CN114015574A - Rapid proliferation culture method for noctiluca alternifolia - Google Patents
Rapid proliferation culture method for noctiluca alternifolia Download PDFInfo
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- 241000200174 Noctiluca Species 0.000 title claims abstract description 42
- 238000012136 culture method Methods 0.000 title claims abstract description 12
- 230000035755 proliferation Effects 0.000 title claims abstract description 10
- 241000195493 Cryptophyta Species 0.000 claims abstract description 35
- 239000013535 sea water Substances 0.000 claims abstract description 31
- 239000001963 growth medium Substances 0.000 claims abstract description 22
- 241000196316 Tetraselmis subcordiformis Species 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 10
- 238000005286 illumination Methods 0.000 claims abstract description 8
- 230000031016 anaphase Effects 0.000 claims abstract description 4
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 230000003203 everyday effect Effects 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims abstract description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 6
- 235000013343 vitamin Nutrition 0.000 claims description 6
- 229930003231 vitamin Natural products 0.000 claims description 6
- 239000011782 vitamin Substances 0.000 claims description 6
- 229940088594 vitamin Drugs 0.000 claims description 6
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 6
- 241000196317 Platymonas Species 0.000 claims description 4
- 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 description 3
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 3
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 3
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 3
- 229930003756 Vitamin B7 Natural products 0.000 claims description 3
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 3
- 239000011565 manganese chloride Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 239000011684 sodium molybdate Substances 0.000 claims description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 239000011735 vitamin B7 Substances 0.000 claims description 3
- 235000011912 vitamin B7 Nutrition 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 239000011686 zinc sulphate Substances 0.000 claims description 3
- 235000016709 nutrition Nutrition 0.000 abstract description 9
- 235000015097 nutrients Nutrition 0.000 abstract description 8
- 230000035764 nutrition Effects 0.000 abstract description 8
- 150000003839 salts Chemical class 0.000 abstract description 7
- 238000000034 method Methods 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 11
- 239000002609 medium Substances 0.000 description 4
- 241000199914 Dinophyceae Species 0.000 description 2
- 241000200173 Noctiluca scintillans Species 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 241000195633 Dunaliella salina Species 0.000 description 1
- 241000242583 Scyphozoa Species 0.000 description 1
- 241000196321 Tetraselmis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
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- 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
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Abstract
The invention discloses a rapid proliferation culture method of noctilucent algae, which is a method for obtaining high-density noctilucent algae in a short time at one time by providing low-level nutrient salt addition and through a chain of inorganic nutrition, bait algae and noctilucent algae, and comprises the following steps: (1) the noctilucent algae bait is prepared from Platymonas subcordiformis, and the Platymonas subcordiformis is cultured for 7 days by using an f/2 seawater culture medium until the late stage of the index. (2) Diluting noctiluca and the anaphase Platymonas subcordiformis of the index in the step (1) into a mixed culture system with final concentrations of 1 cells/mL and 10000 cells/mL by using an f/8 seawater culture medium, then culturing under the conditions that the temperature is 20 +/-1 ℃, the illumination is 2650 +/-100 lux and the illumination period is L: D =14 h: 10 h, shaking up the mixture twice regularly every day, and culturing for 8-10 days to obtain the noctiluca subcordiformis with the density of more than 100 cells/mL.
Description
Technical Field
The invention belongs to the fields of microalgae biology and marine biology, and particularly relates to a culture method for rapidly proliferating noctiluca by artificially enriching nutrients to obtain a large number of algae cells in a short time.
Background
The 'blue tear' landscape is due toThe natural phenomenon of seawater fluorescence caused by mass propagation or aggregation of organisms such as noctiluca edulis, seawater fluorescence and the like in the sea. In Fujian quan, a sea area near shore in Xiamen, blue eye tears become a well-known tourism landscape, and attract a large number of tourists to chase after from 3 months to 5 months every year, thereby having positive significance for the construction of local tourism industry. Night light algae (Noctiluca scintillans) Massive growth and accumulation offshore is the main cause of the local "blue tears" phenomenon. The alga is dinoflagellate widely distributed in the world, can be divided into red noctiluca fulva (RNS) which completely affects heterotrophic life and green noctiluca fulva (GNS) which coexists with photosynthetic microalgae and performs heterotrophic life in both nutrition and nutrition, is common in China coast and is red noctiluca fulva, and is also a main cause of 'blue eye tear' phenomena such as puddle and the like.
