 ℃, controlling the pH value to be 7.2 in the fermentation process, controlling the air flow to be 0.3vvm, controlling the pressure of a fermentation tank to be 0.05MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is about 10% in the fermentation process. After the seed liquid is inoculated into a fermentation tank, the thalli begin to grow and reproduce, and when the Optical Density (OD) of the thalli in the fermentation liquid is620) When the concentration reached 0.5 (diluted 30 times), n-decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 1-2% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. When the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation, and fermenting for 190 h.
And (3) detection results: the average content of decane monitored in real time in fermentation tail gas is 3500mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 145g/L, the conversion rate of the decadic acid is 67%, the content of impurity decadic fatty acid is 2.97%, and the content of impurity decahydroxyl fatty acid is 2.12%.
EXAMPLE 10 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation process was the same as in example 9, except that: in the fermentation process, the fermentation temperature is controlled to be 0.08MPa, and the dissolved oxygen is maintained to be more than 30%. The total amount of decane added as a fermentation substrate was 1300 g. When the fermentation substrate in the fermentation liquid is detected to be 0, the fermentation is stopped, and the fermentation time is 172 h.
And (3) detection results: the average decane content monitored in real time in the fermentation tail gas is 1800mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 183g/L, the conversion rate of the decadic acid is 84.7%, the content of impurity decadic fatty acid is 0.97%, and the content of impurity decahydroxyl fatty acid is 0.52%.
EXAMPLE 11 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation process was the same as in example 9, except that: the fermentation temperature is controlled to be 0.12MPa and the dissolved oxygen is maintained to be more than 50 percent in the fermentation process. The total amount of decane added as a fermentation substrate was 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 150 hours.
And (3) detection results: the average content of decane monitored in real time in the fermentation tail gas is 1000mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 190g/L, the conversion rate of the decadic acid is 87.7%, the content of impurity decadic fatty acid is 0.37%, and the content of impurity decadic hydroxy fatty acid is 0.27%.
EXAMPLE 12 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation process was the same as in example 9, except that: the fermentation temperature is controlled to be 0.15MPa and the dissolved oxygen is maintained to be more than 50 percent in the fermentation process. The total amount of decane added as a fermentation substrate was 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 201 h.
And (3) detection results: the average content of decane monitored in real time in the fermentation tail gas is 700mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 165g/L, the conversion rate of the decadic acid is 76.3%, the content of impurity decadic fatty acid is 1.78%, and the content of impurity decahydroxyl fatty acid is 1.26%.
EXAMPLE 13 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation method comprises the following steps: inoculating Candida tropicalis CATN145 thallus to a seed culture medium, culturing at 30 deg.C, and measuring the Optical Density (OD) of the thallus in the seed solution620) When the concentration reaches 0.5 (diluted by 30 times), the seed solution is inoculated into a fermentation culture medium for fermentation and transformation, and the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 29 ℃, controlling the pH value to be 7.2 in the fermentation process, controlling the air flow to be 0.1vvm, controlling the pressure of a fermentation tank to be 0.08MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is lower than 10% in the fermentation process. After the seed liquid is inoculated into a fermentation tank, the thalli begin to grow and reproduce, and when the Optical Density (OD) of the thalli in the fermentation liquid is 620) When the concentration reached 0.5 (diluted 30 times), n-decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 1-2% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation, and fermenting for 175 h.
And (3) detection results: the average content of decane in the fermentation tail gas is 900mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 135g/L, the conversion rate of the decadic acid is 71%, the content of impurity decadic fatty acid is 4.28%, and the content of impurity decahydroxyl fatty acid is 3.15%.
EXAMPLE 14 fermentative production of Dodecadioic acid by Strain CATN145
The fermentation process was the same as in example 13, except that: controlling the air flow to be 0.5vvm and maintaining the dissolved oxygen to be more than 30 percent in the fermentation process. The total amount of decane added as a fermentation substrate was 1300 g. And when the fermentation substrate in the fermentation liquid is detected to be 0, stopping fermentation for 174 h.
