CN117165454A - Continuous culture method of seed liquid - Google Patents
Continuous culture method of seed liquid Download PDFInfo
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- CN117165454A CN117165454A CN202210583712.5A CN202210583712A CN117165454A CN 117165454 A CN117165454 A CN 117165454A CN 202210583712 A CN202210583712 A CN 202210583712A CN 117165454 A CN117165454 A CN 117165454A
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- 239000007788 liquid Substances 0.000 title claims abstract description 58
- 238000012136 culture method Methods 0.000 title claims abstract description 6
- 239000000243 solution Substances 0.000 claims abstract description 57
- 238000011218 seed culture Methods 0.000 claims abstract description 45
- 150000003839 salts Chemical class 0.000 claims abstract description 39
- 238000000855 fermentation Methods 0.000 claims abstract description 37
- 230000004151 fermentation Effects 0.000 claims abstract description 37
- 239000002253 acid Substances 0.000 claims abstract description 33
- 238000012258 culturing Methods 0.000 claims abstract description 31
- 238000010790 dilution Methods 0.000 claims abstract description 28
- 239000012895 dilution Substances 0.000 claims abstract description 28
- 239000001963 growth medium Substances 0.000 claims abstract description 28
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 13
- 239000001301 oxygen Substances 0.000 claims abstract description 13
- 230000000050 nutritive effect Effects 0.000 claims abstract description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 40
- 235000015097 nutrients Nutrition 0.000 claims description 37
- 241000222178 Candida tropicalis Species 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 28
- 240000008042 Zea mays Species 0.000 claims description 21
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 21
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 21
- 229940041514 candida albicans extract Drugs 0.000 claims description 21
- 239000004202 carbamide Substances 0.000 claims description 21
- 235000005822 corn Nutrition 0.000 claims description 21
- 239000012138 yeast extract Substances 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 16
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 11
- 229930006000 Sucrose Natural products 0.000 claims description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- 238000009423 ventilation Methods 0.000 claims description 10
- BNJOQKFENDDGSC-UHFFFAOYSA-N octadecanedioic acid Natural products OC(=O)CCCCCCCCCCCCCCCCC(O)=O BNJOQKFENDDGSC-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 4
- BTZVDPWKGXMQFW-UHFFFAOYSA-N Pentadecanedioic acid Chemical compound OC(=O)CCCCCCCCCCCCCC(O)=O BTZVDPWKGXMQFW-UHFFFAOYSA-N 0.000 claims description 4
- QQHJDPROMQRDLA-UHFFFAOYSA-N hexadecanedioic acid Chemical compound OC(=O)CCCCCCCCCCCCCCC(O)=O QQHJDPROMQRDLA-UHFFFAOYSA-N 0.000 claims description 4
- CXMXRPHRNRROMY-UHFFFAOYSA-N sebacic acid Chemical compound OC(=O)CCCCCCCCC(O)=O CXMXRPHRNRROMY-UHFFFAOYSA-N 0.000 claims description 4
- HQHCYKULIHKCEB-UHFFFAOYSA-N tetradecanedioic acid Chemical compound OC(=O)CCCCCCCCCCCCC(O)=O HQHCYKULIHKCEB-UHFFFAOYSA-N 0.000 claims description 4
- LWBHHRRTOZQPDM-UHFFFAOYSA-N undecanedioic acid Chemical compound OC(=O)CCCCCCCCCC(O)=O LWBHHRRTOZQPDM-UHFFFAOYSA-N 0.000 claims description 4
- 238000007599 discharging Methods 0.000 claims description 3
- KEMQGTRYUADPNZ-UHFFFAOYSA-N heptadecanoic acid Chemical compound CCCCCCCCCCCCCCCCC(O)=O KEMQGTRYUADPNZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 125000000896 monocarboxylic acid group Chemical group 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- DXNCZXXFRKPEPY-UHFFFAOYSA-N tridecanedioic acid Chemical compound OC(=O)CCCCCCCCCCCC(O)=O DXNCZXXFRKPEPY-UHFFFAOYSA-N 0.000 claims description 2
- BDJRBEYXGGNYIS-UHFFFAOYSA-N nonanedioic acid Chemical compound OC(=O)CCCCCCCC(O)=O BDJRBEYXGGNYIS-UHFFFAOYSA-N 0.000 claims 2
- 241000894007 species Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 27
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 9
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 9
- 230000001133 acceleration Effects 0.000 description 8
- 238000011068 loading method Methods 0.000 description 8
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- 238000003786 synthesis reaction Methods 0.000 description 8
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- 150000001335 aliphatic alkanes Chemical class 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 241000222157 Candida viswanathii Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
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Abstract
The invention provides a continuous culture method of seed liquid, which comprises the following steps: inoculating seed solution into seed tank containing seed culture medium, culturing under conditions of dissolved oxygen of above 3% and pH of 4.0-7.5, and culturing to thallus concentration OD 620 After dilution by 30 times, the solution containing nutritive salt is added into a seed tank for continuous culture, and partial seed liquid is discharged for fermenting and producing long-chain dibasic acid, wherein the dilution rate D of the seed tank is 0.007-0.25h ‑1 . The invention can keep the seed liquid at higher activity by adopting a continuous culture preparation method, and can be used for further improving the yield of the long-chain binary acid fermentation production process.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a continuous culture method of seed liquid.
