CN109943599B - Method for producing long-chain dicarboxylic acid by fermentation - Google Patents
Method for producing long-chain dicarboxylic acid by fermentation Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for producing long-chain dicarboxylic acid by fermentation, which comprises the following steps: in the fermentation process, the acid production rate is controlled to be 1.0-2.0 g/L/h. The method can obviously improve the fermentation conversion rate of the long-chain dicarboxylic acid, has low cost and simple operation, and is suitable for large-scale industrial production.
Description
Technical Field
The invention relates to the field of fermentation, in particular to a method for producing long-chain dicarboxylic acid by fermentation.
Background
The long-chain binary acid has very wide application, and can be used as a raw material to synthesize special polyamide, high-grade spice, high-grade hot melt adhesive, cold-resistant plasticizer, high-grade lubricating oil, high-grade antirust agent, high-grade paint, coating and the like. The long-chain dicarboxylic acid can be synthesized by a chemical method or a biological method. The chemical synthesis method has long synthesis route, high temperature and high pressure required by reaction and strict requirements on the catalyst, so that the long-chain dicarboxylic acid on the industrial scale has fewer varieties, and only a few varieties such as the twelve-carbon long-chain dicarboxylic acid exist. The biological method is to take long-chain alkane as a substrate, obtain the long-chain dibasic acid by microbial transformation, and ferment and prepare the long-chain dibasic acid by taking normal alkane as the substrate by utilizing the special function of microbial double-end oxidation. The mechanism research of the alkane oxidation by the microorganism to generate the long-chain dibasic acid can be known as follows: the main reaction is alpha, omega-oxidation, and the side reaction is alpha-oxidative decarboxylation and beta-oxidation, so that the long-chain dibasic acid is further oxidized and degraded. Beta-oxidation primarily provides energy for microbial growth and maintains the transformation activity of microbial cells, which results in incomplete conversion of the alkane substrate into the dibasic acid product, and the alkane conversion rate is critical to the production cost of the dibasic acid.
Chinese patents CN102808004A and CN1259424C disclose a method for improving the fermentation yield of long-chain dicarboxylic acid. The method improves the fermentation conversion rate by adding an alpha-oxidative decarboxylation or beta-oxidation inhibitor in the fermentation process, the used inhibitor is chlorpromazine hydrochloride, halogenated fatty acid and other reagents, the reagents are expensive, the extraction and refining process and cost of dibasic acid are increased, and the conversion rate is only improved to 89% (w/w) to the maximum extent. Chinese patent CN105755062A discloses a method for producing long-chain dicarboxylic acid by using oxidation-reduction potential to regulate and control fermentation process. The method realizes two-stage ORP control fermentation of long-chain dibasic acid by adjusting ventilation amount and stirring speed and combining an oxidant and a reductant, but does not mention yield of the dibasic acid. Chinese patent No. 103074325A discloses a method for mutagenizing Candida tropicalis with long-chain dibasic acid, which utilizes strain mutagenesis to screen high-yield strains, but does not mention dibasic acid conversion rate. The literature of foreign research on long-chain dibasic acids mostly carries out genetic modification on strains, and improves the yield of products by blocking or weakening related enzyme systems of fatty acid beta oxidation and strengthening enzyme systems of fatty acid alpha-omega oxidation.
In summary, in the prior art, the fermentation conversion rate of the process for producing the long-chain dicarboxylic acid by the fermentation method is generally low, while various methods for improving the fermentation conversion rate of the long-chain dicarboxylic acid have high cost and are not very obvious in effect, and no method which can improve the fermentation conversion rate of the long-chain dicarboxylic acid with high efficiency, is simple to operate and has low cost exists in the prior art.
Disclosure of Invention
In order to overcome the defects that the fermentation conversion rate of the long-chain dibasic acid is not high, a large amount of cost needs to be increased and the effect is not obvious in order to improve the fermentation conversion rate in the prior art, the invention provides the method for producing the long-chain dibasic acid by fermentation.
The invention aims to provide a method for producing long-chain dicarboxylic acid by fermentation, wherein the acid production rate is 1.0-2.0 g/L/h, preferably 1.1-1.7 g/L/h, and more preferably 1.2-1.5 g/L/h in the fermentation process.
As is well known to those skilled in the art, the acid production rate refers to the production rate of the long-chain dibasic acid in the fermentation process, and the acid production rate in the fermentation process is characterized by the mass of the long-chain dibasic acid produced in a unit volume of fermentation liquid per unit time.
