CN116411031A - Process for producing long-chain dibasic acid by continuous fermentation - Google Patents

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

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CN116411031A
CN116411031A CN202111668554.5A CN202111668554A CN116411031A CN 116411031 A CN116411031 A CN 116411031A CN 202111668554 A CN202111668554 A CN 202111668554A CN 116411031 A CN116411031 A CN 116411031A
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fermentation
substrate
groups
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tank
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郝英利
赵宏图
鲍家伟
刘修才
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Cathay R&D Center Co Ltd
CIBT America Inc
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CIBT America Inc
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Abstract

The invention provides a process for producing long-chain dibasic acid by continuous fermentation, which comprises the following steps: after fermentation in the fermentation tank I for 82-147 h, continuously feeding substrate and nutrient salt-containing solution into the fermentation liquid in the fermentation tank I, and discharging part of the fermentation liquid to the fermentation tank II for continuous fermentation, wherein the substrate content in the fermentation liquid fed to the fermentation tank II is more than 0 and less than or equal to 4.6%. Compared with the prior art, the conversion rate of a fermentation system is improved by controlling the substrate content in the fermentation liquid entering the fermentation tank II.

Description

Process 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 process 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 chemical synthesis and biological fermentation, and the chemical synthesis methodThe technology is mature, the synthetic route is long, but the method needs to be carried out under the conditions of high temperature and high pressure, has severe requirements on the catalyst, and is only limited to synthesizing the 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 fermentation in the fermentation tank I for 82-147 h, continuously feeding substrate and nutrient salt-containing solution into the fermentation liquid in the fermentation tank I, and discharging part of the fermentation liquid to the fermentation tank II for continuous fermentation, wherein the substrate content in the fermentation liquid fed to the fermentation tank II is more than 0 and less than or equal to 4.6%.
By the technical scheme, compared with the prior art, the conversion rate of a fermentation system is improved by controlling the substrate content in the fermentation liquid entering the fermentation tank II.
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, the present invention provides a process for continuous fermentation production of long chain dibasic acids comprising: after fermentation in the fermentation tank I for 82-147 h, continuously feeding substrate and nutrient salt-containing solution into the fermentation liquid in the fermentation tank I, and discharging part of the fermentation liquid to the fermentation tank II for continuous fermentation, wherein the substrate content in the fermentation liquid fed to the fermentation tank II is more than 0 and less than or equal to 4.6%.
In some embodiments of the invention, the substrate content of the fermentation broth fed to fermentor ii is greater than 0 and less than or equal to 3%.
In some embodiments of the invention, the dissolved oxygen content of the fermentation tank II is controlled to be more than 10%, more preferably more than 20%, and still more preferably 30% -100% in the fermentation process.
In some embodiments of the invention, the temperature of the fermenter II during fermentation is 28-32deg.C; 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 residual hydrocarbon content of the discharged fermentation liquid after fermentation is below 5%, and further below 3%.
In some embodiments of the invention, the nutrient salt-containing solution has a flow acceleration of 0.003 to 0.35mL/h, more preferably 0.003 to 0.17mL/h, and even more preferably 0.003 to 0.15mL/h, relative to a fermentation initiation volume of 1 mL.
In some embodiments of the invention, the substrate has a flow acceleration of 0.0007 to 0.007mL/h, more preferably 0.0007 to 0.004mL/h, relative to a fermentation start volume of 1 mL.
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 Normal paraffins, straight chain saturated fatty acidsAt least one of the linear saturated fatty acid ester and the 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.
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 volume of the fermentation broth remaining in the fermenter I is controlled to be 0.5 to 2 times, further 1 to 1.8 times, the volume of the fermentation broth before the feeding by controlling the discharge speed.
In some embodiments of the invention, the substrate content of the fermentation broth in fermentor I is above 0.8% (v/v) prior to feeding.
In some embodiments of the invention, the substrate content of the fermentation broth in fermenter I is below 7.5% (v/v) prior to feeding.
In the present invention, the cell concentration is measured by a spectrophotometer.
In some embodiments of the invention, the concentration of the cells in fermentor I is maintained at 0.3-0.8 after 30-fold dilution of OD 620. The concentration of the thalli in the fermentation tank I can be controlled by controlling the feeding and/or discharging speed of the fermentation tank I.
In the invention, the dissolved oxygen amount of the fermentation is measured by an on-line dissolved oxygen electrode.
The dissolved oxygen amount of the fermentation tank II is controlled to be more than 12%, more preferably more than 23%, and still more preferably 30% -100% in the fermentation process.
The temperature of the fermentation tank II is controlled to be 28-32 ℃ in the 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.
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 of fermentation tank I, and fermenting in the presence of substrate;
(2) After fermentation in the fermentation tank I is carried out for 82-147 h, substrate and nutrient salt-containing solution are continuously added into fermentation liquor in the fermentation tank I for feeding, and partial fermentation liquor is discharged and conveyed to the fermentation tank II for continuous fermentation, wherein the substrate content in the fermentation liquor conveyed to the fermentation tank II is more than 0 and less than or equal to 4.6%.
In the invention, the fermentation initial volume is equal to the total volume of the fermentation medium after seed liquid and substrate are added.
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.
Further, the concentration of the cells fermented in the step (1) is 0.3 to 0.8 after 30-fold dilution.
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 fermenter I of more than 0.8% (v/v) and less than 7.5% (v/v).
Preferably, in 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 at least 0.8% (v/v) and at most 7.5% (v/v). 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.
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 shaking bottle seed into seed tank containing seed culture medium, controlling inoculation amount to 10-30% (v/v), controlling temperature to 28-32deg.C, pressure to 0.05-0.14MPa, ventilation amount to 0.3-0.7vvm,and pH value is controlled to be 3.0-7.5 by supplementing 10-40 (w/w)% of liquid alkali, and a certain stirring speed is maintained to ensure that dissolved oxygen DO in the seed culture process is more than 10%, and the culture is carried out for 15-30h to ensure that the 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 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 total yield of the long-chain dibasic acid= (amount of long-chain dibasic acid produced (g)/amount of substrate added (g)) ×100%;
time of continuous fermentation: 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 0.11MPa, the ventilation amount to 0.52vvm, 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 17L, 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 0.11MPa, 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 45.5mL/h, simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 163.5mL/h, and controlling the discharging rate to ensure that the concentration OD of the thallus in the fermentation tank I is controlled 620 The dilution by 30 times is 0.66, and the content of the carbadode alkane in the discharged fermentation liquor is 2.2% (v/v). Fermentation tank IAnd continuously producing 1, 12-dodecadiacid.
(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% -75% 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 0.7vvm, 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 632h and the total yield of 1, 12-dodecanedioic acid was 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 31% (v/v), controlling the temperature to 30deg.C, the pressure to 0.14MPa, the ventilation amount to 0.3vvm, controlling the pH value to 7 by adding 10 (w/w)% liquid alkali, maintaining a certain stirring speed to make the dissolved oxygen DO in the seed culture process be above 10%, and culturing for 30h to make 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 17L, 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 5.7 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 43.5mL/h, simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 161.5mL/h, and controlling the discharging rate to ensure that the concentration OD of the thallus in the fermentation tank I 620 The concentration of the carbon tridecane in the discharged fermentation broth is 1.7% (v/v) after dilution by 30 times is 0.54. And continuously producing 1, 13-tridecyl dicarboxylic acid 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 29 ℃, the pressure to be 0.14MPa, the pH value to be 5.5 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, 13-tridecanedioic acid.
The continuous fermentation time was 622h and the total yield of 1, 13-tridecanedioic acid was recorded or calculated, 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 0.45 (w/v), KH 2 PO 4 The content of (C) was 1.1 (w/v)%, and the content of urea was 0.53 (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 0.75 (w/v), urea content was 0.33 (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 0.16 (w/v) percent corn steep liquor, 0.013 (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 shaking culture at 240rpm for 1.5 days under the conditions that the initial pH value is 6.4 and the temperature is 29 ℃;
(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 0.07MPa, the ventilation rate to 0.6vvm, controlling the pH value to 7.4 by adding 15 (w/w)% 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 28h to ensure that the thallus concentration OD 620 And diluting the mixture by 30 times to obtain 0.9, 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 16L, the inoculation amount of the seed solution is 25% (v/v), the addition amount of the hexadecane is 5.2% (v/v), then controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.51vvm, 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 hexadecane in batches after fermenting 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 43.2mL/h, continuously adding the solution containing nutritive salt into the fermentation liquid at a flow acceleration of 132mL/h, and controlling the discharge rate to ensure that the concentration OD of the thallus in the fermentation tank I is controlled 620 The dilution by 30 times is 0.66, and the content of the carbon hexadecane in the discharged fermentation liquor is 2% (v/v). And continuously producing 1, 16-hexadecanoic dibasic acid 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 0.13MPa, the pH value to be 5.6 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, 16-hexadecanoic dibasic acid.
The continuous fermentation time was 620h and the total yield of 1, 16-hexadecanoic acid was recorded or calculated, 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 17L, 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 carbazate into the fermentation liquid at a flow acceleration of 41mL/h, continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 160.5mL/h, and controlling the discharge rate to ensure that the concentration OD of the thallus in the fermentation tank I is controlled 620 The dilution by 30 times is 0.65, and the content of the carbadode alkane in the discharged fermentation liquor is 0.7% (v/v). And continuously producing 1, 12-dodecadiacid 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% -75% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.14MPa, the pH value to be 5.5 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, 12-dodecadiacid.
The continuous fermentation time was 600 hours and the total yield of 1, 12-dodecanedioic acid was 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 17L, 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 48.5mL/h, simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 147.5mL/h, and controlling the discharging rate to ensure that the concentration of the thalli in the fermentation tank I is maintained at OD 620 The dilution by 30 times is 0.65, and the content of the carbadode alkane in the discharged fermentation liquor is 4.5% (v/v). And continuously producing 1, 12-dodecadiacid 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% -75% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.14MPa, the pH value to be 5.5 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 596 hours and the total yield of 1, 12-dodecanedioic acid was 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 the seed liquid and the substrate into a fermentation tank I containing a fermentation medium, wherein the fermentation initial volume is 17L, 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, the fermentation broth was continuously fed with carbadode-alkane at a flow rate of 43.0mL/h, at 161.The nutrient salt-containing solution is added to the fermentation liquor continuously at the flow acceleration of 0mL/h, and the discharging speed is controlled to maintain the concentration of the thalli in the fermentation tank I at OD 620 The dilution by 30 times is 0.65, and the content of the carbadode alkane in the discharged fermentation liquor is 1.9% (v/v). And continuously producing 1, 12-dodecadiacid 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% -17% when the fermentation culture is carried out, controlling the temperature to be 28 ℃, the pressure to be 0.14MPa, the pH value to be 5.5 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 627 hours, and the total yield of 1, 12-dodecanedioic acid was recorded or calculated, and the results are shown in Table 1.
Comparative example 1 fermentation of dodecanedioic acid
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 17L, 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 50mL/h, simultaneously continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 130mL/h, and controlling the discharging rate to ensure that the concentration OD of the thalli in the fermentation tank I is controlled 620 The concentration of the carbon dodecane in the discharged fermentation liquid is maintained to be 0.70 after the fermentation liquid is diluted by 30 times, and the content of the carbon dodecane in the discharged fermentation liquid is 8% (v/v). In the fermentation tank IContinuously producing 1, 12-dodecadiacid.
(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% -75% when the fermentation culture is carried out, controlling the temperature to be 29 ℃, the pressure to be 0.14MPa, the pH value to be 5.7 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 was recorded or calculated and the results are shown in Table 1.
Comparative example 2 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 17L, 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 5.7 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 97h of fermentation, continuously adding hexadecane into the fermentation liquid at a flow acceleration of 48mL/h, continuously adding the solution containing the nutritive salt into the fermentation liquid at a flow acceleration of 131.5mL/h, and controlling the discharging rate to maintain the concentration of the thallus in the fermentation tank I at OD 620 The dilution by 30 times is 0.57, and the content of the carbadode alkane in the discharged fermentation liquor is 7.7% (v/v). The 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 45% -65% when the fermentation culture is carried out, controlling the temperature to be 29 ℃, the pressure to be 0.14MPa, the pH value to be 5.6 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, 16-hexadecanoic dibasic acid.
The continuous fermentation time was 620h and the total yield of 1, 16-hexadecanoic acid was recorded or calculated, the results are shown in Table 1.
Table 1:
total yield (%)
Example 1 98.7％
Example 2 93.6％
Example 3 83.7％
Example 4 98.2％
Example 5 94.6％
Example 6 94.2％
Comparative example 1 90.7％
Comparative example 2 76.4％
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 fermentation in the fermentation tank I for 82-147 h, continuously feeding substrate and nutrient salt-containing solution into the fermentation liquid in the fermentation tank I, and discharging part of the fermentation liquid to the fermentation tank II for continuous fermentation, wherein the substrate content in the fermentation liquid fed to the fermentation tank II is more than 0 and less than or equal to 4.6%.
2. The process according to claim 1, wherein the substrate content of the fermentation broth fed to fermenter II is greater than 0 and less than or equal to 3%; and/or the number of the groups of groups,
the dissolved oxygen amount of the fermentation tank II is controlled to be more than 12%, more preferably more than 23%, and still more preferably 30% -100% in the fermentation process.
3. The process according to claim 1 or 2, wherein the temperature of the fermenter ii during fermentation 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; and/or the residual hydrocarbon content of the discharged fermentation liquid after fermentation is below 5%, and further below 3%.
4. The method according to claim 1 or 2, wherein 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).
5. The method of claim 1, wherein the step of determining the position of the substrate comprises,
the concentration of thalli in the fermentation tank I is maintained to be 0.3-0.8 after the OD620 is diluted by 30 times; and/or the number of the groups of groups,
before feeding, the substrate content of the fermentation liquor in the fermentation tank I is more than 0.8% (v/v); and/or the number of the groups of groups,
before feeding, the substrate content of the fermentation liquor in the fermentation tank I is below 7.5% (v/v).
6. 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.
7. The method of claim 1, wherein the steps include:
(1) Inoculating seed liquid containing fermentation strain into fermentation medium of fermentation tank I, and fermenting in the presence of substrate;
(2) After fermentation in the fermentation tank I is carried out for 82-147 h, substrate and nutrient salt-containing solution are continuously added into fermentation liquor in the fermentation tank I for feeding, and partial fermentation liquor is discharged and conveyed to the fermentation tank II for continuous fermentation, wherein the substrate content in the fermentation liquor conveyed to the fermentation tank II is more than 0 and less than or equal to 4.6%.
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,
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 broth in batches or continuously in the fermentation process so as to ensure that the substrate content in the fermentation broth of the fermentation tank I is more than 0.8% (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 of the fermentation tank I is below 7.5% (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 7.5% (v/v); and/or the number of the groups of groups,
the feeding mode of the fermentation tank II in the step (2) is continuous feeding or batch feeding; and/or the number of the groups of groups,
and (2) continuously discharging or discharging in batches in a discharging mode of the fermentation tank II.
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 to be 5.5-7.5, further to be 5.5-6.7 and/or the ventilation rate to be 0.3-0.7vvm and/or the dissolved oxygen to be more than 10% in the fermentation process in the step (1), and/or the concentration OD of the fermented thallus 620 9-24, and/or 0.3-0.8 after dilution of the fermented cell concentration by 30 times.
CN202111668554.5A 2021-12-31 2021-12-31 Process for producing long-chain dibasic acid by continuous fermentation Pending CN116411031A (en)

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