CN106811417B - A kind of medium for Alexander microalgae and cultivation method thereof - Google Patents
A kind of medium for Alexander microalgae and cultivation method thereof Download PDFInfo
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- 238000012364 cultivation method Methods 0.000 title 1
- 239000001963 growth medium Substances 0.000 claims abstract description 42
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims abstract description 30
- 241000200031 Alexandrium Species 0.000 claims abstract description 25
- 239000013535 sea water Substances 0.000 claims abstract description 24
- 235000010344 sodium nitrate Nutrition 0.000 claims abstract description 15
- 239000004317 sodium nitrate Substances 0.000 claims abstract description 15
- 238000012136 culture method Methods 0.000 claims abstract description 13
- 239000003053 toxin Substances 0.000 claims abstract description 12
- 231100000765 toxin Toxicity 0.000 claims abstract description 12
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 10
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 10
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 10
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims abstract description 9
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims abstract description 9
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims abstract description 9
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims abstract description 9
- 229910000365 copper sulfate Inorganic materials 0.000 claims abstract description 9
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims abstract description 9
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims abstract description 9
- 239000011565 manganese chloride Substances 0.000 claims abstract description 9
- 235000002867 manganese chloride Nutrition 0.000 claims abstract description 9
- 229940099607 manganese chloride Drugs 0.000 claims abstract description 9
- 239000011684 sodium molybdate Substances 0.000 claims abstract description 9
- 235000015393 sodium molybdate Nutrition 0.000 claims abstract description 9
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims abstract description 9
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 9
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 9
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 9
- 244000005700 microbiome Species 0.000 claims abstract description 3
- 239000011550 stock solution Substances 0.000 claims description 34
- 241000195493 Cryptophyta Species 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 19
- 238000012258 culturing Methods 0.000 claims description 15
- 238000005286 illumination Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 239000011734 sodium Substances 0.000 claims description 8
- 229910021654 trace metal Inorganic materials 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- YZDZYSPAJSPJQJ-UHFFFAOYSA-N samarium(3+);trinitrate Chemical compound [Sm+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O YZDZYSPAJSPJQJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 230000003698 anagen phase Effects 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000003306 harvesting Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 230000001502 supplementing effect Effects 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 abstract 1
- 108700012359 toxins Proteins 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000001769 paralizing effect Effects 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000238 shellfish toxin Substances 0.000 description 3
- 208000005374 Poisoning Diseases 0.000 description 2
- 208000004891 Shellfish Poisoning Diseases 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 206010000370 Accident at home Diseases 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000199914 Dinophyceae Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- CETRDCWBMBILAL-XXKOCQOQSA-N Gonyautoxin 1 Chemical compound N=C1N(O)[C@@H](COC(=O)N)[C@@H]2NC(N)=N[C@@]22C(O)(O)[C@H](OS(O)(=O)=O)CN21 CETRDCWBMBILAL-XXKOCQOQSA-N 0.000 description 1
- ARSXTTJGWGCRRR-XXKOCQOQSA-N Gonyautoxin 2 Chemical compound NC(=O)OC[C@@H]1N=C(N)N2C[C@@H](OS(O)(=O)=O)C(O)(O)[C@@]22N=C(N)N[C@@H]12 ARSXTTJGWGCRRR-XXKOCQOQSA-N 0.000 description 1
- 241000283956 Manis Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 108010052164 Sodium Channels Proteins 0.000 description 1
- 102000018674 Sodium Channels Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- ARSXTTJGWGCRRR-LJRZAWCWSA-N [(3as,4r,9s,10as)-2,6-diamino-10,10-dihydroxy-9-sulfooxy-3a,4,8,9-tetrahydro-1h-pyrrolo[1,2-c]purin-4-yl]methyl carbamate Chemical compound [H+].NC(=O)OC[C@@H]1N=C(N)N2C[C@H](OS([O-])(=O)=O)C(O)(O)[C@@]22N=C(N)N[C@@H]12 ARSXTTJGWGCRRR-LJRZAWCWSA-N 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 229940035674 anesthetics Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003193 general anesthetic agent Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 231100000033 toxigenic Toxicity 0.000 description 1
- 230000001551 toxigenic effect Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- 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
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- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Botany (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Cultivation Of Seaweed (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to a culture medium for Alexandrium mimutum and a culture method thereof. The culture medium specifically comprises the following components in molar concentration: 1766 mu mol/L of sodium nitrate, 18.15 mu mol/L of sodium dihydrogen phosphate, 5 mu mol/L of ferric chloride, 5 mu mol/L of Na2EDTA, 0.02 mu mol/L of copper sulfate, 0.015 mu mol/L of sodium molybdate, 0.04 mu mol/L of zinc sulfate, 0.025 mu mol/L of cobalt chloride, 0.45 mu mol/L of manganese chloride and natural seawater. The Alexandrium mimutum cultured by the culture medium can increase the yield of the toxin to the ground, and the culture method is a continuous culture method, so that the time and the labor are saved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a culture medium for Alexandrium mimutum and a culture method thereof.