Noctiluca is a noctiluca algae family of dinoflagellate, and has a round shape, a diameter of 150-2000 μm, a transparent cell wall and a reddish intracellular protoplasm. The algae has one long flagella and one short flagella, the algae body can emit transient blue-green fluorescence (wavelength is 500 nm, duration is 100 ms) when being stimulated (such as shaking, sea waves and the like), and the luminous algae which is propagated or gathered in a large scale can emit fluorescence when being stimulated under the action of the sea waves or human, which is the reason for forming the 'blue-eye tear' landscape. Although the noctilucent algae bloom is identified as a harmful algae bloom, the noctilucent algae does not produce toxicity, and the fluorescent characteristic of the noctilucent algae not only can form 'blue-eye tear' landscape, but also has certain economic value. However, noctilucent algae is not like small jellyfish in Qingdao (which is also an ecological problem), and can form a commodity similar to an aquarium to promote local travel development, because the fluorescence emitted by noctilucent algae is clear and strong only under the condition of high density, but no method capable of rapidly culturing noctilucent algae exists at present, so that the species cannot be commercialized. At present, the culture of noctiluca is only carried out in a small volume under the laboratory condition. Wuyulin et al, in "indoor culture of noctiluca (marine and Hu marsh 1994 Vol. 25, 2)" mention that Platymonas vernalis can be used: (Platymonassp.) as bait material by continuous feeding inThe population density of the noctiluca alternifolia is improved from 0.75 cells/mL to 6 cells/mL within 20 days, the method needs to continuously provide baits, the operation is complicated, and the growth rate of the noctiluca alternifolia is not high. The slow toughening of the noctiluca alternifolia in' laboratory culture of noctiluca alternifolia and observation of tentacle dichotomy thereof (19 th volume 3 of 1995) also mentions that the panoplasma alternifolia can be used as bait of the noctiluca alternifolia, but the population density of the noctiluca alternifolia is not obviously improved in the culture process of 6 days. The noctiluca is cultured by Dunaliella salina in Cao Xinyu under the conditions of 'influence of temperature and salinity on growth of noctiluca algae population density (No. 1 of 356 volume of 2021 of university of Dalian ocean)', and is continuously cultured for 20 days at 12 ℃, wherein the noctiluca is improved from 1 cell/mL to 15 cells/mL, and the noctiluca is characterized by long time consumption and low growth rate. How to rapidly obtain a large number of noctiluca edulis individuals in a short time is not explained by related technologies at present.
Disclosure of Invention
The invention aims to provide a culture method for obtaining high-density noctilucent algae in a short time (8-10 days), and particularly relates to a method for obtaining the high-density noctilucent algae in a short time at one time by providing low-level nutrient salt addition and a chain of inorganic nutrition, bait algae and noctilucent algae.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a rapid proliferation culture method of noctiluca alternifolia comprises the following steps:
(1) the luminous algae bait is selected from Platymonas subcordiformis, and the Platymonas subcordiformis is cultured with f/2 seawater culture medium for 7 days to the later exponential phase (1.0-1.5 × 10)6cells/mL) for use. Wherein the f/2 seawater culture medium has the following formula:
NaNO3 75 mg/L；
NaH2PO3 5 mg/L；
FeCl3·6H2O 3.15 mg/L；
Na2EDFA 4.36 mg/L；
CuSO4·5H2O 9.8 μg/L；
ZnSO4·7H2O 22 μg/L；
MnCl2·4H2O 0.18 mg/L；
CoCl2·6H2O 10 μg/L；
Na2MoO4·2H2O 6.3 μg/L；
vitamin B10.1 mg/L;
vitamin B120.5 mug/L;
vitamin H is 0.1 mg/L.