And (3) detection results: the average decane content monitored in real time in the fermentation tail gas is 1800mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 165g/L, the conversion rate of the decadic acid is 76.3%, the content of impurity decadic fatty acid is 0.98%, and the content of impurity decahydroxyl fatty acid is 0.67%.
Example 15 fermentative production of Dodecadioic acid by Strain 10468
The fermentation method comprises the following steps: inoculating Candida tropicalis 10468 thallus in seed culture medium, culturing at 30 deg.C, and determining the Optical Density (OD) of thallus in seed liquid620) When the concentration reaches 0.6 (diluted by 30 times), the seed solution is inoculated into a fermentation culture medium for fermentation and transformation, and the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 32 ℃, controlling the pH value to be 7.0 in the fermentation process, controlling the air flow to be 0.8vvm, controlling the pressure of the fermentation tank to be 0.10MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is maintained to be more than 20% in the fermentation process. After the seed liquid is inoculated into a fermentation tank, the thalli begin to grow and reproduce, and when the Optical Density (OD) of the thalli in the fermentation liquid is620) When the concentration reached 0.5 (diluted 30 times), decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 1.0-2.0% (v/v), and the total addition amount of decane serving as a fermentation substrate is 1300 g. When the fermentation substrate in the fermentation liquid is detected to be 0, the fermentation is stopped, and the fermentation time is 165 h.
And (3) detection results: the average decane content monitored in real time in the fermentation tail gas is 2160mg/m3. The yield of the dodecanedioic acid in the fermentation liquid after the fermentation is finished is 184g/L, and the dodecanedioic acid is used as the dodecanedioic acidThe conversion rate of the polybasic acid was 84.9%, the content of the impurity deca-fatty acid was 2.78%, and the content of the impurity deca-hydroxy fatty acid was 1.68%.
EXAMPLE 16 fermentative production of Dodecadioic acid by Strain 10468
The fermentation method comprises the following steps: inoculating Candida tropicalis 10468 thallus in seed culture medium, culturing at 30 deg.C, and determining the Optical Density (OD) of thallus in seed liquid620) When the concentration reaches 0.8 (diluted by 30 times), the seed solution is inoculated into a fermentation culture medium for fermentation and transformation, and the inoculation amount is 10% (v/v). Controlling the fermentation temperature to be 30 ℃, controlling the pH value to be 7.2 in the fermentation process, controlling the air flow to be 0.8vvm, controlling the pressure of the fermentation tank to be 0.08MPa, and keeping a certain stirring speed, wherein the dissolved oxygen is maintained to be more than 20% in the fermentation process. After the seed liquid is inoculated into a fermentation tank, the thalli begin to grow and reproduce, and when the Optical Density (OD) of the thalli in the fermentation liquid is620) When the concentration reached 0.5 (diluted 30 times), decane was added as a fermentation substrate. The addition rate of decane is controlled by adopting a fed-batch mode so that the concentration range of decane in fermentation liquid in the fermentation process is 1.0-1.5% (v/v), and the total addition amount of decane as a fermentation substrate is 1300 g. When the fermentation substrate in the fermentation liquid is detected to be 0, the fermentation is stopped, and the fermentation time is 172 h.
And (3) detection results: the average decane content monitored in real time in the fermentation tail gas is 1120mg/m3. After the fermentation is finished, the yield of the decadic acid in the fermentation liquor is 176g/L, the conversion rate of the decadic acid is 81.23%, the content of impurity decadic fatty acid is 2.46%, and the content of impurity decahydroxyl fatty acid is 1.47%.
The embodiment shows that the method of the invention has better effects in reducing the impurity content and ensuring the fermentation efficiency and the acid yield by controlling the fermentation process parameters such as the concentration, the pressure, the air flow and the like of the fermentation substrate. The reduction of the impurity content reduces the difficulty and the production cost of the later extraction and purification process on one hand, and is more beneficial to the production and the manufacture of downstream nylon, plastic, polyamide hot melt adhesive and other products, and the product quality is improved on the other hand.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for improving fermentation conversion rate of fermentation substrate is characterized in that the concentration of the fermentation substrate is controlled to be 0.5% -3% in the fermentation process.