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 takes long-chain alkane or fatty acid as a substrate, and long-chain dibasic acid is obtained through microbial fermentation and conversion, the production process is carried out at normal temperature and normal pressure, and various long-chain dibasic acids such as C9 to C18 can be produced in large scale. Compared with chemical synthesis methods, the biological fermentation method has more obvious advantages in the aspects of raw material availability, product diversity, production efficiency, production cost, environmental advantages and the like, and in the industrialized development of nearly 20 years, the chemical synthesis method has been gradually replaced by the biological fermentation method.
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 the continuous fermentation process is realized by periodically supplementing fresh seed liquid, so that the cost of membrane separation is increased, and the risk of bacterial contamination is increased.
CN109868294A discloses a low dilution ratio (0.001-0.006 h -1 ) The continuous fermentation process of producing long chain binary acid includes continuous substrate feeding and continuous nutrient salt solution feeding after fermenting for 100-150 hr, so as to ensure continuous production of long chain binary acid. However, the method has low dilution rate, so that the activity of the thalli is low, the subsequent acid production efficiency is affected, and the method is not beneficial to industrialized application.
Disclosure of Invention
The invention aims to provide a continuous culture method of seed liquid, which solves the problem that the growth of long-chain diacid bacteria is not easy to control in the existing continuous fermentation, and can keep higher activity by adopting the seed liquid prepared by continuous culture, thereby being capable of being used for further improving the yield of the long-chain diacid fermentation production process.
The method comprises the following steps:
inoculating seed solution into seed tank containing seed culture medium, culturing under conditions of dissolved oxygen of above 3% and pH of 4.0-7.5, and culturing to thallus concentration OD 620 After dilution by 30 times, the solution containing nutritive salt is added into a seed tank for continuous culture, and partial seed liquid is discharged for fermenting and producing long-chain dibasic acid, wherein the dilution rate D of the seed tank is 0.007-0.25h -1 。
In the invention, the solution containing nutrient salt is fed into the seed liquid in the seed culture process, partial seed liquid is discharged, and the dilution rate of the seed tank is controlled to be 0.007-0.25h -1 Thereby performing continuous culture of the seed liquid. The discharged seed liquid can be conveyed to a fermentation medium of long-chain dibasic acid for fermentation culture of the long-chain dibasic acid.
As a preferred embodiment of the present invention, the dilution rate D of the seed tank is 0.02-0.25h -1 Further for 0.08-0.15h -1 。
As one embodiment of the present invention, the culture is carried out to a cell concentration OD 620 After dilution by 30 times to more than 0.5, the feed of the nutrient salt-containing solution into the seed tank was started.
As one embodiment of the present invention, the culture is carried out to a cell concentration OD 620 After 30-fold dilution, the concentration is 0.4-1.0, further 0.5-0.9, and the feeding of the nutrient salt-containing solution into the seed tank is started.
As an embodiment of the invention, the dissolved oxygen in the seed liquid culture process is 3% -60%, further 3% -38%, further 3% -20% or 22% -38%, such as 5%, 10%, 15%, 17%, 22%, 28%, 33%, 38%, 45%, 50%, 55%.
As an embodiment of the present invention, the temperature during the cultivation is 28 to 32℃and/or the pressure is 0.05 to 0.14MPa and/or the pH is preferably 5.0 to 7.0, more preferably 5.0 to 6.5 and/or the ventilation is 0.3 to 0.7vvm.
As an embodiment of the present invention, the seed medium is inoculated in an amount of more than 0 and less than or equal to 30% (v/v), and further 10 to 30% (v/v).
As an actual embodiment of the inventionEmbodiments, OD of seed fluid inoculated in seed medium 620 More than 0.3, preferably more than 0.5 after dilution by a factor of 30.
As an embodiment of the invention, the solution containing the nutrient salt is fed into the seed solution in a continuous feeding or batch feeding mode.
As an embodiment of the present invention, the seed liquid flowing out of the liquid outlet portion is continuously discharged or batchwise discharged.
The seed liquid contains fermentation strains, and the selection range of the fermentation strains is wider, preferably comprises candida tropicalis and candida vista. 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).
As an embodiment of the present invention, the seed medium contains 1-5% (w/v) sucrose, 0.15-1% (w/v) corn steep liquor, 0.2-1.5% (w/v) yeast extract, and KH 2 PO 4 The content of (C) is 0.4-1.8% (w/v), the content of urea is 0.05-0.5% (w/v), and the balance is water.
As an embodiment of the present invention, the nutrient salt-containing solution contains 0 to 2.5 (w/v) of glucose, 0.05 to 2.0 (w/v) of corn steep liquor, 0 to 1.0 (w/v) of yeast extract, 0.005 to 0.5 (w/v) of monopotassium phosphate, 0.005 to 0.08 (w/v) of urea, 0.01 to 0.15 (w/v) of ammonium sulfate, and the balance of water.
As an embodiment of the present invention, the method further comprises the steps of: inoculating glycerol fermentation strain into seed culture medium, and culturing at 28-32deg.C and optionally initial pH of 6.0-6.5 to OD 620 The seed solution is obtained after dilution by 30 times of more than 0.3, preferably more than 0.5. Further, the culture process adopts shaking culture, and the rotating speed is 100-400rpm. The culture time is 24-72h. The obtained seed solution was used for inoculation and cultivation in the seed tank.
As an embodiment of the invention, the expression of the long-chain dibasic acid is HOOC (CH) 2 ) n COOH, wherein n.gtoreq.7, preferably nonoAny one or a combination of two or more of diacid, 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. The long-chain dibasic acid is obtained by fermenting normal alkane or fatty acid with the same carbon number as a fermentation substrate.
In the present invention, the dilution ratio d=q (nutrient salt-containing solution)/V (seed solution) of the seed tank, wherein Q (nutrient salt-containing solution) represents the flow acceleration of the nutrient salt-containing solution in units of: mL/h, V (seed liquid) represents the volume of seed liquid in the seed tank in units of: and (3) mL. For example, calculated as the dilution ratio d=132 mL/h of the seed tank in example 1 divided by 1240 ml=0.106 h -1 。
The invention has the beneficial effects that:
the seed liquid can be kept at higher activity by adopting a continuous culture preparation method, and can be used for further improving the yield of the long-chain binary acid fermentation production process.
Detailed Description
Example 1
Strain 1
The candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 seed Medium and nutrient salt-containing solution
Seed culture medium: sucrose content of 1.1 (w/v), corn steep liquor content of 0.30 (w/v), yeast extract content of 0.6 (w/v), KH 2 PO 4 The content of (C) was 0.9 (w/v)%, and the content of urea was 0.23 (w/v)%.
The nutrient salt-containing solution contained 0.33 (w/v)% corn steep liquor, 0.22 (w/v)% yeast extract, 0.009 (w/v)% monopotassium phosphate, 0.05 (w/v)% urea, and 0.04 (w/v)% ammonium sulfate.
