CN112457995A - Special culture medium for producing concentrated oocystis and culture method thereof - Google Patents
Special culture medium for producing concentrated oocystis and culture method thereof Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 36
- 241000514008 Oocystis Species 0.000 title claims abstract description 12
- 238000012136 culture method Methods 0.000 title description 4
- 210000003250 oocyst Anatomy 0.000 claims abstract description 52
- 241000195493 Cryptophyta Species 0.000 claims abstract description 50
- 238000000855 fermentation Methods 0.000 claims abstract description 48
- 230000004151 fermentation Effects 0.000 claims abstract description 48
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 42
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 30
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 14
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 10
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 10
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims abstract description 10
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 claims abstract description 7
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000004471 Glycine Substances 0.000 claims abstract description 7
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 claims abstract description 7
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract 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 abstract description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 5
- 235000019270 ammonium chloride Nutrition 0.000 claims abstract description 5
- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 claims abstract description 5
- 235000020958 biotin Nutrition 0.000 claims abstract description 5
- 239000011616 biotin Substances 0.000 claims abstract description 5
- 229960002685 biotin Drugs 0.000 claims abstract description 5
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims abstract description 5
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims abstract description 5
- 239000004202 carbamide Substances 0.000 claims abstract description 5
- QGUAJWGNOXCYJF-UHFFFAOYSA-N cobalt dinitrate hexahydrate Chemical compound O.O.O.O.O.O.[Co+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O QGUAJWGNOXCYJF-UHFFFAOYSA-N 0.000 claims abstract description 5
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims abstract description 5
- 229960004642 ferric ammonium citrate Drugs 0.000 claims abstract description 5
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims abstract description 5
- 239000008103 glucose Substances 0.000 claims abstract description 5
- 239000004313 iron ammonium citrate Substances 0.000 claims abstract description 5
- 235000000011 iron ammonium citrate Nutrition 0.000 claims abstract description 5
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims abstract description 5
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 claims abstract description 5
- 239000001632 sodium acetate Substances 0.000 claims abstract description 5
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 5
- 239000004317 sodium nitrate Substances 0.000 claims abstract description 5
- 235000010344 sodium nitrate Nutrition 0.000 claims abstract description 5
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims abstract description 5
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 30
- 230000001954 sterilising effect Effects 0.000 claims description 24
- 239000000126 substance Substances 0.000 claims description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims description 13
- 239000002028 Biomass Substances 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 229930003270 Vitamin B Natural products 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 235000019156 vitamin B Nutrition 0.000 claims description 8
- 239000011720 vitamin B Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 5
- -1 0.1-0.4g/L Chemical compound 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- QJMBVMLTKGQFKW-UHFFFAOYSA-L disodium dihydrogen phosphate dihydrate Chemical compound O.O.[Na+].[Na+].OP(O)([O-])=O.OP(O)([O-])=O QJMBVMLTKGQFKW-UHFFFAOYSA-L 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 4
- 238000004519 manufacturing process Methods 0.000 claims 4
- 239000000203 mixture Substances 0.000 claims 2
- 239000011780 sodium chloride Substances 0.000 claims 2
- 241000222195 Ascochyta Species 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 10
- 230000005764 inhibitory process Effects 0.000 abstract description 6
- LXAHHHIGZXPRKQ-UHFFFAOYSA-N 5-fluoro-2-methylpyridine Chemical compound CC1=CC=C(F)C=N1 LXAHHHIGZXPRKQ-UHFFFAOYSA-N 0.000 abstract description 2
- 230000006698 induction Effects 0.000 abstract description 2
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 abstract 1
- 229930003451 Vitamin B1 Natural products 0.000 abstract 1
- 229930003779 Vitamin B12 Natural products 0.000 abstract 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract 1
- 235000013877 carbamide Nutrition 0.000 abstract 1
- 229960004106 citric acid Drugs 0.000 abstract 1
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 abstract 1
- 235000001727 glucose Nutrition 0.000 abstract 1
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 abstract 1
- 229960003495 thiamine Drugs 0.000 abstract 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 abstract 1
- 235000010374 vitamin B1 Nutrition 0.000 abstract 1
- 239000011691 vitamin B1 Substances 0.000 abstract 1
- 235000019163 vitamin B12 Nutrition 0.000 abstract 1
- 239000011715 vitamin B12 Substances 0.000 abstract 1
- 238000009360 aquaculture Methods 0.000 description 10
- 244000144974 aquaculture Species 0.000 description 10
- 238000005286 illumination Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 241000195628 Chlorophyta Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000459 effect on growth Effects 0.000 description 1
- 230000001463 effect on reproduction Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
Images
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
- C12N1/12—Unicellular algae; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
Abstract
The invention relates to the technical field of microalgae cultivation, and discloses a special culture medium for producing concentrated oocystis, which comprises a component A and a component B, wherein the component A comprises glucose, sodium acetate, sodium nitrate, urea, ammonium chloride, sodium dihydrogen phosphate dihydrate, EDTA disodium and ferric chloride hexahydrate; the component B comprises citric acid, ferric ammonium citrate, calcium chloride dihydrate, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, cobalt nitrate hexahydrate, vitamin B1, vitamin B12 and biotin. The invention has the following advantages and effects: the A component and the B component are mixed and inoculated with the oocyst algae seed solution, the oocyst algae can grow rapidly during fermentation culture, and the concentrated oocyst algae solution is produced and has the effects of high growth rate and high success rate of expanded culture. The special culture medium is added with glycerol, maltotriose and glycine, and the oocyst algae with the obvious inhibition effect on moss can be obtained by domestication through induction of various factors in the culture process.