2. The process of claim 1, wherein the fermentation substrate comprises decane, a ten carbon fatty acid derivative, or a mixture thereof; the product obtained by fermentation is dodecanedioic acid.
3. The method according to claim 1, wherein the strain used in the fermentation is selected from the group consisting of species of the genus Corynebacterium (Geotechum), Geotrichum (Geotrichum), Candida (Candida), Pichia (Pichia), Rhodotorula (Rhodotorula), Saccharomyces (Saccharomyces), Yarrowia (Yarrowia); preferably a Candida species; more preferably Candida tropicalis (Candida tropicalis) or Candida sake (Candida desake).
4. The method according to claim 2, wherein the pressure is controlled to be 0.05 to 0.15Mpa and/or the air flow is controlled to be 0.1 to 0.7vvm during the fermentation.
5. The method according to claim 2, wherein the fermentation temperature is controlled to be 28 ℃ to 33 ℃ during the fermentation.
6. The method according to claim 2, wherein the concentration of the fermentation substrate in the fermentation broth is controlled to be 1% -2%, the pressure in the fermentation process is controlled to be 0.08-0.12 MPa, and the air flow is controlled to be 0.3-0.5 vvm.
7. A method for increasing fermentation conversion of a fermentation substrate, comprising the steps of:
(1) seed culture: inoculating the strain used for fermentation into a seed culture medium to culture until the seed is mature;
(2) Fermentation culture: transferring the seed liquid obtained in the step (1) into a fermentation culture medium, and diluting the fermentation liquid by 30 times to obtain thallus optical density OD620When the concentration reaches above 0.5 percent, adding a fermentation substrate, and controlling the concentration of the fermentation substrate to be kept at 0.5 to 3 percent.
8. The method of claim 7, wherein the seed medium comprises sucrose 1.0-3.0%, urea 0.1-0.5%, yeast extract 0.1-1.0%, corn steep liquor 0.2-1.0%, and potassium dihydrogen phosphate 0.4-1.2%.
9. The method of claim 7, wherein the fermentation medium comprises the following components: 2.0 to 5.0 percent of glucose, 0.1 to 1.0 percent of yeast extract, 0.2 to 1.0 percent of corn steep liquor, 0.1 to 1 percent of potassium nitrate, 0.1 to 1.0 percent of monopotassium phosphate and 0.1 to 0.5 percent of ammonium sulfate.
10. A dodecanedioic acid prepared according to the process of any one of claims 1-9.
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| CN1351006A (en) * | 2000-10-26 | 2002-05-29 | 中国石油化工股份有限公司 | Process for refining long-chain biatomic acid |
| CN101121653A (en) * | 2006-08-07 | 2008-02-13 | 上海凯赛生物技术研发中心有限公司 | Long carbon-chain dibasic acid prepared from fatty acids or derivatives thereof and preparation method for the long carbon-chain dibasic acid |
| CN107326051A (en) * | 2017-08-02 | 2017-11-07 | 上海凯赛生物技术研发中心有限公司 | A kind of decanedioic acid of Production by Microorganism Fermentation and preparation method thereof |
| CN109652472A (en) * | 2019-02-26 | 2019-04-19 | 成都锦汇科技有限公司 | A kind of long carbochain biatomic acid biofermentation preparation method |
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| CN1351006A (en) * | 2000-10-26 | 2002-05-29 | 中国石油化工股份有限公司 | Process for refining long-chain biatomic acid |
| CN101121653A (en) * | 2006-08-07 | 2008-02-13 | 上海凯赛生物技术研发中心有限公司 | Long carbon-chain dibasic acid prepared from fatty acids or derivatives thereof and preparation method for the long carbon-chain dibasic acid |
| CN107326051A (en) * | 2017-08-02 | 2017-11-07 | 上海凯赛生物技术研发中心有限公司 | A kind of decanedioic acid of Production by Microorganism Fermentation and preparation method thereof |
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