CN116410873A - Method for producing long-chain dibasic acid by continuous fermentation - Google Patents

Method for producing long-chain dibasic acid by continuous fermentation Download PDF

Info

Publication number
CN116410873A
CN116410873A CN202111662222.6A CN202111662222A CN116410873A CN 116410873 A CN116410873 A CN 116410873A CN 202111662222 A CN202111662222 A CN 202111662222A CN 116410873 A CN116410873 A CN 116410873A
Authority
CN
China
Prior art keywords
fermentation
substrate
groups
liquid
acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111662222.6A
Other languages
Chinese (zh)
Inventor
郝英利
赵宏图
鲍家伟
刘修才
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cathay R&D Center Co Ltd
CIBT America Inc
Original Assignee
Cathay R&D Center Co Ltd
CIBT America Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cathay R&D Center Co Ltd, CIBT America Inc filed Critical Cathay R&D Center Co Ltd
Priority to CN202111662222.6A priority Critical patent/CN116410873A/en
Publication of CN116410873A publication Critical patent/CN116410873A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Mycology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention provides a method for producing long-chain dibasic acid by continuous fermentation, which comprises the following steps: after 85-145h of fermentation, continuously feeding substrate and nutrient salt-containing solution into the fermentation liquid, and discharging part of the fermentation liquid for continuous fermentation, wherein the dissolved oxygen is controlled to be more than 10% in the continuous fermentation process. According to the invention, the substrate and the solution containing the nutritive salt are continuously added into the fermentation liquid, part of the fermentation liquid flows out, and the flowing fermentation liquid is continuously fermented, so that the metabolic activity of the thalli is exerted, the continuous production of long-chain dibasic acid can be maintained, the conversion rate of the substrate is improved, the residue of the substrate can be reduced, the quality of the fermentation liquid is improved, and the separation and purification of the downstream process of the fermentation liquid are facilitated.