3 seed culture
(i) Shake flask seed culture: inoculating the glycerol tube strain of Candida tropicalis into 500mL triangular flask (liquid loading amount of 50 mL) containing seed culture medium, and at initial pH of 6.2 and temperatureShaking culture at a rotation speed of 220rpm for 36h at 29℃and OD 620 More than 0.5 after 30 times dilution;
(ii) Continuous culture of seed liquid: inoculating shake flask seed into a seed tank containing 6L seed culture medium, inoculating 20% (v/v), culturing at 29 deg.C under 0.10MPa, ventilation of 0.62vvm, pH of 5.7, and culturing under 35% -50% of dissolved oxygen DO to thallus concentration OD 620 And diluting the solution to be 0.62 after 30 times, continuously adding the nutrient salt-containing solution into the seed tank at a flow acceleration of 132mL/h for continuous culture, and simultaneously flowing out a part of seed liquid for fermentation production of long-chain dibasic acid. The seed liquid volume in the seed tank was maintained at 1.24L.
Example 2
Strain 1
The candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 seed Medium and nutrient salt-containing solution
Seed culture medium: sucrose content of 1.2 (w/v), corn steep liquor content of 0.30 (w/v), yeast extract content of 0.6 (w/v), KH 2 PO 4 The content of (C) was 0.9 (w/v)%, and the content of urea was 0.23 (w/v)%.
The nutrient salt-containing solution contained 0.33 (w/v)% corn steep liquor, 0.22 (w/v)% yeast extract, 0.009 (w/v)% monopotassium phosphate, 0.06 (w/v)% urea, and 0.04 (w/v)% ammonium sulfate.
3 seed culture
(i) Shake flask seed culture: inoculating the glycerol tube strain of Candida tropicalis into 500mL triangular flask (liquid loading amount is 50 mL) filled with seed culture medium, shake culturing at 220rpm for 36h under initial pH value of 6.2 and temperature of 29 deg.C, and OD 620 More than 0.5 after 30 times dilution;
(ii) Continuous culture of seed liquid: inoculating shake flask seed into a seed tank containing 6L seed culture medium, inoculating 20% (v/v), culturing at 29 deg.C under 0.10MPa, ventilation of 0.62vvm, pH of 5.6, and culturing under 25% -35% of dissolved oxygen DO to thallus concentration OD 620 Diluted 30 times to 0.60The continuous flow of 132mL/h is added with the nutrient salt-containing solution to the continuous flow in the seed tank for continuous culture, and a part of seed liquid is discharged for fermentation production of long-chain dibasic acid. The seed liquid volume in the seed tank was maintained at 1.24L.
Example 3
Strain 1
The candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 seed Medium and nutrient salt-containing solution
Seed culture medium: sucrose content of 1.1 (w/v), corn steep liquor content of 0.30 (w/v), yeast extract content of 0.6 (w/v), KH 2 PO 4 The content of (C) was 0.9 (w/v)%, and the content of urea was 0.23 (w/v)%.
The nutrient salt-containing solution contained 0.31 (w/v)% corn steep liquor, 0.22 (w/v)% yeast extract, 0.009 (w/v)% monopotassium phosphate, 0.05 (w/v)% urea, and 0.04 (w/v)% ammonium sulfate.
3 seed culture
(i) Shake flask seed culture: inoculating the glycerol tube strain of Candida tropicalis into 500mL triangular flask (liquid loading amount is 50 mL) filled with seed culture medium, shake culturing at 220rpm for 36h under initial pH value of 6.2 and temperature of 29 deg.C, and OD 620 More than 0.5 after 30 times dilution;
(ii) Continuous culture of seed liquid: inoculating shake flask seed into a seed tank containing 6L seed culture medium, inoculating 20% (v/v), culturing at 29 deg.C, 0.09MPa, 0.62vvm, pH 5.7, and culturing under 5% -15% oxygen-dissolved DO to thallus concentration OD 620 And diluting the solution to be 0.58 after 30 times, continuously adding the solution containing the nutrient salt into the seed tank at a flow acceleration of 132mL/h for continuous culture, and simultaneously, flowing out a part of seed liquid for fermentation production of long-chain dibasic acid. The seed liquid volume in the seed tank was maintained at 1.24L.
Example 4
Strain 1
The candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 seed Medium and nutrient salt-containing solution
Seed culture medium: sucrose content of 1.1 (w/v), corn steep liquor content of 0.30 (w/v), yeast extract content of 0.6 (w/v), KH 2 PO 4 The content of (C) was 0.9 (w/v)%, and the content of urea was 0.23 (w/v)%.
The nutrient salt-containing solution contained 0.33 (w/v)% corn steep liquor, 0.24 (w/v)% yeast extract, 0.009 (w/v)% monopotassium phosphate, 0.05 (w/v)% urea, and 0.04 (w/v)% ammonium sulfate.
3 seed culture
(i) Shake flask seed culture: inoculating the glycerol tube strain of Candida tropicalis into 500mL triangular flask (liquid loading amount is 50 mL) filled with seed culture medium, shake culturing at 220rpm for 36h under initial pH value of 6.2 and temperature of 29 deg.C, and OD 620 More than 0.5 after 30 times dilution;
(ii) Continuous culture of seed liquid: inoculating shake flask seed into a seed tank containing 6L seed culture medium, inoculating 20% (v/v), culturing at 29 deg.C under 0.10MPa, ventilation of 0.62vvm, pH of 5.7, and culturing under 25% -35% of dissolved oxygen DO to thallus concentration OD 620 And diluting the solution to be 0.62 after 30 times, continuously adding the nutrient salt-containing solution into the seed tank at a flow acceleration of 132mL/h for continuous culture, and simultaneously flowing out a part of seed liquid for fermentation production of long-chain dibasic acid. The seed liquid volume in the seed tank was maintained at 2.51L.
Example 5
Strain 1
The candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 seed Medium and nutrient salt-containing solution
Seed culture medium: sucrose content of 1.1 (w/v), corn steep liquor content of 0.30 (w/v), yeast extract content of 0.6 (w/v), KH 2 PO 4 The content of (C) was 0.8 (w/v)%, and the content of urea was 0.23 (w/v)%.
The nutrient salt-containing solution contained 0.33 (w/v)% corn steep liquor, 0.22 (w/v)% yeast extract, 0.009 (w/v)% monopotassium phosphate, 0.05 (w/v)% urea, and 0.04 (w/v)% ammonium sulfate.
3 seed culture
(i) Shake flask seed culture: inoculating the glycerol tube strain of Candida tropicalis into 500mL triangular flask (liquid loading amount is 50 mL) filled with seed culture medium, shake culturing at 220rpm for 36h under initial pH value of 6.2 and temperature of 29 deg.C, and OD 620 More than 0.5 after 30 times dilution;
(ii) Continuous culture of seed liquid: inoculating shake flask seed into a seed tank containing 6L seed culture medium, inoculating 20% (v/v), culturing at 29 deg.C under 0.10MPa, ventilation of 0.62vvm, pH of 5.7, and culturing under 25% -35% of dissolved oxygen DO to thallus concentration OD 620 And diluting the solution to be 0.62 after 30 times, continuously adding the nutrient salt-containing solution into the seed tank at a flow acceleration of 132mL/h for continuous culture, and simultaneously flowing out a part of seed liquid for fermentation production of long-chain dibasic acid. The seed liquid volume in the seed tank was maintained at 0.59L.
Example 6
Strain 1
The candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 seed Medium and nutrient salt-containing solution
Seed culture medium: sucrose content of 3.2 (w/v), corn steep liquor content of 0.45 (w/v), yeast extract content of 0.9 (w/v), KH 2 PO 4 The content of (C) was 0.6 (w/v)%, and the content of urea was 0.37 (w/v)%.
The nutrient salt-containing solution contained 1 (w/v) glucose, 0.85 (w/v) corn steep liquor, 0.82 (w/v) yeast extract, 0.1 (w/v) monopotassium phosphate, 0.01 (w/v) urea and 0.08 (w/v) ammonium sulfate.
3 seed culture
(i) Shake flask seed culture: inoculating the glycerol tube strain of Candida tropicalis into 500mL triangular flask (liquid loading amount is 50 mL) filled with seed culture medium, shake culturing at 220rpm for 36h under initial pH value of 6.2 and temperature of 29 deg.C, and OD 620 More than 0.5 after 30 times dilution;
(ii) Continuous culture of seed liquid: inoculating shake flask seed into a seed tank containing 6L seed culture medium, inoculating 15% (v/v), culturing at 28deg.C under 0.10MPa, ventilation of 0.62vvm, pH of 5.6, and culturing with dissolved oxygen DO at 25% -35% until thallus concentration OD 620 And diluting the solution to be 0.72 after 30 times, continuously adding the nutrient salt-containing solution into the seed tank at a flow acceleration of 132mL/h for continuous culture, and simultaneously, flowing out a part of seed liquid for fermentation production of long-chain dibasic acid. The seed liquid volume in the seed tank was maintained at 1.24L.
Example 7
Strain 1
Candida viscidosa (Candida viswanathii) strain CAES2113 with a preservation number cctccc M2020048.
2 seed Medium and nutrient salt-containing solution
Seed culture medium: sucrose content of 1.5 (w/v), corn steep liquor content of 0.65 (w/v), yeast extract content of 0.25 (w/v), KH 2 PO 4 The content of (C) was 1.3 (w/v)%, and the content of urea was 0.12 (w/v)%.
The nutrient salt-containing solution contained 1 (w/v) glucose, 1.2 (w/v) corn steep liquor, 0.4 (w/v) yeast extract, 0.015 (w/v) monopotassium phosphate, 0.03 (w/v) urea and 0.08 (w/v) ammonium sulfate.
3 seed culture
(i) Shake flask seed culture: inoculating the glycerol tube strain of Candida tropicalis into 500mL triangular flask (liquid loading amount is 50 mL) filled with seed culture medium, shake culturing at 220rpm for 36h under initial pH value of 6.2 and temperature of 29 deg.C, and OD 620 More than 0.5 after 30 times dilution;
(ii) Continuous culture of seed liquid: inoculating shake flask seed into a seed tank containing 6L seed culture medium, inoculating 25% (v/v), culturing at 28deg.C under 0.11MPa, ventilation of 0.62vvm, pH of 5.6, and culturing with dissolved oxygen DO at 25% -35% to thallus concentration OD 620 Diluting 30 times to 0.64, and continuously feeding into the seed tank at a flow acceleration of 132mL/hThe solution containing the nutrient salt is continuously cultured, and a part of seed liquid is discharged for fermenting and producing long-chain dibasic acid. The seed liquid volume in the seed tank was maintained at 1.24L.
Example 8
Strain 1
The candida tropicalis (Candida tropicalis) strain CAT H1614 has a preservation number of CCTCC M2013143.
2 seed Medium and nutrient salt-containing solution
Seed culture medium: sucrose content of 2.2 (w/v), corn steep liquor content of 0.33 (w/v), yeast extract content of 0.6 (w/v), KH 2 PO 4 The content of (C) was 0.9 (w/v)%, and the content of urea was 0.23 (w/v)%.
The nutrient salt-containing solution contained 0.37 (w/v)% corn steep liquor, 0.22 (w/v)% yeast extract, 0.012 (w/v)% monopotassium phosphate, 0.08 (w/v)% urea, and 0.04 (w/v)% ammonium sulfate.
3 seed culture
(i) Shake flask seed culture: inoculating the glycerol tube strain of Candida tropicalis into 500mL triangular flask (liquid loading amount is 50 mL) filled with seed culture medium, shake culturing at initial pH of 6.2 and temperature of 29 deg.C at 300rpm for 36h, and OD 620 More than 0.5 after 30 times dilution;
(ii) Continuous culture of seed liquid: inoculating shake flask seed into a seed tank containing 6L seed culture medium, inoculating 25% (v/v), culturing at 29 deg.C under 0.10MPa, ventilation of 0.62vvm, pH of 5.6, and culturing under 25% -35% of dissolved oxygen DO to thallus concentration OD 620 And diluting the solution to be 0.66 after 30 times, continuously adding the solution containing the nutrient salt into the seed tank at a flow acceleration of 132mL/h for continuous culture, and simultaneously, flowing out a part of seed liquid for fermentation production of long-chain dibasic acid. The seed liquid volume in the seed tank was maintained at 1.24L.
Application examples 1 to 8
The seed solutions cultured in examples 1 to 8 were used for fermentation verification as follows:
fermentation medium of long carbon chain dibasic acid: glucose content of 2.0 (w/v), corn steep liquor content of 0.20 (w/v), yeast extract content of 0.25 (w/v), potassium nitrate content of 0.05 (w/v), potassium dihydrogen phosphate content of 0.07 (w/v), urea content of 0.15 (w/v), ammonium sulfate content of 0.20 (w/v), sodium chloride content of 0.12 (w/v) and the balance of water.
First, seed solution (OD) of mature candida tropicalis (Candida tropicalis) strain CAT H1614 was inoculated into a fermentation medium 620 0.6% after 30 times dilution) and substrate alkane, the inoculation amount of the seed liquid is 25% (v/v), the addition amount of alkane is 5% (v/v), then the fermentation is carried out by controlling the temperature to 29 ℃, the pressure to 0.10MPa, the ventilation amount to 0.5vvm, the pH value to 5.7 and the dissolved oxygen to more than 10%, and the alkane content in the fermentation liquid is maintained to be more than 2% (v/v) all the time through feeding alkane in the fermentation process.
After 125 hours of fermentation, the seed solutions obtained in examples 1 to 8 were fed into the fermentation broth at a flow rate of 132mL/h, respectively, to maintain the cell concentration in the fermenter at OD 620 The dilution is more than 0.50 after 30 times, and the volume of the fermentation liquid in the fermentation tank is maintained at 22.5L by controlling the discharging rate, so that the long carbon chain dibasic acid is continuously produced. The yield results of the long chain dibasic acids are shown in Table 1.
TABLE 1
Claims (10)
1. A continuous culture method of seed liquid, comprising the following steps:
inoculating seed solution into seed tank containing seed culture medium, culturing under conditions of dissolved oxygen of above 3% and pH of 4.0-7.5, and culturing to thallus concentration OD 620 After being diluted by 30 times, the solution containing nutritive salt is added into the seed tank to continue culturing, and partial seed liquid is discharged for fermenting and producing long chainDibasic acid with seed tank dilution rate D of 0.007-0.25h -1 。
2. The method according to claim 1, wherein the seed tank dilution ratio D is 0.02-0.25h -1 Further for 0.08-0.15h -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,
3% -60%, further 3% -38%, further 3% -20% or 22% -38% of dissolved oxygen in the seed liquid culture process; and/or the number of the groups of groups,
the expression of the long-chain dibasic acid is HOOC (CH) 2 ) n COOH, wherein n is not less than 7, preferably any one or a combination of two or more 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.
3. The method according to claim 1, wherein the culture is carried out to a cell concentration OD 620 After dilution by 30 times to more than 0.5, the feed of the nutrient salt-containing solution into the seed tank was started.
4. The method according to claim 1, wherein the culture is carried out to a cell concentration OD 620 After dilution by 30 times, the concentration is 0.4-1.0, and the solution containing nutrient salt is fed into the seed tank.
5. The method of claim 1, wherein the seed medium is inoculated in an amount greater than 0 and less than or equal to 30% (v/v), further 10-30% (v/v).
6. The method according to claim 1, characterized in that the temperature during the cultivation is 28-32 ℃, and/or the pressure is 0.05-0.14MPa, and/or the pH value is preferably 5.0-7.0, further preferably 5.0-6.5, and/or the ventilation is 0.3-0.7vvm.
7. The method of claim 1, wherein the seed solution comprises a fermentation species including, but not limited to, candida tropicalis, candida vista.
8. The method according to claim 1 or 5, wherein the seed medium contains sucrose in an amount of 1-5 (w/v), corn steep liquor in an amount of 0.15-1 (w/v), yeast extract in an amount of 0.2-1.5 (w/v), KH 2 PO 4 The content of (C) is 0.4-1.8% (w/v), the content of urea is 0.05-0.5% (w/v), and the balance is water.
9. The method according to claim 1, 3 or 4, wherein the nutrient salt-containing solution contains 0-2.5 (w/v) glucose, 0.05-2.0 (w/v) corn steep liquor, 0-1.0 (w/v) yeast extract, 0.005-0.5 (w/v) potassium dihydrogen phosphate, 0.005-0.08 (w/v) urea and 0.01-0.15 (w/v) ammonium sulfate, the balance being water.
10. The method according to claim 1, wherein the feeding of the nutrient salt-containing solution into the seed solution is carried out in a continuous or batch mode; and/or the number of the groups of groups,
the way of flowing out part of the seed liquid is continuous discharging or batch discharging.
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