(2) Diluting noctiluca and the anaphase Platymonas subcordiformis of the index in the step (1) into a mixed culture system with final concentrations of 1 cells/mL and 10000 cells/mL by using an f/8 seawater culture medium, then culturing under the conditions that the temperature is 20 +/-1 ℃, the illumination is 2650 +/-100 lux, the illumination period is L: D =14 h: 10 h, shaking up the mixture slightly and uniformly twice every day at regular time (morning and evening), and culturing for 8-10 days to obtain the noctiluca subcordiformis with the density of more than 100 cells/mL.
The formula of the f/8 culture medium is that the f/2 culture medium is diluted by sterile seawater with three times of volume to obtain the f/8 seawater culture medium; wherein the preparation of the sterile seawater comprises the following steps: filtering natural seawater with 0.22 μm filter membrane, and sterilizing at 121 deg.C for 25 min.
Has the advantages that:
(1) the method cultures the noctilucent algae by adding the bait algae and the inorganic nutrition at one time, and has simple and convenient operation and easy execution.
(2) The invention adopts a mode of providing low-concentration nutrition (f/8 seawater culture medium) to stimulate the growth of bait algae of the noctilucent algae, provides continuous nutrition supply for the noctilucent algae, and simultaneously does not cause the mass propagation of the bait algae to inhibit the growth of the noctilucent algae because of too much provided inorganic nutrition, thus obtaining a large amount of high-density noctilucent algae in a short time.
Drawings
FIG. 1 is a graph showing population density increase curves of noctiluca and Platymonas under different nutritional conditions, wherein A is f/2; b is f/4 (f/2 culture medium is diluted by equal volume of sterile seawater); c is f/8 (f/2 medium using three times the volume of sterile seawater dilution); d is f/16 (f/2 medium diluted with seven volumes of sterile seawater); e is f/32 (f/2 medium diluted with fifteen times the volume of sterile seawater); f is a blank seawater control.
Detailed Description
For further disclosure, but not limitation, the present invention is described in further detail below with reference to examples.
Example 1
A rapid proliferation culture method of noctiluca alternifolia comprises the following steps:
(1) the luminous algae bait is selected from Platymonas subcordiformis, and the Platymonas subcordiformis is cultured with f/2 seawater culture medium for 7 days to the later exponential phase (1.0-1.5 × 10)6cells/mL) for use. Wherein the f/2 seawater culture medium has the following formula:
NaNO3 75 mg/L；
NaH2PO3 5 mg/L；
FeCl3·6H2O 3.15 mg/L；
Na2EDFA 4.36 mg/L；
CuSO4·5H2O 9.8 μg/L；
ZnSO4·7H2O 22 μg/L；
MnCl2·4H2O 0.18 mg/L；
CoCl2·6H2O 10 μg/L；
Na2MoO4·2H2O 6.3 μg/L；
vitamin B10.1 mg/L;
vitamin B120.5 mug/L;
vitamin H is 0.1 mg/L.
(2) Diluting noctiluca and the anaphase Platymonas subcordiformis of the index in the step (1) into a mixed culture system with final concentrations of 1 cells/mL and 10000 cells/mL by using an f/8 seawater culture medium, then culturing under the conditions that the temperature is 20 +/-1 ℃, the illumination is 2650 +/-100 lux and the illumination period is L: D =14 h: 12 h, shaking up the mixture slightly and uniformly twice every day at regular time (morning and evening), and culturing for 8-10 days to obtain the noctiluca subcordiformis with the density of more than 100 cells/mL.
The formula of the f/8 culture medium is that the f/2 culture medium is diluted by sterile seawater with three times of volume to obtain the f/8 seawater culture medium; wherein the preparation of the sterile seawater comprises the following steps: filtering natural seawater with 0.22 μm filter membrane, and sterilizing at 121 deg.C for 25 min.