Description

Method for producing long-chain dibasic acid by continuous fermentation
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for producing long-chain dibasic acid by continuous fermentation.
Background
The long-chain dibasic acid has wide application in various fields due to the uniqueness and reactivity of molecular structures, and can be used as a raw material for synthesizing special nylon (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.
At present, the synthesis of long-chain dibasic acid mainly comprises two methods of a chemical synthesis method and a biological fermentation method, the chemical synthesis method is mature in technology and long in synthetic route, but the synthesis needs to be carried out under the conditions of high temperature and high pressure, the requirements on the catalyst are severe, and the synthesis of the long-chain dibasic acid is limited to the synthesis of dibasic acid with specific chain length; the biological fermentation method uses long-chain alkane or fatty acid as substrate, and uses microbial fermentation to obtain long-chain dibasic acid, its production process is implemented under normal temp. and pressure, and can implement large-scale production, for example from C 9 To C 18 And a plurality of long chain dibasic acids.
CN106755146B discloses a method and a device for producing long-chain dibasic acid by continuous fermentation, which are coupled with a seed tank through a membrane filtering device, and a continuous fermentation process is realized by periodically supplementing fresh seed liquid.
Disclosure of Invention
The invention aims to improve the production efficiency of long-chain dicarboxylic acid, and provides a method for producing long-chain dicarboxylic acid by continuous fermentation.
The method comprises the following steps: after 85-145h of fermentation, continuously feeding substrate and nutrient salt-containing solution into the fermentation liquid, and discharging part of the fermentation liquid for continuous fermentation, wherein the dissolved oxygen is controlled to be more than 10% in the continuous fermentation process.
According to the technical scheme, the substrate and the solution containing the nutritive salt are continuously added into the fermentation liquid, and meanwhile, part of the fermentation liquid flows out to continue fermentation, so that the metabolic activity of thalli is improved, the continuous production of long-chain dibasic acid is ensured, and the conversion rate is improved. And the quality of the fermentation liquor can be improved, alkane residues in the fermentation liquor are low, and the fermentation liquor is more easily purified.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
As previously described, a first aspect of the present invention provides a process for continuous fermentation production of long chain dibasic acids, the process comprising: after 85-145h of fermentation, continuously feeding substrate and nutrient salt-containing solution into the fermentation liquid, and discharging part of the fermentation liquid for continuous fermentation, wherein the dissolved oxygen is controlled to be more than 10% in the continuous fermentation process.
In the present invention, the cell concentration is measured by a spectrophotometer.
In some embodiments of the invention, the means for tapping off a portion of the fermentation broth is continuous or batch wise.
In some embodiments of the invention, the concentration of the cells in the fermentation broth, OD620, remaining after the partial fermentation broth is tapped off is controlled to be 0.3-0.8 after dilution by a factor of 30.
In some embodiments of the invention, the volume of the remaining fermentation broth is controlled to be 0.5-2 times, further 1-1.8 times the volume of the fermentation broth before feeding by controlling the discharging speed.
In some embodiments of the invention, the substrate is present in the fermentation broth at a level of 1% (v/v) or more prior to feeding.
In some embodiments of the invention, the substrate content in the fermentation broth prior to feeding is less than 8% (v/v).
In the invention, the dissolved oxygen amount of the fermentation is measured by an on-line dissolved oxygen electrode.
As a preferred embodiment of the invention, the dissolved oxygen content in the continuous fermentation process is controlled to be more than 20%, and further 30% -100%.
When the dissolved oxygen is controlled in the above range in the continuous fermentation process, the fermentation yield and the equipment utilization efficiency can be improved.
The temperature is controlled to be 28-32 ℃ in the continuous fermentation process; and/or, the pressure is 0.05-0.14MPa; and/or a pH of 5.5 to 7.5, further 5.5 to 6.7; and/or the ventilation is 0.3-0.7vvm. The residual hydrocarbon content of the discharged fermentation liquid after fermentation is 5% (v/v) or less, and further 3% (v/v) or less. In the invention, the pressure is gauge pressure.
In some embodiments of the invention, the acceleration of flow of the substrate, the acceleration of flow of the nutrient salt-containing solution, and the volume of the fermentation broth satisfy the following relationship:
0.005h -1 * V (fermentation liquor) is less than or equal to Q (nutrient salt-containing solution) is less than or equal to 0.3h -1 * V (fermentation broth);
0.001h -1 * V (fermentation liquor) is less than or equal to Q (substrate) and less than or equal to 0.006h -1 * V (fermentation broth);
wherein V (fermentation broth) represents the volume of fermentation broth in units of: and (3) mL. Q (nutrient salt-containing solution) represents the flow acceleration of the nutrient salt-containing solution in units of: mL/h, Q (substrate) represents the flow acceleration of the substrate in units of: mL/h.
Preferably, the acceleration of flow of the nutrient salt-containing solution and the volume of the fermentation broth satisfy: 0.006h -1 * V (fermentation liquor) is less than or equal to Q (solution containing nutrient salt) is less than or equal to 0.15h -1 * V (fermentation broth).
Preferably, the flow acceleration of the substrate and the volume of the fermentation broth satisfy: 0.001h -1 * V (fermentation liquor) is less than or equal to Q (substrate) and less than or equal to 0.004h -1 * V (fermentation broth).
Dilution ratio d= [ Q (nutrient salt-containing solution) +q (substrate) ]/V (fermentation broth).
In some embodiments of the invention, the dilution rate D also has a certain influence on the bacterial activity, the yield and the yield of the dibasic acid, and the bacterial activity, the yield and the yield of the dibasic acid can be further improved by optimizing the dilution rate. Preferably, the dilution rate D of the fermentation broth is maintained at 0.002 to 0.3h by feeding the substrate and the nutrient salt-containing solution into the fermentation broth -1 Further 0.002-0.15h -1 Further for 0.002-0.10h -1 Further 0.002-0.05h -1 。
In some embodiments of the invention, the nutrient salt-containing solution contains 0-2.5 (w/v) glucose, 0.07-2.0 (w/v) corn steep liquor, 0-1.0 (w/v) yeast extract, 0.01-0.5 (w/v) monopotassium phosphate, 0.01-0.5 (w/v) urea, and 0.05-0.3 (w/v) ammonium sulfate. In the present invention, the unit "(w/v)%" means a mass-to-volume ratio, i.e., a ratio of the mass of each component to the volume of the nutrient salt-containing solution, the mass being in g and the volume being in 100 mL.
The range of selection of the substrate according to the invention is relatively broad, preferably the substrate is selected from C 9 -C 18 At least one of the normal alkane, linear saturated fatty acid ester and linear saturated fatty acid salt, preferably C 10 -C 16 For example, including but not limited to, undecane, dodecene, tridecane, hexadecane, and the like. In this preferred case, the yield and the yield of the long-chain dibasic acid can be further improved.
In some embodiments of the present invention, preferably, the long chain dibasic acid has the expression HOOC (CH 2 ) n COOH, wherein n is not less than 7, more preferably at least one of azelaic acid, sebacic acid, 1, 11-undecanoic acid, 1, 12-dodecanoic acid, 1, 13-tridecanoic acid, 1, 14-tetradecanoic acid, 1, 15-pentadecanoic acid, 1, 16-hexadecanoic acid, 1, 17-heptadecanoic acid and 1, 18-octadecanoic acid.
The equipment used in the continuous fermentation production of the long-chain dibasic acid is not particularly limited, and may be conventional equipment in the art, and fermentation in a fermenter is preferred.
Further, the method comprises the steps of:
(1) Inoculating seed liquid containing fermentation strain into fermentation medium, fermenting in the presence of substrate;
(2) After the fermentation in step (1) is carried out for 85-145h, feeding is carried out by continuously feeding substrate and nutrient salt-containing solution into the fermentation broth, and part of the fermentation broth is discharged for continuous fermentation.
In the step (1), the seed liquid is inoculated in an amount of 10 to 30% (v/v) relative to the fermentation initiation volume.
The temperature is controlled to be 28-32 ℃ in the fermentation process in the step (1); and/or, the pressure is 0.05-0.14MPa; and/or a pH of 5.5 to 7.5, further 5.5 to 6.7; and/or the ventilation is 0.3-0.7vvm; and/or, the dissolved oxygen amount is more than 10%; and/or, the concentration of the fermented cells OD 620 9-24. The pressure is gauge pressure.
Further, the concentration of the cells fermented in the step (1) is 0.3 to 0.8 after 30-fold dilution.
Further, the fermentation in the step (1) is carried out in a fermentation tank I.
In the step (1), the substrate is added in an amount of 10% (v/v) or less, and further 1 to 5% (v/v) relative to the fermentation initiation volume.
According to some embodiments of the invention, in step (1), the substrate is fed to the fermentation broth in batches or continuously during the fermentation process to ensure a substrate content in the fermentation broth of 1% (v/v) or more and 8% (v/v) or less.
Preferably, in the step (1), after fermentation for 10 to 100 hours, further 10 to 50 hours, further 15 to 40 hours, a substrate is added to the fermentation broth to ensure that the concentration thereof is 1% (v/v) or more and 8% (v/v) or less. The mode of adding the substrate is not particularly limited, and may be added in portions, continuously or in whole, and may be selected as desired by those skilled in the art according to actual conditions.
The continuous fermentation in the step (2) is carried out in a fermentation tank II; the fermentation tank II is fed continuously or batchwise. The discharging mode of the fermentation tank II adopts continuous discharging or batch discharging.
Because the alkane substrate and the dibasic acid obtained by conversion are of long carbon chain structures, when alkane residues in the fermentation broth are low, the dibasic acid in the fermentation broth is more easily purified.
In one embodiment, the residual hydrocarbon content of the fermentation broth discharged from fermentor II is less than 5% (v/v), and more preferably less than 3% (v/v).
As will be appreciated by those skilled in the art, the seed solution is incubated in the seed medium prior to inoculation into the fermentation medium, when the cell concentration OD is determined 620 Stopping culturing when the dilution is 0.5-1.0, namely the seed liquid is mature seed liquid, and then inoculating the mature seed liquid into the fermentation culture medium in the step (1). The culturing process can be a culturing mode of two or more stages. For example, the fermentation cells may be cultured in a shake flask, and the seed solution obtained by the shake flask culture may be transferred into a seed tank for culturing to a cell concentration OD 620 Diluting by 30 times to 0.5-1.0 to obtain mature seed liquid.
In the present invention, the method of culturing the seed liquid is not particularly limited, and a seed liquid culturing method conventional in the art may be employed, and for example, includes but is not limited to culturing by employing the following methods:
(i) The shake flask seed culture process comprises the following steps: inoculating fermentation strain into triangular flask containing seed culture medium, shake culturing at 28-32deg.C and optional initial pH of 6.0-6.5 at 200-250rpm for 1-2 days;
(ii) The seed tank culture process comprises the following steps: inoculating shake flask seeds into a seed tank filled with seed culture medium, controlling inoculation amount to 10-30% (v/v), controlling temperature to 28-32deg.C, pressure (gauge pressure) to 0.05-0.14MPa, ventilation amount to 0.3-0.7vvm, controlling pH value to 3.0-7.5 by adding 10-40% (w/w) liquid alkali, maintaining stirring speed to make dissolved oxygen DO in seed culture process be above 10%, and culturing for 15-30 hr to make thallus concentration OD 620 And diluting the mixture by 30 times to obtain 0.5-1.0, namely mature seed liquid. Mature seed liquid is used for producing long chain dibasic acid.
In the present invention, the fermentation process may be performed under stirring.
The selection range of the fermentation strain is wider, and candida tropicalis or candida viscidosa are preferred. For example, candida tropicalis (Candida tropicalis) strain CAT H1614, which is deposited under the accession number cctccc M2013143 (see CN110218661 a); candida viscidosa (Candida viswanathii) strain CAES2113, accession number cctccc M2020048 (see CN111748480 a).
In the present invention, three media of a seed medium, a fermentation medium and a fermentation feed medium are mainly used, and the components of each medium are not particularly limited, and medium components commonly used in the art may be employed, for example, including but not limited to employing the following 3 media:
seed culture medium: sucrose content of 1-3.5 (w/v), corn steep liquor content of 0.15-1.5 (w/v), yeast extract content of 0.2-1.5 (w/v), KH 2 PO 4 The content of (C) is 0.4-1.5 (w/v)%, and the content of urea is 0.05-0.7 (w/v)%.
Fermentation medium: glucose content of 1-6.0 (w/v), corn steep liquor content of 0.05-0.9 (w/v), yeast extract content of 0.1-0.5 (w/v), potassium nitrate content of 0.05-1.5 (w/v), potassium dihydrogen phosphate content of 0.05-1.0 (w/v), urea content of 0.05-0.5 (w/v), ammonium sulfate content of 0.05-0.3 (w/v), sodium chloride content of 0.05-0.4 (w/v).
Feed medium: comprises a substrate and a nutrient salt-containing solution, wherein the nutrient salt-containing solution contains 0-2.5 (w/v) percent of glucose, 0.05-2.0 (w/v) percent of corn steep liquor, 0-1.0 (w/v) percent of yeast extract, 0.005-0.5 (w/v) percent of monopotassium phosphate, 0.005-0.08 (w/v) percent of urea and 0.01-0.2 (w/v) percent of ammonium sulfate. All the above culture media were sterilized at 121℃for 20min for use.
In order to clearly describe the production method of long-chain dibasic acid according to the present invention, the following provides a preferred embodiment for explanation:
(1) Inoculating seed liquid and substrate containing fermentation strain into a fermentation tank I containing fermentation culture medium, wherein the inoculation amount of the seed liquid is 10-30% (v/v) based on the initial volume of fermentation, adding substrate into the seed liquid before or after inoculation, and relative to the volume of the fermentation culture medium or the initial volume of fermentation, the addition amount of the substrate is 1-5% (v/v), then controlling the temperature to be 28-32 ℃, the pressure to be 0.05-0.14MPa, the ventilation amount to be 0.3-0.7vvm, controlling the pH value of the fermentation liquid to be 5.5-7.5 for fermentation in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding substrate into the fermentation liquid after fermentation for 10-100h to ensure that the content of the substrate in the fermentation liquid is more than 1% (v/v);
(2) After 80-150h of fermentation, continuously adding substrate and nutrient salt-containing solution into the fermentation broth, and controlling the concentration OD of the fermented thallus 620 The dilution is 0.3-0.8 after 30 times, part of fermentation liquor flowing out of the fermentation tank I enters the fermentation tank II for continuous fermentation, and the volume of the fermentation liquor in the fermentation tank I is maintained to be 1-2 times, further 1-1.8 times of the fermentation initial volume before feeding by controlling the discharging rate of the fermentation tank I.
The fermentation liquor flowing out of the fermentation tank I is continuously fermented in the fermentation tank II, the dissolved oxygen amount is controlled to be more than 10% in the continuous fermentation process, the temperature is 28-32 ℃, the pressure (gauge pressure) is 0.05-0.14MPa, the pH value in the fermentation process is 5.5-7.5, and the ventilation rate is 0.3-0.7vvm. The residual hydrocarbon content of the discharged material after fermentation is below 5%, and is further below 3%.