In the prior art, a plurality of researches are carried out on a method for improving the fermentation conversion rate of long-chain dicarboxylic acid, but no report is provided for improving the fermentation conversion rate by controlling the acid production speed in the fermentation process. The inventor discovers that the long-chain dicarboxylic acid fermentation process comprises the following steps: the acid production speed has great influence on the fermentation conversion rate of the long-chain dibasic acid. The fermentation conversion rate of the long-chain dicarboxylic acid can be effectively improved by controlling the acid production rate within a specific range; on the basis, the inventors have studied this phenomenon in a deep level from the mechanism aspect and found that: the acid production rate has obvious influence on the fermentation conversion rate because the thallus density has influence on the conversion of a substrate, the generation of intermediate products, byproducts and the like, and the high fermentation conversion rate of the long-chain dicarboxylic acid can be realized only under certain balanced conditions; on one hand, the acid production speed is too high, the substrate conversion speed is too high, the supply of coenzyme NADPH is insufficient, the beta oxidation is enhanced, the substrate conversion rate is low, and the quality of the product is reduced due to excessive accumulation of the intermediate product hydroxycarboxylic acid; on the other hand, the acid production speed is too slow, the fermentation period is prolonged, the production intensity is reduced, and the production cost is increased. Therefore, the fermentation conversion rate of the long-chain dicarboxylic acid can be effectively improved by controlling the reasonable acid production speed.
The preferred embodiments of the above-described embodiments are described in detail above.
The control method of the acid production speed is not limited, and the acid production speed only needs to be controlled within a specific range; generally, conventional methods for controlling the acid production rate include, for example: the stirring operation, the fermentation temperature or the ventilation quantity are controlled, or the combination of several control means and the like are all suitable for the invention, and the technical effects of the invention can be realized by matching with other process operations and coordinating together.
According to a preferable technical scheme of the invention, the acid production speed can be controlled by controlling the stirring speed and matching with other process conditions. Those skilled in the art will appreciate that the rate of agitation is determined depending on the type of fermenter vessel, the shape of the paddles, the number of paddles, and the like.
In a preferable technical scheme of the invention, the stirring speed during fermentation is 500-1000 rpm. The stirring speed is characterized by the stirring rotating speed of the two layers of turbine disk blade stirring paddles, and more specifically, the stirring speed is characterized by the stirring rotating speed of the two layers of turbine disk blade stirring paddles used on a 10L stainless steel fermentation tank.
It should be noted that the present invention is not limited to the use of the stirring blade and the stirring speed, and any stirring blade that is conventional in the art can achieve a specific acid production rate in combination with a specific speed is within the scope of the present invention.
According to another preferred technical scheme, the acid production speed can be controlled by controlling the ventilation quantity and matching with other process conditions.
According to a preferable technical scheme of the invention, the ventilation volume in the fermentation process is 0.1-1 vvm, preferably 0.2-0.6 vvm; the aeration is characterized by the volume of air (L) introduced into the fermentation broth per unit volume (L) per unit time (min).
The invention can control the acid production speed not only by the above mode, but also by the combination of several modes.
According to a preferable technical scheme, the effect of better improving the fermentation conversion rate can be realized by controlling the joint regulation of the stirring speed and the ventilation quantity and matching with other fermentation process parameters. The stirring speed and the ventilation rate meet the following conditions:
0.6-0.00042A≤F≤0.8-0.00038A；
wherein: a represents the value of the stirring speed, which is a value in rpm; the stirring speed is characterized by the stirring rotating speed of the stirring paddles of the two layers of turbine disc blades;
f represents the value of the ventilation, which is a value in vvm.
In a preferred embodiment of the present invention, the fermented strain includes Candida Tropicalis (Candida Tropicalis) or Candida sake (Candida sake). For example: the yeast strain can be Candida Tropicalis (with the preservation number of CCTCC M203052), candida Tropicalis (with the preservation number of CCTCC M2011192), candida sake (CATN 145) or Candida sake (with the preservation number of CCTCC M2011486), candida sake (Candida sake) CATH4013 (with the preservation number of CCTCC M2011486), candida sake (Candida sake) CATH4014 (with the preservation number of CCTCC M2011), candida sake (Candida sake) CATH4012 (with the preservation number of CCTCC M2011485), candida sake (Candida sake) CATH4016 (with the preservation number of CCTCC M2011488), or Candida sake (Candida sake) CATH430 (with the preservation number of CCTCC M2011489).