Background
Alexandrium mimutum belongs to Protactysta, Dinovellata and Alexandrium, is a marine planktonic dinoflagellate with squama Manis, has a cell size of 13-30 μm, is widely distributed, and is related to the production of Paralytic Shellfish Toxins (PSTs). Goodyearia toxins (gonyautoxin) GTX1, GTX2, GTX3 and GTX4 generated in the body are enriched by mollusks and finally transmitted to shellfish, fish and human through food chains, can cause severe paralytic shellfish poisoning (paralytic shellfish poisoning PSP) of human, and have many domestic accidents caused by death due to eating of the paralytic shellfish toxin-containing textured snails.
Paralytic shellfish toxins are alkaloids with neurotoxic action, wherein the poisoning mechanism is that the toxins are combined with a sodium ion channel structure to block the flow of sodium ions. No medicine is released after poisoning, and the only rescue methods are artificial respiration and transfusion. PSTs are strongly toxic, colorless, tasteless and paralytic, so that the PSTs have the possibility of being applied to biochemical weapons in military, are used as precursor substances for exploring novel anesthetics in medical treatment, have been sold in part of PSTs toxin standards abroad in the aspects of environment and food detection, but are expensive in price.
The preparation of gonyautoxins requires large amounts of raw materials, usually obtained by culturing toxigenic microalgae. The toxic yield of the Alexandrium mimutum is influenced by environmental factors such as light intensity, temperature and salinity, and is also influenced by nutritional formulas such as nitrogen and phosphorus and culture modes. It is known that Alexandrium mimutum is usually cultured in f/2 seawater culture medium and K seawater culture medium, but they are not the most toxic nutrient formulation, and if it is used for large-scale cultivation of algae, it will cause waste of resources and increase of cost.
Disclosure of Invention
The present invention has been made to solve the problems of Alexandrium mimutum, and an object of the present invention is to provide a culture medium for Alexandrium mimutum and a culture method thereof.
In addition, as the growth of the Alexandrium is sensitive to disturbance, the scheme adopts a semi-continuous culture mode, so that the culture period is short, and time and labor are saved. Is a culture medium which is very suitable for the virus production of the Alexandrium mimutum, and the method is an efficient culture method.
Herein, a medium and a semi-continuous culture method for culturing Alexandrium mimutum are disclosed, which can significantly increase the toxic content.
In order to achieve the purpose, the invention adopts the following technical scheme:
a culture medium for Alexandrium mimutum comprisesThe following molar concentrations of the components: 1766 mu mol/L of sodium nitrate, 18.15 mu mol/L of sodium dihydrogen phosphate, 5 mu mol/L of ferric chloride and Na2EDTA5 mu mol/L, copper sulfate 0.02 mu mol/L, sodium molybdate 0.015 mu mol/L, zinc sulfate 0.04 mu mol/L, cobalt chloride 0.025 mu mol/L, manganese chloride 0.45 mu mol/L, and natural seawater.
Further preferably, the culture medium further comprises samarium nitrate of 0.01. mu. mol/L.
More preferably, the method for preparing the culture medium is as follows:
(1) standing natural seawater, filtering supernatant seawater with 0.22 μm filter membrane, placing in transparent culture container, and sterilizing with high pressure steam at 121 deg.C for 20 min;
(2) preparing stock solutions from the components, wherein the concentration of the sodium nitrate stock solution is 1766mmol/L, the concentration of the sodium dihydrogen phosphate stock solution is 18.15mmol/L, preparing the stock solutions of trace metal elements into a combined stock solution containing 5mmol/L ferric chloride and Na2EDTA5mmol/L, copper sulfate 0.02mmol/L, sodium molybdate 0.015mmol/L, zinc sulfate 0.04mmol/L, cobalt chloride 0.025mmol/L, manganese chloride 0.45mmol/L, the stock solution is also autoclaved at 121 ℃ for 20 minutes;
(3) and (3) adding 1 ml of nitrogen stock solution, 1 ml of phosphorus stock solution and 1 ml of trace metal element stock solution into sterilized seawater in an ultra-clean workbench or a culture room after ultraviolet sterilization for 30 minutes, and fixing the volume to 1L to prepare the culture medium with the concentration specified in the formula, thereby completing the preparation of the culture medium.