Further, the flow acceleration is controlled such that the flow acceleration of the substrate and the volume of the fermentation broth satisfy: 0.001h -1 * V (fermentation liquor) is less than or equal to Q (substrate) and less than or equal to 0.004h -1 * V (fermentation broth); the flow acceleration of the nutrient salt-containing solution and the volume of the fermentation broth are as follows: 0.006h -1 * V (fermentation liquor) is less than or equal to Q (solution containing nutrient salt) is less than or equal to 0.15h -1 * V (fermentation broth).
The volume of the fermentation liquor in the fermentation tank I is maintained at 15-25L by controlling the discharging rate.
In the present invention, the method for measuring the concentration of the dibasic acid in the fermentation broth is not particularly limited, and a technique known to those skilled in the art can be used, for example, a measurement method disclosed in chinese patent ZL 95117436.3. Specifically, the pH value of the fermentation broth is regulated to 3.0 by using hydrochloric acid solution, then 100mL of diethyl ether is added for extracting the dibasic acid in the fermentation broth, and evaporation is adopted to remove the diethyl ether, so as to obtain dibasic acid powder; and dissolving the obtained diacid powder in ethanol, and titrating with 0.1mol/L NaOH solution to finally obtain the diacid titration amount in the fermentation broth.
The present invention will be described in detail by examples. In the examples below, various raw materials used were available from commercial sources without particular explanation. In the invention, the pressure is gauge pressure.
The initial volume of fermentation is equal to the total volume of the fermentation medium after inoculating the seed liquid and adding the substrate.
The total yield of the long-chain dibasic acid= (amount of long-chain dibasic acid produced (g)/amount of substrate added (g)) ×100%;
yield of long chain diacid = amount of long chain diacid produced (g)/broth starting volume (L)/continuous fermentation time (h);
dilution ratio d= [ Q (nutrient salt-containing solution) +q (substrate)]V (broth), wherein V (broth) broth volume (mL), Q (nutrient salt-containing solution) represents nutrient salt-containing solution flow acceleration (mL/h), and Q (substrate) represents substrate flow acceleration (mL/h). For example: calculated, dilution ratio d= (45.5 mL/h+163.5 mL/h)/(22000 mL) =0.0095 h in example 1 -1 。
Continuous fermentation time: inoculating seed liquid in a fermentation tank I until stopping the fermentation tank.
EXAMPLE 1 fermentation of dodecanedioic acid
Strain 1
A candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 Medium
Seed culture medium: sucrose content of 1.1 (w/v), corn steep liquor content of 0.30 (w/v), yeast extract content of 0.62 (w/v), KH 2 PO 4 The content of (C) was 0.8 (w/v)%, and the content of urea was 0.24 (w/v)%.
Fermentation medium: glucose content was 2.1 (w/v), corn steep liquor content was 0.20 (w/v), yeast extract content was 0.26 (w/v), potassium nitrate content was 0.05 (w/v), potassium dihydrogen phosphate content was 0.07 (w/v), urea content was 0.16 (w/v), ammonium sulfate content was 0.20 (w/v), and sodium chloride content was 0.11 (w/v).
Feed medium: comprises a substrate and a solution containing nutritive salt, wherein the substrate is the carbadodecyl benzene; the nutrient salt-containing solution contained 0.3 (w/v) percent corn steep liquor, 0.21 (w/v) percent yeast extract, 0.008 (w/v) percent monopotassium phosphate, 0.06 (w/v) percent urea and 0.05 (w/v) percent ammonium sulfate.
3 culture method
(i) The shake flask seed culture process comprises the following steps: inoculating a glycerol tube strain of candida tropicalis into a 500mL triangular flask (the liquid loading amount is 62 mL) filled with a seed culture medium, and performing shake culture at a rotating speed of 250rpm for 1 day under the condition that the temperature is 32 ℃;
(ii) The seed tank culture process comprises the following steps: taking shake flask seeds, inoculating into a 10L seed tank (containing 5L of seed culture medium), controlling the inoculation amount to 10% (v/v), controlling the temperature to 32 ℃, the pressure to 0.10MPa, the ventilation amount to 0.5vvm, controlling the pH value to 6.2 by adding 40 (w/w)% of liquid alkali, and maintaining a certain stirring speed to ensure that the dissolved oxygen DO in the seed culture process is above 10%, and culturing for 15h to ensure that the thallus concentration OD 620 And diluting the mixture by 30 times to obtain 0.8, namely mature seed liquid.
4 method for producing long chain dibasic acid
(1) Inoculating the seed liquid and the substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 15L, the inoculation amount of the seed liquid is 25% (v/v), the addition amount of the carbadodecyl benzene is 5.1% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.52vvm, controlling the pH value of the fermentation liquid to 6.1 in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding the carbadodecyl benzene in batches after the fermentation for 25h so that the content of the carbadodecyl benzene in the fermentation liquid is more than 2% (v/v); the inoculum size (v/v) of the seed liquid and the addition amount (v/v) of the carbadode alkane are relative to the fermentation starting volume.
(2) Continuously adding the laurocapram into the fermentation liquid at a flow acceleration of 45.5mL/h after 125h fermentation, simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 163.5mL/h, and controlling the concentration OD of the thallus in the fermentation tank I 620 The dilution is 0.65 after 30 times, and the volume of the fermentation liquid in the fermentation tank I is kept at 22L by controlling the discharging rate, so that the 1, 12-dodecadiacid is continuously produced in the fermentation tank I.
(3) And (3) conveying the fermentation liquor discharged in the step (2) into a fermentation tank II for fermentation culture, controlling the dissolved oxygen in the fermentation tank II to be 55% -70% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.14MPa, the pH value to be 6.1 and the ventilation to be 0.7vvm, controlling the volume of the fermentation liquor in the fermentation tank II to be within 20L through continuous or batch discharging, and continuously fermenting in the fermentation tank II to produce the 1, 12-dodecadiacid.
The continuous fermentation time was 625 hours, and the total yield of 1, 12-dodecanedioic acid, the yield and the residual hydrocarbon content in the discharged material were recorded or calculated, and the results are shown in Table 1.
EXAMPLE 2 fermentation of tridec dibasic acid
Strain 1
A candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 Medium
Seed culture medium: sucrose content of 3.0 (w/v), corn steep liquor content of 1.0 (w/v), yeast extract content of 0.21 (w/v), KH 2 PO 4 The content of (C) was 0.92 (w/v)%, and the content of urea was 0.51 (w/v)%.
Fermentation medium: glucose content was 5.0 (w/v), corn steep liquor content was 0.7 (w/v), yeast extract content was 0.33 (w/v), potassium nitrate content was 0.53 (w/v), potassium dihydrogen phosphate content was 0.60 (w/v), urea content was 0.19 (w/v), ammonium sulfate content was 0.2 (w/v), and sodium chloride content was 0.15 (w/v).
Feed medium: comprises a substrate and a solution containing nutrient salt, wherein the substrate is carbon tridecane; the nutrient salt-containing solution contained 0.5 (w/v) glucose, 0.11 (w/v) corn steep liquor, 0.008 (w/v) monopotassium phosphate, 0.007 (w/v) urea and 0.16 (w/v) ammonium sulfate.
3 culture method
(i) The shake flask seed culture process comprises the following steps: inoculating a glycerol tube strain of candida tropicalis into a 500mL triangular flask (the liquid loading amount is 100 mL) filled with a seed culture medium, and performing shake culture at 200rpm for 1 day under the conditions that the initial pH value is 6 and the temperature is 30 ℃;
(ii) The seed tank culture process comprises the following steps: taking shake flask seeds, inoculating into a 10L seed tank (containing 8L of seed culture medium), controlling the inoculation amount to 30% (v/v), controlling the temperature to 30 ℃, the pressure to 0.14MPa, the ventilation amount to 0.3vvm, controlling the pH value to 7 by adding 10 (w/w)% of liquid alkali, and maintaining a certain stirring speed to ensure that the dissolved oxygen DO in the seed culture process is above 10%, and culturing for 30h to ensure that the thallus concentration OD 620 And diluting the mixture by 30 times to obtain 1.0, namely mature seed liquid.
4 method for producing long chain dibasic acid