In a preferred technical scheme of the invention, the substrate for fermentation comprises one or more of C9-C18 n-alkane, linear saturated fatty acid ester and linear saturated fatty acid salt; preferably comprises one or more of C11-C16 n-alkanes, linear saturated fatty acids, linear saturated fatty acid esters and linear saturated fatty acid salts; more preferably one of C11, C12, C13, C15, C14 or C16 n-alkane, linear saturated fatty acid ester and linear saturated fatty acid salt.
In a preferred embodiment of the present invention, the fermentation medium comprises: carbon source, nitrogen source, phosphorus source, trace metal element source and growth factor.
Wherein the carbon source comprises or is one or more of glucose, sucrose, maltose, molasses, methanol and ethanol; more preferably, the carbon source comprises or is one or more of glucose, sucrose. The concentration of the carbon source is preferably 20 to 60g/L.
Wherein, the nitrogen source comprises or is organic nitrogen and/or inorganic nitrogen, the organic nitrogen comprises but is not limited to one or more of yeast extract, peptone and corn steep liquor, and the inorganic nitrogen comprises but is not limited to one or more of urea, ammonium sulfate and potassium nitrate; preferably, the nitrogen source comprises or is ammonium sulfate and/or potassium nitrate. The concentration of the nitrogen source is preferably 0.5 to 10g/L. Preferably, the concentration of the ammonium sulfate is preferably 0.5 to 5g/L. Preferably, the concentration of the potassium nitrate is 0.5-5 g/L.
Wherein the phosphorus source comprises or is one or more of phosphate, monohydrogen phosphate and dihydrogen phosphate; preferably, the phosphate comprises or is one or more of potassium phosphate salt, sodium phosphate salt, ammonium phosphate salt; more preferably, the phosphate comprises or is monopotassium phosphate. The concentration of the phosphorus source is preferably 1 to 5g/L.
Wherein the trace metal element source comprises one or more of sulfate, hydrochloride and nitrate of potassium, calcium, magnesium, iron, copper, zinc and manganese. The concentration of the trace metal element source is preferably 0.1 to 50ppm.
Wherein the growth factor comprises one or more of amino acids, citric acid and vitamins; more preferably, the growth factor comprises or is one or a mixture of two of citric acid and biotin. The concentration of the growth factor is preferably 0.01-1 ppm.
According to a preferable technical scheme of the invention, the tank temperature during fermentation is 28-32 ℃.
In a preferable technical scheme of the invention, the tank pressure during fermentation is 0.05-0.14 Mpa, and the pressure is gauge pressure.
According to a preferable technical scheme of the invention, the fermentation is controlled to have pH of 5.0-8.5.
The stage of fermentation can be judged by the person skilled in the art on the basis of the general knowledge in the art. Generally, the early stage of the fermentation is from the beginning of the fermentation to 30 to 60 hours of fermentation. The later stage of fermentation is from the beginning of fermentation for 30-60 h to the end of fermentation.
According to a preferable technical scheme of the invention, the fermentation period is 100-180 h.
Detailed Description
The invention provides a method for improving the fermentation conversion rate of long-chain dibasic acid by controlling the acid production rate in the fermentation process. The acid production rate refers to the production rate of the long-chain dibasic acid in the fermentation process, and the fermentation acid production rate is characterized by the mass of the long-chain dibasic acid produced by fermentation liquor in unit time and unit volume.
. The invention controls the acid production rate in the fermentation process within a proper range by controlling the ventilation quantity and/or the stirring speed in the fermentation process and matching with other process parameters and conditions, thereby improving the fermentation conversion rate.
A method for producing long-chain dicarboxylic acid by fermentation comprises the following steps:
a) Activating strains;
b) Preparing a seed solution in a seed tank by using a seed culture medium;
c) Inoculating the seed liquid into a fermentation tank containing a fermentation culture medium, wherein the acid production rate is 1.0-2.0 g/L/h, preferably 1.1-1.7 g/L/h, and more preferably 1.2-1.5 g/L/h in the fermentation process; the acid production rate refers to the production rate of the long-chain dicarboxylic acid in the fermentation process, and is calculated by the mass of the long-chain dicarboxylic acid produced by unit volume of fermentation liquid in unit time.
In one embodiment of the present invention, a) strain activation comprises the steps of: taking the glycerol of Candida Tropicalis (Candida Tropicalis) or Candida sake (Candida sake) to be placed in a shake flask of a YPD culture medium, and carrying out shake culture at the rotating speed of 200-250 rpm and the culture temperature of 28-30 ℃ for 24-48 h.