As a further preference, the salinity of the natural seawater is adjusted to 24% by adding sodium chloride or pure water.
A culture method for culturing Alexandrium mimutum by adopting the culture medium specifically comprises the following steps:
(1) inoculating the algae in logarithmic growth phase into the culture medium, adjusting the initial density to be 5000 +/-200 cells/ml, the temperature to be 25 ℃, and the illumination to be 125 +/-12.5 mu mol/(m)2S) standing and culturing for 8-9 days under the condition that the illumination time ratio of day and night is 12 h: 12 h;
(2) adding the same volume of the new culture medium, performing amplification culture for 4-5 days;
(3) repeating the culture process in the step (2) to continuously increase the volume of the culture solution;
(4) after the repetition process of the step (2) is completed, taking out part of the algae liquid in the incubator, placing the part at the temperature of 20 ℃, and illuminating the part at 125 +/-12.5 mu mol/(m)2S), standing and culturing under the condition that the day and night illumination time ratio is 12 h: 12h, harvesting algae cells after 3-5 days, simultaneously supplementing an equal-volume fresh culture medium of the residual algae liquid into the culture device, mixing with the residual algae liquid in the culture device, and repeating the culture process in the step (2);
(5) and (5) continuously repeating the step (2) and the step (4) to obtain the Alexandrium mimutum with high toxin content.
More preferably, in the step (4), sodium nitrate is added to the taken-out algal solution in an amount of 1766umol per liter of algal solution.
Compared with the prior art, the invention has the beneficial effects that:
1. the toxin yield is improved by 48.6 percent compared with the prior toxin yield (5.31 mu mol/L), and can be as high as 7.89 mu mol/L of gonyautoxin;
2. the method adopts semi-continuous culture mode, can continuously obtain algae cells, shortens culture period, and improves production efficiency. The culture time is shortened from the prior 22-30 days to 7-10 days.
3. The nutrient variety is reduced, the operation is simplified, and the expenditure is saved, for example, the invention omits the addition of vitamins compared with the existing culture medium.
Detailed Description
The technical solution of the present invention is further described below by means of specific examples.
The raw materials used in the examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art, unless otherwise specified.
Example 1:
a culture medium for Alexandrium mimutum comprises the following components in molar concentration: the content of sodium nitrate is 1766 mu mol/L,sodium dihydrogen phosphate 18.15. mu. mol/L, ferric chloride 5. mu. mol/L, Na2EDTA5 mu mol/L, copper sulfate 0.02 mu mol/L, sodium molybdate 0.015 mu mol/L, zinc sulfate 0.04 mu mol/L, cobalt chloride 0.025 mu mol/L, manganese chloride 0.45 mu mol/L, and natural seawater.
The preparation method of the culture medium comprises the following specific steps:
(1) standing natural seawater, filtering supernatant seawater with 0.22 μm filter membrane, placing in transparent culture container, and sterilizing with high pressure steam at 121 deg.C for 20 min; the salinity of the natural seawater is adjusted to 24 per mill by adding sodium chloride or pure water;
(2) preparing stock solutions from the components, wherein the concentration of the sodium nitrate stock solution is 1766mmol/L, the concentration of the sodium dihydrogen phosphate stock solution is 18.15mmol/L, preparing the stock solutions of trace metal elements into a combined stock solution containing 5mmol/L ferric chloride and Na2EDTA5mmol/L, copper sulfate 0.02mmol/L, sodium molybdate 0.015mmol/L, zinc sulfate 0.04mmol/L, cobalt chloride 0.025mmol/L, manganese chloride 0.45mmol/L, the stock solution is also autoclaved at 121 ℃ for 20 minutes;
(3) and (3) adding 1 ml of nitrogen stock solution, 1 ml of phosphorus stock solution and 1 ml of trace metal element stock solution into sterilized seawater in an ultra-clean workbench or a culture room after ultraviolet sterilization for 30 minutes, and fixing the volume to 1L to prepare the culture medium with the concentration specified in the formula, thereby completing the preparation of the culture medium.