Description
Technical Field
The invention relates to the technical field of microalgae cultivation, in particular to a special culture medium for producing concentrated oocystis and a culture method thereof.
Background
With the use of a large amount of chemical fertilizers in agricultural production and the discharge of industrial wastewater, after a large amount of nitrogen and phosphorus elements enter a water body, the environment of the aquaculture water body is gradually eutrophicated, and undesirable algae such as blue-green algae and moss in the aquaculture water body often have the capability of rapid growth, so that cyanobacterial bloom or other undesirable algae phases often appear in the aquaculture water body, which leads to the deterioration of the environment of the aquaculture water body, the poisoning of aquaculture animals and finally the failure of aquaculture.
In the prior art, beneficial algae preparations are used for artificially intervening the algae phase of the aquaculture water body, and the beneficial algae is directionally cultured to improve the aquaculture water environment and improve the aquaculture rate. In addition, the algae preparation not only has the effect of directional water fertilization, but also has the functions of increasing dissolved oxygen in water and absorbing ammonia nitrogen and nitrite, so that the algae preparation is widely applied to aquaculture. The oocysts are relatively stable green algae, can keep the phenomenon of algae falling for 40-50 days, and have relatively strong capacity of absorbing ammonia nitrogen and nitrite, so the oocysts are the microalgae for well improving water quality strips and cultivating water algae phases.
Most of the existing oocysts are cultured by autotrophic illumination in the culture process, the culture efficiency is low, and the concentration of the cultured algae solution is low, so that the use is influenced.
Disclosure of Invention
The invention aims to provide a special culture medium for producing the concentrated oocyst algae, which has the effects of strong activity and high growth speed of the oocyst algae produced and cultured.
The technical purpose of the invention is realized by the following technical scheme: a special culture medium for producing concentrated oocystis comprises a component A and a component B, wherein the component A comprises 15-55g/L of glucose, 5-10g/L of sodium acetate, 5-10g/L of sodium nitrate, 1-3g/L of urea, 2-5g/L of ammonium chloride, 0.1-0.4g/L, EDTA disodium dihydrogen phosphate dihydrate 0.1-0.3g/L and 0.2-0.4g/L ferric chloride hexahydrate; the component B comprises 3-8g/L of citric acid, 3-8g/L of ferric ammonium citrate, 20-50g/L of calcium chloride dihydrate, 0.5-3g/L of manganese chloride tetrahydrate, 0.2-0.6g/L of zinc sulfate heptahydrate, 0.3-0.8g/L of sodium molybdate dihydrate, 0.5-0.8g/L of copper sulfate pentahydrate, 0.3-0.6g/L of cobalt nitrate hexahydrate, 10.1-0.3 g/L of vitamin B, 120.001-0.01 g/L of vitamin B and 0.003-0.005g/L of biotin; the volume ratio of the component A to the component B is 200:1-50: 1.
The invention further provides a preparation method of the component A, which comprises the following steps: taking the substances in the component A, dissolving the substances in water in a fermentation tank in proportion, and sterilizing at high temperature; the preparation method of the component B comprises the following steps: dissolving the components in the component B in sterile water, and sterilizing.
The invention is further provided with: the component B is prepared by filtering and sterilizing.
The invention is further provided with: the volume ratio of the component A to the component B is 100: 1.
The invention is further provided with: the component A also comprises 0.5-0.8g/L of maltotriose and 1-1.5g/L of glycine, the special culture medium for producing the concentrated oocyst algae also comprises glycerol, and the mass ratio of the citric acid to the glycerol is 1: 1.1-1.3.
The invention also aims to provide a method for culturing the oocyst algae by using the special culture medium, which comprises the steps of taking the substances in the component A, dissolving the substances in water in a fermentation tank according to a proportion, adjusting the pH value by using a NaOH solution, sterilizing at 121 ℃ for 20min, cooling to 30 ℃, adding the filtered and sterilized component B into the fermentation tank, inoculating the oocyst algae seed liquid into the fermentation tank, and ending fermentation when the fermentation biomass reaches 35000-50000mg/L to obtain the concentrated oocyst algae.
The invention is further provided with: during fermentation, when the culture temperature is 25-35 ℃, the fermentation biomass reaches 20000-.
The invention has the beneficial effects that: .
1. The special culture medium comprises the component A and the component B, the oocyst algae seed solution is inoculated after mixing, the oocyst algae can grow rapidly during fermentation culture, and the concentrated oocyst algae solution is produced and has the effects of high growth rate and high success rate of expanded culture.
2. At present, no oocyst algae specially inhibiting moss is domesticated and cultured in the prior art, glycerol, maltotriose and glycine are added into the special culture medium, and the temperature is reduced to 10-15 ℃ in the culture process, and then the illumination treatment is carried out, and the oocyst algae with the obvious inhibition effect on the moss can be obtained through induction of various factors.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a graph showing the growth of oocysts in example 1 and comparative example.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention.
Example 1: a special culture medium for producing concentrated oocystis comprises a component A and a component B, wherein the component A comprises 15g/L of glucose, 5g/L of sodium acetate, 5g/L of sodium nitrate, 1g/L of urea, 2g/L of ammonium chloride, 0.1g/L, EDTA g/L of disodium dihydrogen phosphate dihydrate, and 0.2g/L of ferric chloride hexahydrate. The preparation method of the component A comprises the following steps: dissolving the components in component A in water at a certain proportion in a fermentation tank, adjusting pH to 7.5 + -0.2 with NaOH solution, and sterilizing at 121 deg.C for 20 min.
The component B comprises 3g/L of citric acid, 20g/L of ferric ammonium citrate, 20g/L of calcium chloride dihydrate, 0.5g/L of manganese chloride tetrahydrate, 0.2g/L of zinc sulfate heptahydrate, 0.3g/L of sodium molybdate dihydrate, 0.5g/L of copper sulfate pentahydrate, 0.3g/L of cobalt nitrate hexahydrate, 10.1g/L of vitamin B, 120.001g/L of vitamin B and 0.003g/L of biotin. The preparation method of component B comprises dissolving the materials in component B in sterile water, and filtering for sterilization.
The volume ratio of the component A to the component B is 200: 1.
when the special culture medium for producing the concentrated oocyst algae is used for culturing the oocyst algae, the substances in the component A are taken and dissolved in water in proportion in a fermentation tank, the pH value is adjusted by NaOH solution, the pH value is maintained within the range of 7.5 +/-0.2, the sterilization is carried out at 121 ℃ for 20min, the temperature is cooled to 30 ℃, the component B after filtration and sterilization is added into the fermentation tank, the oocyst algae seed liquid is inoculated into the fermentation tank, the culture temperature is 25-35 ℃, the illumination is not carried out, the fermentation is finished until the fermentation biomass reaches 35000-.
Example 2: a special culture medium for producing concentrated oocystis comprises a component A and a component B, wherein the component A comprises 55g/L of glucose, 10g/L of sodium acetate, 10g/L of sodium nitrate, 3g/L of urea, 5g/L of ammonium chloride, 0.4g/L, EDTA g/L of disodium dihydrogen phosphate dihydrate, and 0.4g/L of ferric chloride hexahydrate. The preparation method of the component A comprises the following steps: dissolving the components in component A in water at a certain proportion in a fermentation tank, adjusting pH with NaOH solution, maintaining at 7.5 + -0.2, and sterilizing at 121 deg.C for 20 min.