In step A), the YPD medium comprises: 10g/kg of peptone, 5g/kg of yeast extract, 10g/kg of glucose and natural pH.
In one embodiment of the invention, the parameters of the seed tank in step B) are: the culture temperature is 28-30 ℃, the ventilation rate is 0.1-1 vvm, and the optimization is 0.2-0.6 vvm; the tank pressure is 0.08-0.1 MPa (gauge pressure), the culture time is 12-36 h, and the seed maturity index is OD 620 Is 15 to 30.
The seed medium in step B) is an aqueous medium, preferably comprising the following components: 10-30 g/L of cane sugar, 1.5-10 g/L of corn steep liquor, 1-10 g/L of yeast extract, 4-12 g/L of monopotassium phosphate, 0.5-5 g/L of urea and 0-30 ml/L of alkane, and preparing an aqueous solution culture medium by using water and sterilizing.
In one embodiment of the invention, in the step C), the temperature is controlled to be 28-32 ℃ in the fermentation process, the ventilation rate is 0.1-1 vvm, preferably 0.2-0.6 vvm (the volume of air introduced into the fermentation liquid in unit volume of unit time), the tank pressure is 0.05-0.14 MPa (gauge pressure), and the two layers of turbine disc blades are used for stirring at the stirring speed of 500-1000 rpm; adding 10-40% (w/v) NaOH solution to control the pH value of the fermentation liquor to be 5.0-8.5; adding the substrate in batches when the fermentation period is 10-20 h, wherein the total fermentation period is 100-180 h;
in one embodiment of the invention, in step C), the substrate comprises one or more of C9 to C18 n-alkanes, linear saturated fatty acids, linear saturated fatty acid esters, and linear saturated fatty acid salts; preferably comprises one or more of C11-C16 n-alkane, linear saturated fatty acid ester and linear saturated fatty acid salt; more preferably one of a C11, C12, C13, C15, C14 or C16 n-alkane, a linear saturated fatty acid ester and a linear saturated fatty acid salt;
in one embodiment of the invention, the fermentation medium in step C) comprises: carbon source, nitrogen source, phosphorus source, trace metal element source and growth factor;
the carbon source comprises or is one or more of glucose, sucrose, maltose, molasses, methanol and ethanol; more preferably, the carbon source comprises or is one or a mixture of glucose and sucrose; the carbon source concentration is 20-60 g/L.
The nitrogen source can be organic nitrogen and/or inorganic nitrogen, the organic nitrogen comprises but is not limited to one or more of yeast extract, peptone and corn steep liquor, and the inorganic nitrogen comprises but is not limited to one or more of urea, ammonium sulfate and potassium nitrate; preferably, the nitrogen source comprises or is one or a mixture of ammonium sulfate and potassium nitrate; the concentration of the nitrogen source is 0.5-5 g/L.
The phosphorus source comprises or is one or more of an ortho-phosphate salt, a mono-hydrogen phosphate salt and a di-hydrogen phosphate salt, preferably the phosphate comprises or is one or more of a potassium phosphate salt, a sodium phosphate salt, an ammonium phosphate salt; more preferably, the phosphate comprises or is monopotassium phosphate. The concentration of the phosphorus source is 1-5 g/L.
The trace metal source comprises one or more of sulfate, hydrochloride and nitrate of potassium, calcium, magnesium, iron, copper, zinc and manganese. The concentration of the trace element source is 0.1-50 ppm.
The growth factor comprises one or more of amino acid, citric acid and vitamin; preferably comprises or is one or the mixture of two of citric acid and biotin. The concentration of the growth factor is 0.01-1 ppm.
The present invention will be described in detail below with reference to examples to make the features and advantages of the present invention more apparent. It should be noted that the examples are for understanding the concept of the present invention and the scope of the present invention is not limited to only the examples listed herein.
Unless otherwise specified, the concentrations in the present invention are mass percent concentrations.
In the present invention, the concentration of the dibasic acid in the culture solution can be measured by a technique known to those skilled in the art, for example, the measurement method disclosed in Chinese patent ZL 95117436.3. Specifically, adjusting the pH of the fermentation broth to 3.0 by using a hydrochloric acid solution, adding 100mL of diethyl ether for extracting the dibasic acid in the fermentation broth, and removing the diethyl ether by evaporation to obtain dibasic acid powder; and dissolving the obtained dibasic acid powder in ethanol, and titrating by using 0.1mol/L NaOH solution to finally obtain the titration amount of the dibasic acid in the fermentation liquid.
In the present invention, "about" is used to modify numerical values in parameters of components, reaction conditions, concentrations, and the like, unless otherwise specified. The numerical parameters set forth in the specification and claims are therefore approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, this is not to be taken as a limitation on the application of the doctrine of equivalents to the scope of the claims. At the very least, numerical values for each of the parameters are determined by the number of significant digits recited. Although the numerical values in the specific examples are as accurate as possible, any data will have certain errors due to systematic errors in the experimental test methods.
Example 1
(1) Activating strains:
inoculating the glycerol tube strain of the Candida tropicalis into a shake flask filled with 30ml of seed culture medium, wherein the pH is natural, and the shake culture is carried out for 24 hours at the temperature of 29 ℃ and the rpm of 220; YPD medium comprises: 10g/kg of peptone, 5g/kg of yeast extract and 10g/kg of glucose;
(2) Preparing a seed solution:
taking shake flask seeds, inoculating the seeds into a seed tank filled with a seed culture medium, and inoculating the initial pH value of the systemAt 29 deg.C, ventilation rate of 0.5vvm, tank pressure of 0.1MPa, culturing for 18h, naturally reducing pH to 3 620 Up to 15;
(3) Fermentation:
inoculating the seeds into a fermentation tank containing 6L of fermentation medium (50 g/L glucose, 3g/L potassium nitrate, 4g/L potassium dihydrogen phosphate, 0.3g/L ammonium sulfate, 50ppm magnesium sulfate and 1ppm citric acid), controlling the temperature at 30 ℃, the ventilation amount at 0.5vvm and the tank pressure at 0.1MPa (gauge pressure) in the fermentation process, and stirring by using two layers of turbine disc blades at the stirring speed of 1000rpm; 30% (w/v) of liquid caustic soda is added in the fermentation process to control the pH value of the fermentation liquor; adding substrate n-dodecyl alkane in batches when the fermentation period is 10-20 h, and controlling the alkane content in the fermentation liquor to be below 10% (v/v); controlling the pH value of the fermentation to be 5.0-8.5, controlling the total fermentation period to be 130 hours, and basically controlling the content of residual hydrocarbon to be 0 when the fermentation is finished;
the product concentration of LCDA is 130mg/g, the acid production rate is 1.9g/L/h, and the alkane mass conversion rate is 85%.
Example 2
(1) Activating strains:
inoculating the glycerol tube strain of the Candida tropicalis into a shake flask filled with 30ml of seed culture medium, wherein the pH is natural, and the shake culture is carried out for 24 hours at the temperature of 29 ℃ and the rpm of 220; YPD medium comprises: 10g/kg of peptone, 5g/kg of yeast extract and 10g/kg of glucose;
(2) Preparing a seed solution:
inoculating shake flask seeds into a seed tank containing seed culture medium, inoculating to the seed tank, adjusting initial pH to 6.0, ventilating amount to 0.5vvm at 29 deg.C, tank pressure to 0.1MPa, culturing for 18 hr, naturally lowering pH to 3 620 Up to 15;
(3) And (3) fermentation:
inoculating seeds into a fermentation tank containing a fermentation culture medium (20 g/L of glucose, 2g/L of potassium nitrate, 3g/L of monopotassium phosphate, 2g/L of ammonium sulfate, 50ppm of magnesium sulfate and 1ppm of citric acid), controlling the temperature at 30 ℃, the ventilation quantity at 1.0vvm and the tank pressure at about 0.12MPa (gauge pressure) in the fermentation process, stirring by using a two-layer turbine disc blade stirring paddle, stirring at the rotating speed of 600rpm, and supplementing 30% (w/v) of liquid caustic soda in the fermentation process to control the pH value of the fermentation liquor to be 5.0-8.5; adding substrate n-dodecyl alkane in 6 batches when the fermentation period is 10-20 h, and controlling the alkane content in the fermentation liquor to be below 10% (v/v); the total fermentation period is 140 hours, and the residual hydrocarbon content is basically 0 at the end of fermentation;
the acid production concentration of LCDA is 132mg/g, the acid production rate is 1.8g/L/h, and the alkane mass conversion rate is 88%.
Example 3
(1) Activating strains:
inoculating the glycerol tube strain of the Candida tropicalis into a shake flask filled with 30ml of seed culture medium, wherein the pH is natural, and the shake culture is carried out for 24 hours at the temperature of 29 ℃ and the rpm of 220; YPD medium comprises: 10g/kg of peptone, 5g/kg of yeast extract and 10g/kg of glucose;
(2) Preparing a seed solution:
inoculating shake flask seeds into a seed tank containing seed culture medium, inoculating to the seed tank, adjusting initial pH to 6.0, ventilating amount to 0.5vvm at 29 deg.C, tank pressure to 0.1MPa, culturing for 18 hr, naturally lowering pH to 3 620 Up to 15;
(3) Fermentation:
inoculating seeds into a fermentation tank containing a fermentation medium (30 g/L of glucose, 2g/L of potassium nitrate, 3g/L of monopotassium phosphate, 2g/L of ammonium sulfate, 50ppm of magnesium sulfate and 1ppm of citric acid), controlling the temperature at 30 ℃, the ventilation quantity at 0.2vvm and the tank pressure at about 0.12MPa (gauge pressure) in the fermentation process, stirring by a two-layer turbine disc paddle stirring paddle, and stirring at the rotating speed of 1000rpm; 30% (w/v) of liquid caustic soda is added in the fermentation process to control the pH value of the fermentation liquor to be 5.0-8.5; adding substrate n-dodecyl alkane in batches when the fermentation period is 10-20 h, and controlling the alkane content in the fermentation liquid to be below 10% (v/v); the total fermentation period is 145 hours, and the residual hydrocarbon content at the end of the fermentation is basically 0;
the acid production concentration of LCDA is 150mg/g, the acid production rate is 1.45g/L/h, and the alkane mass conversion rate is 92%.
Example 4
(1) Activating strains:
inoculating the glycerol tube strain of the Candida tropicalis into a shake flask filled with 30ml of seed culture medium, wherein the pH is natural, and the shake culture is carried out for 24 hours at the temperature of 29 ℃ and the rpm of 220; YPD medium comprises: 10g/kg of peptone, 5g/kg of yeast extract and 10g/kg of glucose;
(2) Preparing a seed solution:
inoculating shake flask seed into seed tank containing seed culture medium, inoculating to seed tank, adjusting initial pH of inoculated system to 6.0, ventilating amount to 0.5vvm at 29 deg.C, tank pressure to 0.1MPa, culturing for 18h, naturally lowering pH to 3 in the culture process 620 Up to 15;
(3) Fermentation:
inoculating seeds into a fermentation tank containing a fermentation medium (30 g/L of glucose, 2g/L of potassium nitrate, 2.5g/L of monopotassium phosphate, 1.5g/L of ammonium sulfate, 50ppm of magnesium sulfate and 1ppm of citric acid), controlling the temperature of 30 ℃, the ventilation quantity to be 0.4vvm and the tank pressure to be about 0.12MPa (gauge pressure) in the fermentation process, stirring by a two-layer turbine disc paddle stirring paddle, and stirring at the rotating speed of 600rpm; 30% (w/v) of liquid caustic soda is added in the fermentation process to control the pH value of the fermentation liquor to be 5.0-8.5, and substrate n-dodecyl alkane is added in batches when the fermentation period is 10-20 h, so that the alkane content in the fermentation liquor is controlled to be below 10% (v/v); the total fermentation period is 160 hours, and the residual hydrocarbon content is basically 0 at the end of fermentation;
the acid production concentration of LCDA is 160mg/g, and the acid production speed is 1.3g/L/h. The alkane mass conversion rate was 94%.
Example 5
(1) Activating strains:
inoculating the glycerol tube strain of the Candida tropicalis into a shake flask filled with 30ml of seed culture medium, wherein the pH is natural, and the shake culture is carried out for 24 hours at the temperature of 29 ℃ and the rpm of 220; the YPD medium comprises: 10g/kg of peptone, 5g/kg of yeast extract and 10g/kg of glucose;
(2) Preparing a seed solution:
inoculating shake flask seed into seed tank containing seed culture medium, inoculating to seed tank, adjusting initial pH of inoculated system to 6.0, ventilating amount to 0.5vvm at 29 deg.C, tank pressure to 0.1MPa, culturing for 18h, naturally lowering pH to 3 in the culture process 620 Up to 15;
(3) And (3) fermentation:
inoculating seeds into a fermentation tank containing a fermentation medium (20 g/L of glucose, 2g/L of potassium nitrate, 2.5g/L of monopotassium phosphate, 1.5g/L of ammonium sulfate, 50ppm of magnesium sulfate and 1ppm of citric acid), controlling the temperature of 30 ℃, the ventilation amount to be 0.3vvm and the tank pressure to be about 0.12MPa (gauge pressure) in the fermentation process, stirring by a two-layer turbine disc paddle stirring paddle, and stirring at the rotating speed of 900rpm; 30% (w/v) of liquid caustic soda is added in the fermentation process to control the pH value of the fermentation liquor to be 5.0-8.5, and when the fermentation period is 10-20 h, the substrate n-dodecyl alkane is added in 7 batches, and the alkane content in the fermentation liquor is controlled to be below 10% (v/v); the total fermentation period is 160 hours, and the residual hydrocarbon content is basically 0 at the end of fermentation;
the acid production concentration of LCDA is 160mg/g, the acid production rate is 1.4g/L/h, and the alkane mass conversion rate is 95%.