A culture method for culturing Alexandrium mimutum by adopting the culture medium specifically comprises the following steps:
(1) inoculating the algae in logarithmic growth phase into the culture medium, adjusting the initial density to be 5000 +/-200 cells/ml, the temperature to be 25 ℃, and the illumination to be 125 +/-12.5 mu mol/(m)2S) standing and culturing for 8-9 days under the condition that the illumination time ratio of day and night is 12 h: 12 h;
(2) adding the same volume of the new culture medium, performing amplification culture for 4-5 days;
(3) repeating the culture process in the step (2) to continuously increase the volume of the culture solution;
(4) after the repetition of one step (2) is completed, the culture vessel is placedTaking out part of the algae liquid, and adding sodium nitrate into the part, wherein the addition amount is 1766umol per liter of algae liquid; then the mixture is placed at the temperature of 20 ℃ and the illumination is 125 +/-12.5 mu mol/(m)2S), standing and culturing under the condition that the day and night illumination time ratio is 12 h: 12h, harvesting algae cells after 3-5 days, simultaneously supplementing an equal-volume fresh culture medium of the residual algae liquid into the culture device, mixing with the residual algae liquid in the culture device, and repeating the culture process in the step (2);
(5) and (5) continuously repeating the step (2) and the step (4) to obtain the Alexandrium mimutum with high toxin content.
Sodium nitrate was added to the taken-out algal solution in an amount of 1766umol per liter of algal solution.
Example 2:
a culture medium for Alexandrium mimutum comprises the following components in molar concentration: 1766 mu mol/L of sodium nitrate, 18.15 mu mol/L of sodium dihydrogen phosphate, 5 mu mol/L of ferric chloride and Na25 mu mol/L of EDTA, 0.02 mu mol/L of copper sulfate, 0.015 mu mol/L of sodium molybdate, 0.04 mu mol/L of zinc sulfate, 0.025 mu mol/L of cobalt chloride, 0.45 mu mol/L of manganese chloride, 0.01 mu mol/L of samarium nitrate and natural seawater.
The preparation method of the culture medium comprises the following specific steps:
(1) standing natural seawater, filtering supernatant seawater with 0.22 μm filter membrane, placing in transparent culture container, and sterilizing with high pressure steam at 121 deg.C for 20 min; the salinity of the natural seawater is adjusted to 24 per mill by adding sodium chloride or pure water;
(2) preparing stock solutions from the components, wherein the concentration of the sodium nitrate stock solution is 1766mmol/L, the concentration of the sodium dihydrogen phosphate stock solution is 18.15mmol/L, preparing the stock solutions of trace metal elements into a combined stock solution containing 5mmol/L ferric chloride and Na2EDTA5mmol/L, copper sulfate 0.02mmol/L, sodium molybdate 0.015mmol/L, zinc sulfate 0.04mmol/L, cobalt chloride 0.025mmol/L, manganese chloride 0.45mmol/L, samarium nitrate 0.01mmol/L, the stock solution is also autoclaved at 121 ℃ for 20 minutes;
(3) and (3) adding 1 ml of nitrogen stock solution, 1 ml of phosphorus stock solution and 1 ml of trace metal element stock solution into sterilized seawater in an ultra-clean workbench or a culture room after ultraviolet sterilization for 30 minutes, and fixing the volume to 1L to prepare the culture medium with the concentration specified in the formula, thereby completing the preparation of the culture medium.
A culture method for culturing Alexandrium mimutum by adopting the culture medium specifically comprises the following steps:
(1) inoculating the algae in logarithmic growth phase into the culture medium, adjusting the initial density to be 5000 +/-200 cells/ml, the temperature to be 25 ℃, and the illumination to be 125 +/-12.5 mu mol/(m)2S) standing and culturing for 8-9 days under the condition that the illumination time ratio of day and night is 12 h: 12 h;
(2) adding the same volume of the new culture medium, performing amplification culture for 4-5 days;
(3) repeating the culture process in the step (2) to continuously increase the volume of the culture solution;
(4) after the repetition process of the step (2) is completed, taking out a part of the algae liquid in the incubator, and adding sodium nitrate into the part, wherein the adding amount is 1766umol per liter of algae liquid; then the mixture is placed at the temperature of 20 ℃ and the illumination is 125 +/-12.5 mu mol/(m)2S), standing and culturing under the condition that the day and night illumination time ratio is 12 h: 12h, harvesting algae cells after 3-5 days, simultaneously supplementing an equal-volume fresh culture medium of the residual algae liquid into the culture device, mixing with the residual algae liquid in the culture device, and repeating the culture process in the step (2);
(5) and (5) continuously repeating the step (2) and the step (4) to obtain the Alexandrium mimutum with high toxin content.