(1) Inoculating the seed solution and the substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 15L, the inoculation amount of the seed solution is 25% (v/v), the addition amount of the tridecane is 5.1% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.11MPa, the ventilation amount to 0.54vvm, controlling the pH value of the fermentation liquid to 5.7 in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding the tridecane in batches after fermenting for 25h so that the content of the tridecane in the fermentation liquid is more than 2% (v/v); the inoculum size (v/v) of the seed liquid and the addition amount (v/v) of the carbon tridecane are relative to the fermentation starting volume.
(2) After 115h of fermentation, continuously adding the tridecane alkane into the fermentation liquid at a flow acceleration of 25.8mL/h, simultaneously continuously adding the solution containing the nutrient salt into the fermentation liquid at a flow acceleration of 117.5mL/h, and controlling the concentration OD of the thallus in the fermentation tank I 620 The dilution is 0.50 after 30 times, and the volume of the fermentation liquid in the fermentation tank I is kept at 22L by controlling the discharging rate, so that the 1, 13-tridecanedioic acid is continuously produced in the fermentation tank I.
(3) And (3) conveying the fermentation liquor discharged in the step (2) into a fermentation tank II for fermentation culture, controlling the dissolved oxygen in the fermentation tank II to be 45% -65% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.14MPa, the pH value to be 5.7 and the ventilation to be 0.75vvm, controlling the volume of the fermentation liquor in the fermentation tank II to be within 20L through continuous or batch discharging, and continuously fermenting in the fermentation tank II to produce the 1, 13-tridecanedioic acid.
The continuous fermentation time was 617h, and the total yield of 1, 13-tridecanedioic acid, the yield and the residual hydrocarbon content in the discharged material were recorded or calculated, and the results are shown in Table 1.
EXAMPLE 3 fermentation of hexadecanoic dibasic acid
Strain 1
A candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 Medium
Seed culture medium: sucrose content of 1.3 (w/v), corn steep liquor content of 0.35 (w/v), yeast extract content of 0.46 (w/v), KH 2 PO 4 The content of (C) was 1.1 (w/v)%, and the content of urea was 0.52 (w/v)%.
Fermentation medium: glucose content was 4.4 (w/v), corn steep liquor content was 0.1 (w/v), yeast extract content was 0.15 (w/v), potassium nitrate content was 1.2 (w/v), potassium dihydrogen phosphate content was 0.76 (w/v), urea content was 0.31 (w/v), ammonium sulfate content was 0.18 (w/v), and sodium chloride content was 0.09 (w/v).
Feed medium: comprises a substrate and a solution containing nutrient salt, wherein the substrate is carbon hexadecane; the nutrient salt-containing solution contained 0.15 (w/v) percent corn steep liquor, 0.015 (w/v) percent monopotassium phosphate, 0.014 (w/v) percent urea and 0.15 (w/v) percent ammonium sulfate.
3 culture method
(i) The shake flask seed culture process comprises the following steps: inoculating a glycerol tube strain of candida tropicalis into a 500mL triangular flask (the liquid loading amount is 50 mL) filled with a seed culture medium, and performing shake culture for 1.5 days at a rotating speed of 240rpm under the conditions that the initial pH value is 6.3 and the temperature is 28 ℃;
(ii) The seed tank culture process comprises the following steps: taking shake flask seeds, inoculating into a 10L seed tank (containing 5L of seed culture medium), controlling the inoculum size to 30v%, controlling the temperature to 28 ℃, the pressure to 0.08MPa, the ventilation to 0.6vvm, controlling the pH value to 7.4 by adding 15 (w/w)% liquid alkali, maintaining a certain stirring speed, so that the dissolved oxygen DO in the seed culture process is above 10%, and culturing for 28h, so that the thallus concentration OD 620 And diluting the mixture by 30 times to obtain 0.92 which is mature seed liquid.
4 method for producing long chain dibasic acid
(1) Inoculating the seed solution and the substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 15L, the inoculation amount of the seed solution is 25% (v/v), the addition amount of the hexadecane is 5.1% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.52vvm, controlling the pH value of the fermentation liquid to 5.9 in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding the hexadecane in batches after the fermentation for 25h so that the content of the hexadecane in the fermentation liquid is more than 2% (v/v); the inoculum size (v/v) of the seed liquid and the addition amount (v/v) of the hexadecane were relative to the fermentation starting volume.
(2) Continuously adding hexadecane into the fermentation liquid at a flow acceleration of 47.2mL/h after fermenting for 95h, simultaneously continuously adding the solution containing nutritive salt into the fermentation liquid at a flow acceleration of 132mL/h, and controlling the thallus concentration OD in the fermentation tank I 620 The dilution is 0.65 after 30 times, and the volume of the fermentation liquid in the fermentation tank I is kept at 22L by controlling the discharging rate, so that 1, 16-hexadecanoic dibasic acid is continuously produced in the fermentation tank I.
(3) And (3) conveying the fermentation liquor discharged in the step (2) into a fermentation tank II for fermentation culture, controlling the dissolved oxygen in the fermentation tank II to be 35% -60% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.14MPa, the pH value to be 5.9 and the ventilation to be 0.82vvm, controlling the volume of the fermentation liquor in the fermentation tank II to be within 20L through continuous or batch discharging, and continuously fermenting in the fermentation tank II to produce the 1, 16-hexadecanoic dibasic acid.
The continuous fermentation time was 621h, and the total yield of 1, 16-hexadecanoic acid, the yield and the residual hydrocarbon content in the discharged material were recorded or calculated, and the results are shown in Table 1.
EXAMPLE 4 fermentation of dodecadiacid
The strain, medium and seed culture method of this example are the same as in example 1, except that the method for producing long chain dibasic acid in step 4 is as follows:
(1) Inoculating the seed liquid and the substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 15L, the inoculation amount of the seed liquid is 25% (v/v), the addition amount of the carbadodecyl benzene is 5.1% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.52vvm, controlling the pH value of the fermentation liquid to 5.7 in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding the carbadodecyl benzene in batches after the fermentation for 25h so that the content of the carbadodecyl benzene in the fermentation liquid is more than 2% (v/v); the inoculum size (v/v) of the seed liquid and the addition amount (v/v) of the carbadode alkane are relative to the fermentation starting volume.
(2) After 125h of fermentation, continuously adding the laurocapram into the fermentation liquid at a flow acceleration of 146.2mL/h, and simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 318.5mL/h, and controlling the concentration OD of the thalli in the fermentation tank I 620 The dilution is 0.67 after 30 times, and the volume of the fermentation liquid in the fermentation tank I is kept at 22L by controlling the discharging rate, so that the 1, 12-dodecadiacid is continuously produced in the fermentation tank I.
(3) And (3) conveying the fermentation liquor discharged in the step (2) into a fermentation tank II for fermentation culture, controlling the dissolved oxygen in the fermentation tank II to be 55% -70% when the fermentation culture is carried out, controlling the temperature to be 29 ℃, the pressure to be 0.12MPa, the pH value to be 5.7 and the ventilation to be 0.77vvm, controlling the volume of the fermentation liquor in the fermentation tank II to be within 20L through continuous or batch discharging, and continuously fermenting in the fermentation tank II to produce the 1, 12-dodecadiacid.
The continuous fermentation time was 620 hours, and the total yield of 1, 12-dodecanedioic acid, the yield and the residual hydrocarbon content in the discharged material were recorded or calculated, and the results are shown in Table 1.
EXAMPLE 5 fermentation of dodecadiacid
The strain, medium and seed culture method of this example are the same as in example 1, except that the method for producing long chain dibasic acid in step 4 is as follows:
(1) Inoculating the seed liquid and the substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 15L, the inoculation amount of the seed liquid is 25% (v/v), the addition amount of the carbadodecyl benzene is 5.3% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.52vvm, controlling the pH value of the fermentation liquid to 5.8 in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding the carbadodecyl benzene in batches after the fermentation for 25h so that the content of the carbadodecyl benzene in the fermentation liquid is more than 2% (v/v); the inoculum size (v/v) of the seed liquid and the addition amount (v/v) of the carbadode alkane are relative to the fermentation starting volume.
(2) After 125h of fermentation, continuously adding the laurocapram into the fermentation liquid at a flow acceleration of 521.2mL/h, and simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 1053.7mL/h, so as to control the concentration OD of the thalli in the fermentation tank I 620 The dilution is 0.62 after 30 times, and the volume of the fermentation liquid in the fermentation tank I is kept at 22L by controlling the discharging rate, so that the 1, 12-dodecadiacid is continuously produced in the fermentation tank I.
(3) And (3) conveying the fermentation liquor discharged in the step (2) into a fermentation tank II for fermentation culture, controlling the dissolved oxygen in the fermentation tank II to be 55% -70% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.13MPa, the pH value to be 5.8 and the ventilation to be 0.76vvm, controlling the volume of the fermentation liquor in the fermentation tank II to be within 20L through continuous or batch discharging, and continuously fermenting in the fermentation tank II to produce the 1, 12-dodecadiacid.
The continuous fermentation time was 615 hours, and the total yield of 1, 12-dodecanedioic acid, the yield and the residual hydrocarbon content in the discharged material were recorded or calculated, and the results are shown in Table 1.
EXAMPLE 6 fermentation of dodecadiacid
The strain, medium and seed culture method of this example are the same as in example 1, except that the method for producing long chain dibasic acid in step 4 is as follows:
(1) Inoculating seed liquid and a substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 15L, the inoculation amount of the seed liquid is 26% (v/v), the addition amount of the carbadodecyl benzene is 5.1% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.52vvm, controlling the pH value of the fermentation liquid to 5.7 in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding the carbadodecyl benzene in batches after fermenting for 25h so that the content of the carbadodecyl benzene in the fermentation liquid is more than 2% (v/v); the inoculum size (v/v) of the seed liquid and the addition amount (v/v) of the carbadode alkane are relative to the fermentation starting volume.
(2) After 125h of fermentation, continuously adding the laurocapram into the fermentation liquid at a flow acceleration of 43.5mL/h, and simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 161.2mL/h, so as to control the concentration OD of the thalli in the fermentation tank I 620 The dilution is 0.67 after 30 times, and the volume of the fermentation liquid in the fermentation tank I is kept at 22L by controlling the discharging rate, so that the 1, 12-dodecadiacid is continuously produced in the fermentation tank I.
(3) And (3) conveying the fermentation liquor discharged in the step (2) into a fermentation tank II for fermentation culture, controlling the dissolved oxygen in the fermentation tank II to be 12% -16% when the fermentation culture is carried out, controlling the temperature to be 29 ℃, the pressure to be 0.15MPa, the pH value to be 5.7 and the ventilation to be 0.72vvm, controlling the volume of the fermentation liquor in the fermentation tank II to be within 20L through continuous or batch discharging, and continuously fermenting in the fermentation tank II to produce the 1, 12-dodecadiacid.
The continuous fermentation time was 617h, and the total yield of 1, 12-dodecanedioic acid, the yield and the residual hydrocarbon content in the discharged material were recorded or calculated, and the results are shown in Table 1.
EXAMPLE 7 fermentation of tridec dibasic acid
The strain, medium, seed culture method of this example are the same as example 2, except that the method for producing long chain dibasic acid in step 4 is as follows:
(1) Inoculating the seed solution and the substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 15L, the inoculation amount of the seed solution is 25% (v/v), the addition amount of the tridecane is 5.1% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.52vvm, controlling the pH value of the fermentation liquid to 5.9 in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding the tridecane in batches after fermenting for 25h so that the content of the tridecane in the fermentation liquid is more than 2% (v/v); the inoculum size (v/v) of the seed liquid and the addition amount (v/v) of the carbon tridecane are relative to the fermentation starting volume.
(2) After fermentation for 95h, continuously adding tridecane alkane into the fermentation liquid at a flow acceleration of 48.2mL/h, and simultaneously continuously adding the nutrient salt-containing solution into the fermentation liquid at a flow acceleration of 132mL/hLiquid, control of the concentration OD of the cells in the fermenter I 620 The dilution is 0.65 after 30 times, and the volume of the fermentation liquid in the fermentation tank I is kept at 22L by controlling the discharging rate, so that the 1, 13-tridecanedioic acid is continuously produced in the fermentation tank I.
(3) And (3) conveying the fermentation liquor discharged in the step (2) into a fermentation tank II for fermentation culture, controlling the dissolved oxygen in the fermentation tank II to be 13% -18% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.14MPa, the pH value to be 5.9 and the ventilation to be 0.77vvm, controlling the volume of the fermentation liquor in the fermentation tank II to be within 20L through continuous or batch discharging, and continuously fermenting in the fermentation tank II to produce the 1, 13-tridecanedioic acid.
The continuous fermentation time was 619h, and the total yield of 1, 13-tridecanedioic acid, the yield and the residual hydrocarbon content in the discharged material were recorded or calculated, and the results are shown in Table 1.
EXAMPLE 8 fermentation of hexadecanoic dibasic acid
The strain, medium, seed culture method of this example are the same as in example 3, except that the method for producing long chain dibasic acid in step 4 is as follows:
(1) Inoculating the seed solution and the substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 15L, the inoculation amount of the seed solution is 25% (v/v), the addition amount of the hexadecane is 5.1% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.52vvm, controlling the pH value of the fermentation liquid to 5.9 in the fermentation process, controlling the dissolved oxygen amount in the fermentation process to be more than 10%, and adding the hexadecane in batches after the fermentation for 25h so that the content of the hexadecane in the fermentation liquid is more than 2% (v/v); the inoculum size (v/v) of the seed liquid and the addition amount (v/v) of the hexadecane were relative to the fermentation starting volume.
(2) After fermentation for 95h, continuously adding hexadecane into the fermentation liquid at a flow acceleration of 146.2mL/h, and simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 318.5mL/h, and controlling the concentration OD of the thallus in the fermentation tank I 620 Dilution by 30 times is 0.65, and the volume of fermentation liquid in the fermentation tank I is maintained at 22L by controlling the discharging rate, thereby continuously producing 1, 16-sixteen carbon in the fermentation tank IAnd (3) dibasic acid.
(3) And (3) conveying the fermentation liquor discharged in the step (2) into a fermentation tank II for fermentation culture, controlling the dissolved oxygen in the fermentation tank II to be 35% -60% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.13MPa, the pH value to be 5.9 and the ventilation to be 0.82vvm, controlling the volume of the fermentation liquor in the fermentation tank II to be within 20L through continuous or batch discharging, and continuously fermenting in the fermentation tank II to produce the 1, 16-hexadecanoic dibasic acid.
The continuous fermentation time was 622h, and the total yield of 1, 16-hexadecanoic acid, yield and residual hydrocarbon content in the effluent were recorded or calculated, and the results are shown in Table 1.
Table 1:
Figure BDA0003449949460000181
Figure BDA0003449949460000191
the preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims (10)