Example 6
(1) Activating strains:
inoculating the glycerol tube strain of the Candida tropicalis into a shake flask filled with 30ml of seed culture medium, performing shake culture at 220rpm and 29 ℃ for 24 hours under natural pH; YPD medium comprises: 10g/kg of peptone, 5g/kg of yeast extract and 10g/kg of glucose;
(2) Preparing a seed solution:
inoculating shake flask seed into seed tank containing seed culture medium, inoculating to seed tank, adjusting initial pH of inoculated system to 6.0, ventilating amount to 0.5vvm at 29 deg.C, tank pressure to 0.1MPa, culturing for 18h, naturally lowering pH to 3 in the culture process 620 Up to 15;
(3) Fermentation:
inoculating seeds into a fermentation tank containing a fermentation medium (20 g/L of glucose, 2g/L of potassium nitrate, 2.5g/L of monopotassium phosphate, 1.5g/L of ammonium sulfate, 50ppm of magnesium sulfate and 1ppm of citric acid), adding 10% (v/v) of alkane, controlling the temperature of the fermentation process to be 30 ℃, controlling the ventilation quantity to be 0.3vvm and controlling the tank pressure to be about 0.12MPa (gauge pressure), and stirring by using a two-layer turbine disc paddle stirring paddle at the stirring speed of 800rpm; 30% (w/v) of liquid caustic soda is added in the fermentation process to control the pH value of the fermentation liquor to be 5.0-8.5, and the substrate n-tridecane is added in batches when the fermentation period is 10-20 h, so that the alkane content in the fermentation liquor is controlled to be less than or equal to 10% (v/v); the total fermentation period is 160 hours, and the residual hydrocarbon content is basically 0 at the end of fermentation;
the acid production concentration of LCDA is 150mg/g, and the acid production speed is 1.25g/L/h; alkane mass conversion was 93%.
Example 7
(1) Activating strains:
inoculating the glycerol tube strain of the Candida tropicalis into a shake flask filled with 30ml of seed culture medium, wherein the pH is natural, and the shake culture is carried out for 24 hours at the temperature of 29 ℃ and the rpm of 220; the YPD medium comprises: 10g/kg of peptone, 5g/kg of yeast extract and 10g/kg of glucose;
(2) Preparing a seed solution:
inoculating shake flask seed into seed tank containing seed culture medium, inoculating to seed tank, adjusting initial pH of inoculated system to 6.0, ventilating amount to 0.5vvm at 29 deg.C, tank pressure to 0.1MPa, culturing for 18h, naturally lowering pH to 3 in the culture process 620 Up to 15;
(3) Fermentation:
inoculating seeds into a fermentation tank containing a fermentation medium (20 g/L of glucose, 2g/L of potassium nitrate, 2.5g/L of monopotassium phosphate, 1.5g/L of ammonium sulfate, 50ppm of magnesium sulfate and 1ppm of citric acid), adding 10% (v/v) of alkane, controlling the temperature of the fermentation process to be 30 ℃, controlling the ventilation quantity to be 0.3vvm and controlling the tank pressure to be about 0.12MPa (gauge pressure), and stirring by using a two-layer turbine disc paddle stirring paddle at the stirring speed of 800rpm; 30% (w/v) of liquid caustic soda is added in the fermentation process to control the pH value of the fermentation liquor to be 5.0-8.5, the substrate n-hexadecane is added in batches when the fermentation period is 10-20 h, and the alkane content in the fermentation liquor is controlled to be less than or equal to 10% (v/v); the total fermentation period is 160 hours, and the residual hydrocarbon content is basically 0 at the end of fermentation;
the acid production concentration of LCDA is 120mg/g, and the acid production speed is 1.25g/L/h; the alkane mass conversion rate is 80 percent.
Claims (8)
1. A method for producing long-chain dicarboxylic acid by fermentation is characterized by comprising the following steps: the fermentation strain is Candida Tropicalis (Candida Tropicalis); in the fermentation process, the acid production rate is 1.1-1.7 g/L/h; the ventilation rate in the fermentation process is 0.2-0.6 vvm, the stirring speed during fermentation is 500-1000 rpm, and the stirring speed and the ventilation rate meet the following conditions:
0.6-0.00042A≤F≤0.8-0.00038A，
wherein A represents a value of a stirring speed in rpm, said stirring speed being characterized by a stirring rotation speed of a two-layer turbine disc blade stirring paddle, and F represents a value of a ventilation amount in vvm.
2. The method of claim 1, wherein: in the fermentation process, the acid production rate is 1.2-1.5 g/L/h.
3. The method of claim 1 or 2, wherein: the fermented strain is Candida tropicalis with a preservation number of CCTCC M203052 or Candida tropicalis CATN145 with a preservation number of CCTCC M2011192.
4. The method of claim 1, wherein: the substrate for fermentation comprises C11-C16 n-alkanes.
5. The method of claim 1, wherein: the tank temperature during fermentation is 28-32 ℃.
6. The method of claim 1, wherein: the tank pressure during fermentation is 0.05-0.14 Mpa, and the pressure is gauge pressure.
7. The method of claim 1, wherein: the pH value during fermentation is 5.0-8.5.
8. The method of claim 1, wherein: the fermentation period is 100-180 h.
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| CN1257126A (en) * | 1998-12-16 | 2000-06-21 | 中国石油化工集团公司 | Process for producing alpha, omega-long chain binary acid by using microorganism fermentation |
| CN1570124A (en) * | 2004-05-12 | 2005-01-26 | 上海凯赛生物技术研发中心有限公司 | Long chain normal dibasic acid production method |
| CN104862348A (en) * | 2014-02-26 | 2015-08-26 | 中国科学院过程工程研究所 | Method for fermentation-membrane separation combined production of long-chain dicarboxylic acid |
| CN104862347A (en) * | 2014-02-26 | 2015-08-26 | 中国科学院过程工程研究所 | Method for producing long-chain binary acid through fermentation separation coupling |
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| CN1257126A (en) * | 1998-12-16 | 2000-06-21 | 中国石油化工集团公司 | Process for producing alpha, omega-long chain binary acid by using microorganism fermentation |
| CN1570124A (en) * | 2004-05-12 | 2005-01-26 | 上海凯赛生物技术研发中心有限公司 | Long chain normal dibasic acid production method |
| CN104862348A (en) * | 2014-02-26 | 2015-08-26 | 中国科学院过程工程研究所 | Method for fermentation-membrane separation combined production of long-chain dicarboxylic acid |
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| PB01 | Publication | ||
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Address after: 4 / F, building 5, No. 1690, Cailun Road, Shanghai Free Trade Zone Applicant after: CATHAY R&D CENTER Co.,Ltd. Applicant after: CATHAY INDUSTRIAL BIOTECH Ltd. Address before: 200120 Shanghai Zhangjiang High Tech Park of Pudong New Area Cailun Road No. 5 No. 1690 Applicant before: CATHAY R&D CENTER Co.,Ltd. Applicant before: CATHAY INDUSTRIAL BIOTECH Ltd. |
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| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191030 Address after: 4 / F, building 5, No. 1690, Cailun Road, Shanghai Free Trade Zone Applicant after: CATHAY R&D CENTER Co.,Ltd. Applicant after: CIBT USA Address before: 201203 floor 4, building 5, No. 1690, Cailun Road, Shanghai pilot Free Trade Zone Applicant before: CATHAY R&D CENTER Co.,Ltd. Applicant before: CATHAY INDUSTRIAL BIOTECH Ltd. |
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| GR01 | Patent grant | ||
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| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231130 Address after: 4 / F, building 5, No. 1690, Cailun Road, free trade zone, Pudong New Area, Shanghai Patentee after: CATHAY R&D CENTER Co.,Ltd. Patentee after: CIBT USA Patentee after: Kasai (Shanghai) Biotechnology Co.,Ltd. Address before: 4 / F, building 5, No. 1690, Cailun Road, Shanghai pilot Free Trade Zone, 201203 Patentee before: CATHAY R&D CENTER Co.,Ltd. Patentee before: CIBT USA |