The results of the measurements of the Alexandrium mimutum cultured in examples 1 and 2 were as follows:
the yield of toxin of the harvested algal cells in example 1 was 6.63umol gonyautoxin per liter of algal fluid. The yield of toxin of the harvested algal cells in example 2 was 7.89umol gonyautoxin per liter of algal fluid. The addition of samarium nitrate is positive to the subsequent influence, and is obviously improved compared with the addition of no samarium nitrate.
Claims (3)
1. A method for culturing Alexandrium mimutum with culture mediumAlexandrium minutum ) The method of (1) for culturing a microorganism,the culture medium is characterized by comprising the following components in molar concentration: 1766 mu mol/L of sodium nitrate, 18.15 mu mol/L of sodium dihydrogen phosphate, 5 mu mol/L of ferric chloride and Na2EDTA5 mu mol/L, copper sulfate 0.02 mu mol/L, sodium molybdate 0.015 mu mol/L, zinc sulfate 0.04 mu mol/L, cobalt chloride 0.025 mu mol/L, manganese chloride 0.45 mu mol/L, samarium nitrate 0.01 mu mol/L and natural seawater; the salinity of the natural seawater is adjusted to 24 per mill by adding sodium chloride or pure water;
the method specifically comprises the following steps:
(1) inoculating algae in logarithmic growth phase into the culture medium, adjusting initial density to 5000 + -200 cell/ml, temperature to 25 deg.C, and illumination to 125 + -12.5 μmol/(m)2S) standing and culturing for 8-9 days under the condition that the illumination time ratio of day and night is 12 h: 12 h;
(2) adding the same volume of the new culture medium, performing amplification culture for 4-5 days;
(3) repeating the culture process in the step (2) to continuously increase the volume of the culture solution;
(4) after the repetition of step (2), the algae liquid in the incubator is partially removed and placed at a temperature of 20 ℃ under light of 125 + -12.5 μmol/(m)2S), standing and culturing under the condition that the day and night illumination time ratio is 12 h: 12h, harvesting algae cells after 3-5 days, simultaneously supplementing an equal-volume fresh culture medium of the residual algae liquid into the culture device, mixing with the residual algae liquid in the culture device, and repeating the culture process in the step (2);
(5) and (5) continuously repeating the step (2) and the step (4) to obtain the Alexandrium mimutum with high toxin content.
2. The culture method according to claim 1, wherein the culture medium is prepared by the following method:
(1) standing natural seawater, filtering supernatant seawater with 0.22 μm filter membrane, placing in transparent culture container, sterilizing with 121 deg.C high pressure steam for 20 min, and adjusting salinity of the natural seawater to 24 ‰byadding sodium chloride or pure water;
(2) the components are firstly prepared intoThe concentration of the stock solution of sodium nitrate is 1766mmol/L, the concentration of the stock solution of sodium dihydrogen phosphate is 18.15mmol/L, and the stock solution of trace metal elements contains 5mmol/L of ferric chloride and Na2EDTA5mmol/L, copper sulfate 0.02mmol/L, sodium molybdate 0.015mmol/L, zinc sulfate 0.04mmol/L, cobalt chloride 0.025mmol/L, manganese chloride 0.45mmol/L, samarium nitrate 0.01. mu. mol/L, stock solution also at 121 deg.C steam sterilization for 20 minutes;
(3) and (3) adding 1 ml of sodium nitrate stock solution, 1 ml of sodium dihydrogen phosphate stock solution and 1 ml of trace metal element stock solution into sterilized seawater in an ultra-clean workbench or a culture room after ultraviolet sterilization for 30 minutes, and fixing the volume to 1L to prepare the medium with the concentration specified in the formula, thus completing the preparation of the culture medium.
3. The culture method according to claim 1, wherein in the step (4), sodium nitrate is added to the taken-out algal solution in an amount of 1766. mu. mol per liter of algal solution.
Priority Applications (1)
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| "Toxic Alexandrium minutum (Dinophyceae) from Vietnam with new gonyautoxin analogue";Po-Teen Lim等;《Harmful Algae》;20070430;第6卷(第3期);第321-331页 * |
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