1. A method for producing long chain dibasic acid by continuous fermentation, comprising:
after 85-145h of fermentation, continuously feeding substrate and nutrient salt-containing solution into the fermentation liquid, and discharging part of the fermentation liquid for continuous fermentation, wherein the dissolved oxygen is controlled to be more than 10% in the continuous fermentation process.
2. The process according to claim 1, wherein the dilution D of the fermentation broth is maintained at 0.002% to over0.3h -1 Further 0.002-0.15h -1 Further for 0.002-0.10h -1 Further 0.002-0.05h -1 The method comprises the steps of carrying out a first treatment on the surface of the And/or the number of the groups of groups,
the flow acceleration of the substrate, the flow acceleration of the nutrient salt-containing solution and the volume of the fermentation broth satisfy the following relationship:
0.005h -1 * V (fermentation liquor) is less than or equal to Q (nutrient salt-containing solution) is less than or equal to 0.3h -1 * V (fermentation broth);
0.001h -1 * V (fermentation liquor) is less than or equal to Q (substrate) and less than or equal to 0.006h -1 * V (fermentation broth);
wherein V (fermentation broth) represents the volume of fermentation broth, Q (nutrient salt-containing solution) represents the flow acceleration of the nutrient salt-containing solution, and Q (substrate) represents the flow acceleration of the substrate.
3. The method of claim 1, wherein the step of determining the position of the substrate comprises,
controlling the concentration OD620 of the thallus in the fermentation liquid which is remained after the partial fermentation liquid flows out to be 0.3-0.8 after being diluted by 30 times; and/or the number of the groups of groups,
the mode of flowing out part of fermentation liquor adopts continuous discharging or batch discharging; and/or the number of the groups of groups,
the volume of the residual fermentation liquid is 0.5-2 times of the volume of the fermentation liquid before feeding by controlling the discharging speed, and is further 1-1.8 times; and/or the number of the groups of groups,
before feeding, the content of the substrate in the fermentation broth is more than 1% (v/v); and/or the number of the groups of groups,
before feeding, the substrate content in the fermentation broth is below 8% (v/v).
4. The method of claim 2, wherein the step of determining the position of the substrate comprises,
the flow acceleration of the substrate, the flow acceleration of the nutrient salt-containing solution and the volume of the fermentation broth satisfy the following relationship:
0.006h -1 * V (fermentation liquor) is less than or equal to Q (solution containing nutrient salt) is less than or equal to 0.15h -1 * V (fermentation broth); and/or the number of the groups of groups,
0.001h -1 * V (fermentation liquor) is less than or equal to Q (substrate) and less than or equal to 0.004h -1 * V (fermentation broth).
5. The method according to any one of claims 1 to 4, wherein the nutrient salt-containing solution contains 0-2.5 (w/v) of glucose, 0.07-2.0 (w/v) of corn steep liquor, 0-1.0 (w/v) of yeast extract, 0.01-0.50 (w/v) of monopotassium phosphate, 0.01-0.50 (w/v) of urea and 0.05-0.3 (w/v) of ammonium sulfate; and/or the number of the groups of groups,
the substrate is selected from C 9 -C 18 At least one of the normal alkane, linear saturated fatty acid ester and linear saturated fatty acid salt, preferably C 10 -C 16 N-alkanes of (a); and/or
The chemical formula of the long-chain dibasic acid is HOOC (CH) 2 ) n COOH, wherein n is not less than 7, preferably at least one of azelaic acid, sebacic acid, 1, 11-undecanedioic acid, 1, 12-dodecadioic acid, 1, 13-tridecanedioic acid, 1, 14-tetradecanedioic acid, 1, 15-pentadecanoic acid, 1, 16-hexadecanedioic acid, 1, 17-heptadecanoic acid and 1, 18-octadecanedioic acid.
6. The method of claim 1, wherein the step of determining the position of the substrate comprises,
controlling the dissolved oxygen to be more than 20% and further 30% -100% in the continuous fermentation process; and/or, the temperature is 28-32 ℃; and/or, the pressure is 0.05-0.14MPa; and/or a pH of 5.5 to 7.5, further 5.5 to 6.7; and/or the ventilation is 0.3-0.7vvm.
7. The method of claim 1, wherein the steps include:
(1) Inoculating seed liquid containing fermentation strain into fermentation medium, fermenting in the presence of substrate;
(2) After the fermentation in step (1) is carried out for 85-145h, feeding is carried out by continuously feeding substrate and nutrient salt-containing solution into the fermentation broth, and part of the fermentation broth is discharged for continuous fermentation.
8. The method of claim 7, wherein the step of determining the position of the probe is performed,
in the step (1), the inoculation amount of the seed liquid is 10-30% (v/v) relative to the fermentation initial volume; and/or the number of the groups of groups,
the fermentation in the step (1) is carried out in a fermentation tank I; and/or the number of the groups of groups,
in the step (1), the substrate is added in an amount of 10% (v/v) or less, and further 1 to 5% (v/v) relative to the fermentation initiation volume; and/or the number of the groups of groups,
in the step (1), substrate is added into the fermentation liquid in batches or continuously in the fermentation process so as to ensure that the substrate content in the fermentation liquid is more than 1% (v/v); and/or the number of the groups of groups,
in the step (1), substrate is added to the fermentation broth in batches or continuously during the fermentation process to ensure that the substrate content in the fermentation broth is below 8% (v/v).
9. The method according to claim 7 or 8, wherein,
in the step (1), after fermentation for 10-100h, further 10-50 h and further 15-40 h, adding substrate into the fermentation liquid to ensure that the content of the substrate in the fermentation liquid is more than 1% (v/v); and/or the number of the groups of groups,
in the step (1), after fermentation for 10-100h, further 10-50 h and further 15-40 h, adding substrate into the fermentation liquid to ensure that the substrate content in the fermentation liquid is below 8% (v/v); and/or the number of the groups of groups,
the continuous fermentation in the step (2) is carried out in a fermentation tank II, and the feeding mode is continuous feeding or batch feeding;
the continuous fermentation in the step (2) is carried out in a fermentation tank II, and the discharging mode adopts continuous discharging or batch discharging; and/or the number of the groups of groups,
the residual hydrocarbon content of the discharged fermentation liquid of the fermentation tank II is below 0.5% (v/v), and further below 0.3% (v/v).
10. The method according to any one of claims 7-9, wherein,
controlling the temperature to be 28-32 ℃ and/or the pressure to be 0.05-0.14MPa and/or the pH value in the fermentation process in the step (1)5.5-7.5, further 5.5-6.7, and/or the ventilation is 0.3-0.7vvm, and/or the dissolved oxygen is above 10%, and/or the concentration of fermented thallus OD 620 9-24, and/or 0.3-0.8 after dilution of the fermented cell concentration by 30 times.
CN202111662222.6A 2021-12-31 2021-12-31 Method for producing long-chain dibasic acid by continuous fermentation Pending CN116410873A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111662222.6A CN116410873A (en) 2021-12-31 2021-12-31 Method for producing long-chain dibasic acid by continuous fermentation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111662222.6A CN116410873A (en) 2021-12-31 2021-12-31 Method for producing long-chain dibasic acid by continuous fermentation