The component B comprises 8g/L of citric acid, 8g/L of ferric ammonium citrate, 50g/L of calcium chloride dihydrate, 3g/L of manganese chloride tetrahydrate, 0.6g/L of zinc sulfate heptahydrate, 0.8g/L of sodium molybdate dihydrate, 0.8g/L of copper sulfate pentahydrate, 0.6g/L of cobalt nitrate hexahydrate, 0.3g/L of vitamin B, 120.01g/L of vitamin B and 0.005g/L of biotin. The preparation method of component B comprises dissolving the materials in component B in sterile water, and filtering for sterilization.
The volume ratio of the component A to the component B is 100: 1.
when the special culture medium for producing the concentrated oocyst algae is used for culturing the oocyst algae, taking substances in the component A, dissolving the substances in water in a fermentation tank according to a proportion, adjusting the pH value by using NaOH solution, maintaining the pH value within the range of 7.5 +/-0.2, sterilizing at 121 ℃ for 20min, cooling to 30 ℃, adding the component B subjected to filtration sterilization into the fermentation tank, inoculating the oocyst algae seed solution into the fermentation tank, adjusting the culture temperature to 25-35 ℃, adjusting the culture temperature to 10-15 ℃ when the fermentation biomass reaches 20000 and 25000mg/L, illuminating for 30min, culturing for 2h, illuminating to 5000lux, adjusting the culture temperature to 25-35 ℃, not illuminating, and continuing to culture until the fermentation biomass reaches 35000 and 50000mg/L, and ending the fermentation to obtain the concentrated oocyst algae.
Example 3: a special culture medium for producing concentrated oocyst algae is different from that in example 1 in that when the special culture medium for producing the concentrated oocyst algae is used for culturing the oocyst algae, all substances in the component A are taken and dissolved in water in proportion in a fermentation tank, the pH is adjusted by NaOH solution and maintained within the range of 7.5 +/-0.2, the sterilization is carried out for 20min at the temperature of 121 ℃, the filtration and sterilization are carried out to the component B, the filtration and sterilization are carried out, the component B is added into the fermentation tank, the oocyst algae seed liquid is inoculated into the fermentation tank, the culture temperature is 25-35 ℃, the fermentation is finished until the fermentation biomass reaches 35000-plus-material 50000mg/L, and the concentrated oocyst algae is obtained.
Example 4: a special culture medium for producing the concentrated oocystis is different from the culture medium in example 1 in that maltotriose and glycine are also included in the component A.
Example 5: a special culture medium for producing concentrated oocystis is different from that in example 1 in that raw materials also comprise glycerol, the mass ratio of citric acid to glycerol is 1:1, the substances in the component A are taken and dissolved in water in a fermentation tank in proportion, the pH is adjusted by NaOH solution and maintained within the range of 7.5 +/-0.2, the sterilization is carried out for 20min at 121 ℃, the glycerol and the component B after filtration sterilization are added into the fermentation tank after cooling to 30 ℃.
Example 6: a special culture medium for producing the concentrated oocystis is different from the culture medium in example 1 in that maltotriose and glycine are also contained in the component A, glycerol is also contained in the component B, and the mass ratio of citric acid to the glycerol is 1:1.
Example 7: a special culture medium for producing concentrated oocyst algae is characterized in that a component A also comprises maltotriose and glycine, the special culture medium also comprises glycerol, the mass ratio of citric acid to glycerol is 1:1, when the special culture medium for producing the concentrated oocyst algae is used for culturing the oocyst algae, all substances in the component A are taken and dissolved in water in proportion in a fermentation tank, the pH value is adjusted by NaOH solution and maintained within the range of 7.5 +/-0.2, the sterilization is carried out for 20min at 121 ℃, the temperature is cooled to 30 ℃, the glycerol and the B component after filtration sterilization are added into the fermentation tank, the oocyst algae seed liquid is inoculated into the fermentation tank, the culture temperature is 25-35 ℃, when the fermentation biomass reaches 2000000 mg/L, the illumination is 15min, the illumination is 6000lux, the culture is carried out for 1h, the culture temperature is adjusted to 25-35 ℃, the illumination is not carried out, the culture is continued until the fermentation biomass reaches 35000-, obtaining the concentrated oocystis.
Comparative example: a culture method of oocyst algae comprises the steps of selecting a conventional culture medium as a BG11 culture medium, adding the culture medium into a fermentation tank, adjusting the pH value with NaOH solution to maintain the pH value in the fermentation tank within the range of 7.5 +/-0.2, inoculating a seed solution of the oocyst algae into the fermentation tank, culturing at the temperature of 25-35 ℃, not carrying out illumination, and finishing fermentation until the fermentation biomass reaches 35000-.
Experiment one is to determine the growth curve of the oocyst algae in example 1 and comparative example 1 during culture, and as shown in fig. 1, the growth rate of the oocyst algae in example 1 is significantly higher than that of the oocyst algae in the comparative example.
Example Co-culture inhibition experiment of Oocystolonia and lichen
Cleaning and draining moss with clear water, spin-drying water by using a centrifuge at 4000rpm, cleaning the moss with the clear water for 2 times, accurately weighing 0.2g of moss in each 1L triangular flask, and adding 500mL of water. Experimental group 0.1g of each of the oocysts obtained in examples 1 to 6 was weighed and put into three triangular flasks, respectively. The blank group was prepared by adding 0.1g of oocyst algae cultured in a conventional medium (BG11) into a flask as a control. And placing illumination culture on the illumination culture shelf. After 7 days, the moss is filtered out, the moss is dried by a centrifuge at 4000rpm, and is washed by clean water for 2 times, and then the moss is weighed, observed in shape and observed under a microscope.
And (4) analyzing results: compared with the comparative example, the examples 1 to 6 have stronger inhibition effect on moss, and the growth and reproduction speed of the oocyst algae is high through analysis. Example 7 compared with examples 1-6, the inhibition effect on moss is obviously enhanced, and through analysis, the oocyst algae obtained through domestication culture in example 7 can have a strong inhibition effect on growth and reproduction of moss.
TABLE 1 results of weighing after co-cultivation of oocysts and lichens
Claims (7)
1. A special culture medium for producing concentrated oocystis is characterized in that: the composition comprises a component A and a component B, wherein the component A comprises 15-55g/L of glucose, 5-10g/L of sodium acetate, 5-10g/L of sodium nitrate, 1-3g/L of urea, 2-5g/L of ammonium chloride, 0.1-0.4g/L, EDTA g/L of disodium dihydrogen phosphate dihydrate, 0.1-0.3g/L of ferric chloride hexahydrate and a mixture of sodium chloride and sodium chloride;
the component B comprises 3-8g/L of citric acid, 3-8g/L of ferric ammonium citrate, 20-50g/L of calcium chloride dihydrate, 0.5-3g/L of manganese chloride tetrahydrate, 0.2-0.6g/L of zinc sulfate heptahydrate, 0.3-0.8g/L of sodium molybdate dihydrate, 0.5-0.8g/L of copper sulfate pentahydrate, 0.3-0.6g/L of cobalt nitrate hexahydrate, 10.1-0.3 g/L of vitamin B, 120.001-0.01 g/L of vitamin B and 0.003-0.005g/L of biotin;
the volume ratio of the component A to the component B is 200:1-50: 1.
2. The special culture medium for the production of oocysts concentrated according to claim 1, characterized in that: the preparation method of the component A comprises the following steps: taking the substances in the component A, dissolving the substances in water in a fermentation tank in proportion, and sterilizing at high temperature; the preparation method of the component B comprises the following steps: dissolving the components in the component B in sterile water, and sterilizing.
3. The special culture medium for the production of oocysts concentrated according to claim 2, characterized in that: the component B is prepared by filtering and sterilizing.
4. The special culture medium for the production of oocysts concentrated according to claim 1, characterized in that: the volume ratio of the component A to the component B is 100: 1.
5. The special culture medium for the production of oocysts concentrated according to claim 1, 2 or 3, characterized in that: the component A also comprises 0.5-0.8g/L of maltotriose and 1-1.5g/L of glycine, the special culture medium for producing the concentrated oocyst algae also comprises glycerol, and the mass ratio of the citric acid to the glycerol is 1: 1.1-1.3.
6. A method for culturing oocysts using the special culture medium for producing concentrated oocysts according to any one of claims 1 to 5, wherein: taking all substances in the component A, dissolving the substances in water in proportion in a fermentation tank, adjusting the pH value to be within the range of 7.5 +/-0.2 by using NaOH solution, sterilizing for 20min at 121 ℃, cooling to 30 ℃, adding the component B subjected to filtration sterilization into the fermentation tank, inoculating the ascochyta seed liquid into the fermentation tank, and ending the fermentation when the fermentation biomass reaches 35000-.
7. The method of claim 6, wherein the culture medium comprises: during fermentation, when the culture temperature is 25-35 ℃, the fermentation biomass reaches 20000-.
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