The results in FIG. 1 show that the co-cultivation system of noctiluca and Platymonas varies with the concentration of the nutrient salts. The concentration of the nutrient salt is increased, the growth rate of the noctiluca alternifolia is gradually increased, and the highest algae density is gradually increased. The trend reaches a peak when the density of the nutrient salt reaches f/8 and then gradually decreases, and when the nutrient salt is at a higher density (f/2 or f/4), the growth of the noctiluca is gradually inhibited by mass propagation of the tetraselmis. This indicates that medium level nutrient salt enrichment is more beneficial to the growth of noctiluca scintillans in the noctiluca scintillans-tetraselmis coculture system.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (5)
1. A rapid proliferation culture method of noctiluca alternifolia is characterized by comprising the following steps:
(1) selecting Platymonas subcordiformis as noctilucent algae bait, and culturing the Platymonas subcordiformis for 7 days by using an f/2 seawater culture medium until the index later stage for later use;
(2) diluting noctiluca and the anaphase Platymonas subcordiformis of the index in the step (1) into a mixed culture system with final concentrations of 1 cells/mL and 10000 cells/mL by using an f/8 seawater culture medium, then culturing under the conditions that the temperature is 20 +/-1 ℃, the illumination is 2650 +/-100 lux and the illumination period is L: D =14 h: 10 h, shaking up the mixture slightly and uniformly twice every day at regular time (morning and evening), and culturing for 8-10 days to obtain the noctiluca subcordiformis with the density of more than 100 cells/mL.
2. The rapid proliferation culture method of noctiluca according to claim 1, characterized in that: the late exponential phase in the step (1) means that the concentration of the Platymonas is 1.0-1.5 multiplied by 106 cells/mL。
3. The rapid proliferation culture method of noctiluca according to claim 1, characterized in that: the f/2 seawater culture medium in the step (1) has the following formula:
NaNO3 75 mg/L；
NaH2PO3 5 mg/L；
FeCl3·6H2O 3.15 mg/L；
Na2EDFA 4.36 mg/L；
CuSO4·5H2O 9.8 μg/L；
ZnSO4·7H2O 22 μg/L；
MnCl2·4H2O 0.18 mg/L；
CoCl2·6H2O 10 μg/L；
Na2MoO4·2H2O 6.3 μg/L；
vitamin B10.1 mg/L;
vitamin B120.5 mug/L;
vitamin H is 0.1 mg/L.
4. The rapid proliferation culture method of noctiluca according to claim 1, characterized in that: the f/8 culture medium formula in the step (2) is that f/2 culture medium is diluted by using sterile seawater with three times of volume to obtain the f/8 seawater culture medium.
5. The rapid proliferation culture method of noctiluca according to claim 1, characterized in that: the preparation of the sterile seawater in the step (4): filtering natural seawater with 0.22 μm filter membrane, and sterilizing at 121 deg.C for 25 min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114874913A (en) * | 2022-05-12 | 2022-08-09 | 江苏海洋大学 | Method for manufacturing luminous algae fluorescent ecological bottle |
| CN116904318A (en) * | 2023-08-04 | 2023-10-20 | 江苏海洋大学 | Method for stably proliferating noctilucent algae based on mixed culture |
| CN118109552A (en) * | 2024-04-16 | 2024-05-31 | 江苏海洋大学 | Method for judging proliferation of noctilucent algae population based on cell size |
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| CN102643750A (en) * | 2012-04-25 | 2012-08-22 | 王培磊 | Platymonas subcordiformis medium formula and platymonas subcordiformis three-level culturing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114874913A (en) * | 2022-05-12 | 2022-08-09 | 江苏海洋大学 | Method for manufacturing luminous algae fluorescent ecological bottle |
| CN116904318A (en) * | 2023-08-04 | 2023-10-20 | 江苏海洋大学 | Method for stably proliferating noctilucent algae based on mixed culture |
| CN116904318B (en) * | 2023-08-04 | 2024-02-06 | 江苏海洋大学 | Method for stably proliferating noctilucent algae based on mixed culture |
| CN118109552A (en) * | 2024-04-16 | 2024-05-31 | 江苏海洋大学 | Method for judging proliferation of noctilucent algae population based on cell size |
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