Publications (1)

Publication Number Publication Date
CN116410873A true CN116410873A (en) 2023-07-11

Family

ID=87049934

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111662222.6A Pending CN116410873A (en) 2021-12-31 2021-12-31 Method for producing long-chain dibasic acid by continuous fermentation

Country Status (1)

Country Link
CN (1) CN116410873A (en)

Similar Documents

Publication Publication Date Title
US11802298B2 (en) Decanedioic acid produced by microbial fermentation process and preparation method thereof
Lee et al. Citric acid production by Aspergillus niger immobilized on polyurethane foam
EP3533879A1 (en) Method for producing a long chain dicarboxylic acid by fermentation
SE462394B (en) PROCEDURES FOR THE PREPARATION OF CELLULOLYTIC ENZYM
CN110218661A (en) A kind of Candida tropicalis and its application and a kind of production method of long-chain biatomic acid
CN110218745A (en) The method of fermenting and producing long-chain biatomic acid and its obtained long-chain biatomic acid
CN108285915B (en) Fermentation method of gibberellic acid
EP4047081A1 (en) Strain for producing long-chain dicarboxylic acids and fermentation method therefor
CN111748480B (en) Candida virginiana and application thereof
CN116410873A (en) Method for producing long-chain dibasic acid by continuous fermentation
CN112501221A (en) Method for improving conversion rate of threonine and saccharic acid
CN111378696B (en) Fermentation substrate and method for producing long-chain dicarboxylic acid by fermentation of fermentation substrate
CN116411031A (en) Process for producing long-chain dibasic acid by continuous fermentation
CN116376792A (en) Directional transformation method of tyrosine production strain, production strain and tyrosine fermentation method
CN117165635A (en) Method for producing long-chain dibasic acid by continuous fermentation
CN106591401B (en) Fermentation promoter for increasing yield of gentamicin C1a and addition method thereof
CN117165633A (en) Method for producing long-chain dibasic acid by continuous fermentation
CN117165454A (en) Continuous culture method of seed liquid
CN115678926A (en) Method for producing long-chain dicarboxylic acid by continuous fermentation and production method of long-chain dicarboxylic acid
CN110982705B (en) Fermentation process of schizochytrium limacinum
KR20220088933A (en) Long-chain compositions, combinations of long-chain compositions, methods for their preparation and applications thereof
CN109868294A (en) A method of continuously ferment and produces long-chain biatomic acid
CN109868296A (en) A method of continuously ferment and produces long-chain biatomic acid
CN109868295B (en) Method for producing long-chain dicarboxylic acid by continuous fermentation
CN109868226A (en) A kind of nutrient salt solution and its application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination