CN1710062A - Scaphoid algae open culture method and its special culture medium - Google Patents
Scaphoid algae open culture method and its special culture medium Download PDFInfo
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- CN1710062A CN1710062A CN 200410047886 CN200410047886A CN1710062A CN 1710062 A CN1710062 A CN 1710062A CN 200410047886 CN200410047886 CN 200410047886 CN 200410047886 A CN200410047886 A CN 200410047886A CN 1710062 A CN1710062 A CN 1710062A
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Abstract
This invention discloses the Na-vicula Bory trained method and special-purpose Culture medium openly. This invention offers Culture medium is named ZWN, its prescription is: Sodium nitrate, 180-220mg; potassium dihydrogen phosphate 18-22mg; Sodium silicate, 270-300mg; Vitamin B1, 350-450microng; Ethylenediamine four Acetic acid sodium, 5-7mg; Urea, 7-9mg; FeCl2, 5-7mg;The sea water holds to 1000ml definitely. Including the following step: 1)Dispose the culture medium with the sea water; 2) Na-vicula Bory of density 3*l05 cell / milliliter, according to make trigonometry brown to mean for1:5-9's proportions algae inoculate reach the culture compared with volume of culture, in 19-25 DEG C, illumination intensity 3000-6000 Lux, illumination time is 8-12 h/day terms keeps in touch with the medium.
Description
Technical field
The present invention relates to cultural method and the special culture media thereof of algae, particularly relate to cultural method and the special culture media thereof of boat-shaped algae.
Background technology
Boat-shaped algae (Navicula sp) is the marine diatom of dwelling the end that a class is rich in polyunsaturated fatty acid, is the common primary productivity population of Marine ecosystems.Because the boat-shaped algae contains gamma-linolenic acid (GLA), timnodonic acid (EPA) and docosahexenoic acid (DHA), has become one of important open-mouthed bait of a lot of precious aquatic animals.Also to a large amount of polyose composition of exocytosis, preliminary pharmacological tests shows that the outer total polysaccharides of boat-shaped gonidium contains 21.24% SO to the boat-shaped algae in process of growth
4 2-, the inhibiting rate of S-180 sarcoma is reached more than 60%, have pharmacy value, make the boat-shaped algae become the important source material of marine organisms pharmacy.
Boat-shaped algae and other benthic algaes mix growth in the natural sea-water, and population density is very low, can't satisfy the needs of aquaculture.At present, the artificial culture of boat-shaped algae adopts the f/2 substratum more, and the boat-shaped algae speed of growth is slow in this substratum, especially is difficult to form the population advantage when open cultivation, is very easily polluted by unicell green algas such as chlorella, salt algaes.These unicell green alga fast growths are also secreted the growth that extracellular products suppresses the boat-shaped algae in the time of mass consumption substratum nutrient, cause cultivating failure easily.
Summary of the invention
The purpose of this invention is to provide a kind of substratum that the boat-shaped algae is grown fast.
Boat-shaped algae culture medium called after ZWN provided by the present invention, its prescription is: saltpetre, 180-220mg; SODIUM PHOSPHATE, MONOBASIC, 18-22mg; Water glass, 270-330mg; The urea element, 7-9mg; Vitamins B
1, 350-450 μ g; Disodium ethylene diamine tetraacetate, 5-7mg; Iron(ic) chloride, 5-7mg; Seawater is settled to 1L.
The screening formulation of substratum is: saltpetre, 200mg; SODIUM PHOSPHATE, MONOBASIC, 20mg; Water glass, 300mg; The urea element, 8mg; Vitamins B
1, 400 μ g; Disodium ethylene diamine tetraacetate, 6mg; Iron(ic) chloride, 6mg; Seawater is settled to 1L.
In order to prevent that in culturing process boat-shaped algae is subjected to the pollution of assorted algaes such as green alga, also is added with the 350-450mg/L potassiumiodide in substratum; Be preferably 400mg/L.
Seawater is handled before the preparation substratum as follows: 1) regulating natural sea-water pH with hydrochloric acid is 2-4, leaves standstill 12-24h; 2) regulate pH to 7.0-8.0 with NaOH.
Second purpose of the present invention provides a kind of method of using ZWN culture medium culturing boat-shaped algae provided by the present invention.
Use the method for ZWN culture medium culturing boat-shaped algae, comprise the steps: 1) dispose substratum with seawater; 2) be 1 in Nitzschia closterium minutissima with the culture volume ratio: the ratio of 5-9 is inoculated into Nitzschia closterium minutissima in the substratum, at 19-25 ℃, and intensity of illumination 3000-6000Lux, aerated culture under the light application time 8-12h/ days conditions.
The air flow of culturing process is 0.5m
3/ min; Cultivate and to reach the cultivation terminal point in 6-8 days.Preferred culture condition is: temperature is 25 ℃; Intensity of illumination is 5000Lux; Light application time is 10h/ days.
Artificial culture boat-shaped algae relates generally to the gordian technique of two aspects: i.e. the control of the composition of substratum and green alga pollution.ZWN substratum provided by the present invention can make the boat-shaped frustule utilize the nutritive ingredient of substratum fast, reaching cell divides fast, growth cycle shortens 3-4 days than traditional f/2 substratum, and can be beneficial to the accumulation of boat-shaped algae biomass, exceeds 0.4-1 doubly than traditional substratum; In the ZWN substratum, add potassiumiodide, can effectively reach the purpose of control green alga pollution, can adopt open training method to obtain boat-shaped algae and target product thereof on a large scale, culture fishery and marine organisms pharmacy are had crucial meaning.
Description of drawings
Fig. 1 is the growth curves of three kinds of boat-shaped algaes at ZWN substratum and f/2 substratum
Fig. 2 is containing potassiumiodide and is not containing the growth curve of open culture in the potassiumiodide ZWN substratum for the boat-shaped algae
Fig. 3 is containing potassiumiodide and is not containing the growth curve of sterile culture in the potassiumiodide ZWN substratum for the boat-shaped algae
Embodiment
1, ZWN substratum preparation
1) get the natural seawater that leaves standstill of 1L, adding 1N hydrochloric acid adjusting seawater pH is 3, leaves standstill 12h, disgorging; Adding 1N NaOH then, to be adjusted to pH be about 7.5, leaves standstill disgorging.
2) quantitatively take by weighing various nutritive salt in the ZWN prescription: saltpetre (KNO
3), 200mg; SODIUM PHOSPHATE, MONOBASIC (NaH
2PO4), 20mg; Water glass (Na
2SiO
3L0H
2O), 300mg; Urea element (NH
2)
2CO, 8mg; Vitamins B
1, 400 μ g; Disodium ethylene diamine tetraacetate (Na
2EDTA), 6mg; Iron(ic) chloride (FeCl
3), 6mg.
3) above-mentioned substance is dissolved in 1) in the gained seawater, and be settled to 1000 milliliters, add the 400mg potassiumiodide then.
2, the cultivation of boat-shaped algae
The used boat-shaped algae of present embodiment has three kinds, is respectively boat-shaped algae YT01, boat-shaped algae LY01, boat-shaped algae LY02.These three kinds of diatoms be the contriver at 2001-2002 respectively from Yantai, beach, Lianyun Harbour gathers, and separates the back in the laboratory and obtains, and preserves the algae kind as the contriver laboratory.
Boat-shaped algae algae kind density is 3 * 10
5Cells/ml is inoculated in the ZWN substratum by 1: 5 volume ratio, is that 25 ℃, light application time are under 10h/ days, light intensity 5000Lux condition, with 0.5m in temperature
3/ min air flow aerated culture.
With f/2 substratum substratum in contrast, training method is with last identical.
Three kinds of boat-shaped algaes in ZWN substratum and f/2 substratum growth curve as shown in Figure 1, A, B, C are respectively boat-shaped algae YT01, LY01, the LY02 that cultivates among the figure in the ZWN substratum; D, E, F are respectively boat-shaped algae YT01, LY01, the LY02 that cultivates in the f/2 substratum.
The result shows that these three kinds of boat-shaped algaes growth cycle in the ZWN substratum is 6-8 days, and is different and variant with ratio with the inoculum density of initial incubation.Generally working as algae kind density is 3 * 10
3Cells/ml, inoculative proportion are 1: 5 o'clock, cultivate promptly to enter exponential phase of growth on the 2nd day, and algae cell density reached maximum value in 6-7 days; Then be 3-5 days stationary phase, the frustule of this moment divides, growth is slack-off, growth ending.Compare with the f/2 substratum, the boat-shaped algae entered logarithmic phase in advance in 4 days, and biomass exceeds 0.4-1 respectively doubly.
The composition of embodiment 2, boat-shaped algae frond lipid acid in the ZWN substratum
According to following formulated ZWN substratum: saltpetre, 180mg; SODIUM PHOSPHATE, MONOBASIC, 22mg; Water glass, 270mg; The urea element, 9mg; Vitamins B
1, 350 μ g; Disodium ethylene diamine tetraacetate, 7mg; Iron(ic) chloride, 5mg; Seawater is settled to 1 liter, and it is 400mg/L that the adding potassiumiodide makes its concentration.
With ZWN is substratum, is that 25 ℃, light application time are that 8h, inoculum size are aerated culture boat-shaped algae 8 days under 1: 9, light intensity 5000Lux condition in temperature, and other operations are identical with embodiment 1.After cultivating end, collect the frond cell, press the composition that the GC-MS method is measured frond lipid acid, the result is as shown in table 1, and wherein i-14:0 represents ten tetra-carbonics in the table; 15:0 represents 15 carbonic acid; 16:2 (n-6) represents hexadecadienoic acid; Ai-17:0 represents 17 carbonic acid; All the other similarly.
The result shows, is main component with its frond polyunsaturated fatty acid of boat-shaped algae of ZWN culture medium culturing with EPA, GLA and DHA, is respectively 31.8-11.1%, 15-18% and the 1.84-7.82% of total fatty acid content.
Table 1. is formed (accounting for total fatty acids per-cent) with several boat-shaped frond lipid acid that ZWN cultivates
| Lipid acid is formed | Boat-shaped algae YT01 | Boat-shaped algae LY01 | Boat-shaped algae LY02 |
| ??i-14:0 ??15:0 ??i-17:0 ??16:2(n-6) ??ai-17:0 ??17:0 ??18:3(n-6) ??20:5(n-3) ??22:6(n-3) ??24:1(n-9) | ??5.1 ??1.4 ??20.68 ??2.18 ??0.23 ??3.86 ??11.1 ??18.32 ??7.82 ??1.27 | ??2.4 ??5.5 ??27.39 ??5.14 ??1.39 ??9.14 ??3.18 ??15.39 ??1.84 ??2.91 | ??2.19 ??6.4 ??29.84 ??1.97 ??5.98 ??8.89 ??6.79 ??17.94 ??2.39 ??10.14 |
| Other lipid components of 24:0 | ??3.85 ??10.1 | ??3.3 ??22.42 | ??3.66 ??13.95 |
According to following formulated ZWN substratum: saltpetre, 220mg; SODIUM PHOSPHATE, MONOBASIC, 18mg; Water glass, 330mg; The urea element, 7mg; Vitamins B
1, 450 μ g; Disodium ethylene diamine tetraacetate, 5mg; Iron(ic) chloride, 7mg; Seawater is settled to 1 liter.
Adding potassiumiodide in the part substratum, to make its concentration be 450mg/L, and another part does not add potassiumiodide, is that 19 ℃, light application time are that 12h/ days, inoculum size are that (algae kind density was 3 * 10 in 1: 9 in temperature
5Cells/ml), aerated culture boat-shaped algae 10 days under the light intensity 5000Lux condition, other operations are identical with embodiment 1.The growth curve of boat-shaped algae YT01 in two kinds of substratum as shown in Figure 2, A is ZWN substratum+400mg/L potassiumiodide among the figure; B is the ZWN substratum.
The result shows, in the ZWN substratum, and postvaccinal the 5th day, the quantity of unicell green alga begins rapid rising, these unicell green algas discharge boat-shaped algae amicine to substratum in the process of growth, thereby the growth of boat-shaped algae is suppressed, and cause cultivating failure; In nutrient solution, add the potassiumiodide of 400mg/L, can effectively prevent the pollution of green alga; Also can effectively contain unicell green alga in the boat-shaped algae culturing liquid that has polluted, be embodied in unicell green alga class population quantity and increase and to stop and reducing gradually, on the many walls of the unicell green alga of survival attached to the vessel level top.
The influence that embodiment 4, potassiumiodide are cultivated for the boat-shaped algae
According to following formulated ZWN substratum: saltpetre (KNO
3), 200mg; SODIUM PHOSPHATE, MONOBASIC (NaH
2PO4), 20mg; Water glass (Na
2SiO
310H
2O), 300mg; Urea element (NH
2)
2CO, 8mg; Vitamins B
1, 400 μ g; Disodium ethylene diamine tetraacetate (Na
2EDTA), 6mg; Iron(ic) chloride (FeCl
3), 6mg; Seawater is settled to 1 liter.
Adding potassiumiodide in the part substratum, to make its concentration be 400mg/L, another part does not add potassiumiodide, then with two kinds of substratum with 0.2 μ m filtering with microporous membrane degerming, under aseptic condition, inoculate, cultivate boat-shaped algae algae kind YT01 by 1: 9 inoculum size, culture condition is identical with embodiment 1.
Boat-shaped algae YT01 at the growth curve of two kinds of substratum as shown in Figure 3, A is the ZWN substratum among the figure; B is ZWN substratum+400mg/L potassiumiodide.The growth indexes such as latent period, growth cycle and biomass that the boat-shaped algae is cultivated are as shown in table 2.
The result shows that accumulation does not have restraining effect to the potassiumiodide of this concentration to the boat-shaped algae biomass.
Table 2. potassiumiodide is to the influence of boat-shaped algae growth indexes
| Evaluation index | ZWN+ potassiumiodide 400mg/L | ??ZWN |
| Latent |
1 |
1 |
| Biomass | ??1.5～1.7g/L | ??1.6～1.7g/L |
After cultivating end, collect frond, measure the unsaturated fatty acid content of frond cell according to a conventional method, the result is as shown in table 3, and method for expressing is identical with embodiment 2 in the table.The result shows that after potassiumiodide added the ZWN substratum, the content of polyunsaturated fatty acid of boat-shaped algae is not significant to be changed, and EPA content slightly increases.
Table 3. potassiumiodide is cultivated the influence of boat-shaped algae content of polyunsaturated fatty acid to ZWN
| Lipid acid is formed | The ZWN substratum | ZWN substratum+potassiumiodide 400mg/L |
| Other lipid components of i-14:0 15:0 i-17:0 16:2 (n-6) ai-17:0 17:0 GLA 18:3 (n-6) EPA 20:5 (n-3) DHA 22:6 (n-3) 24:1 (n-9) 24:0 | ??5.1 ??14 ??20.68 ??2.18 ??0.23 ??3.86 ??11.1 ??18.32 ??7.82 ??1.27 ??3.85 ??10.1 | ??4.9 ??1.6 ??21.68 ??1.17 ??0.24 ??4.1 ??10.5 ??18.1 ??8.42 ??1.48 ??2.85 ??12.1 |
Claims (10)
1, a kind of boat-shaped algae culture medium, its prescription is: saltpetre, 180-220mg; SODIUM PHOSPHATE, MONOBASIC, 18-22mg; Water glass, 270-330mg; The urea element, 7-9mg; Vitamins B
1, 350-450 μ g; Disodium ethylene diamine tetraacetate, 5-7mg; Iron(ic) chloride, 5-7mg; Seawater is settled to 11.
2, substratum according to claim 1 is characterized in that: described saltpetre is 200mg; Described SODIUM PHOSPHATE, MONOBASIC is 20mg; Described water glass is 300mg; Described urea element is 8mg; Described vitamins B
1Be 400 μ g; Described disodium ethylene diamine tetraacetate is 6mg; Described iron(ic) chloride is 6mg.
3, substratum according to claim 1 and 2 is characterized in that: described substratum also contains the 350-450mg/L potassiumiodide.
4, substratum according to claim 1 and 2 is characterized in that: described seawater is handled as follows:
1) regulating natural sea-water pH with hydrochloric acid is 2-4, leaves standstill 12-24h; 2) regulate pH to 7.0-8.0 with NaOH.
5, a kind of method of cultivating the boat-shaped algae comprises the steps: 1) dispose substratum with seawater, described culture medium prescription is: saltpetre, 180-220mg; SODIUM PHOSPHATE, MONOBASIC, 18-22mg; Water glass, 270-330mg; The urea element, 7-9mg; Vitamins B
1, 350-450 μ g; Disodium ethylene diamine tetraacetate, 5-7mg; Iron(ic) chloride, 5-7mg; Seawater is settled to 1 liter; 2) be 1 in Nitzschia closterium minutissima with the culture volume ratio: the ratio of 5-9 is inoculated into Nitzschia closterium minutissima in the substratum, at 19-25 ℃, and intensity of illumination 3000-6000Lux, aerated culture under the light application time 8-12h/ days conditions.
6, cultural method according to claim 5 is characterized in that: the described saltpetre of step 1) is 200mg; Described SODIUM PHOSPHATE, MONOBASIC is 20mg; Described water glass is 300mg; Described urea element is 8mg; Described vitamins B
1Be 400 μ g; Described disodium ethylene diamine tetraacetate is 6mg; Described iron(ic) chloride is 6mg.
7, according to claim 5 or 6 described cultural methods, it is characterized in that: also contain the 350-450mg/L potassiumiodide in the described substratum of step 1).
8, according to claim 5 or 6 described cultural methods, it is characterized in that: described seawater is handled before the configuration substratum as follows: 1) regulating natural sea-water pH with hydrochloric acid is 2-4, leaves standstill 12-24h; 2) regulate pH to 7.0-8.0 with NaOH.
9, according to claim 5 or 6 described cultural methods, it is characterized in that: step 2) air flow of described aerated culture is 0.5m
3/ min.
10, according to claim 5 or 6 described cultural methods, it is characterized in that: step 2) described culture temperature is 25 ℃; Intensity of illumination is 5000Lux; Light application time is 10h/ days.
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| WO2012028040A1 (en) * | 2010-11-03 | 2012-03-08 | 深圳市兆凯生物工程研发中心有限公司 | Method for open diatom cultivation |
| CN102388126A (en) * | 2009-04-10 | 2012-03-21 | 电源开发株式会社 | Micro-alga belonging to genus navicula, process for production of oil by culture of the micro-alga, and oil collected from the micro-alga |
| CN103408338A (en) * | 2013-07-23 | 2013-11-27 | 大连海洋大学 | Nutrition formula used for promoting rapid growth of benthic naviculaceae |
| CN105039166A (en) * | 2015-08-28 | 2015-11-11 | 天津海友佳音生物科技股份有限公司 | Culture solution for promoting growth of navicula tenera in open pond and culture method |
| CN105130119A (en) * | 2015-08-28 | 2015-12-09 | 天津海友佳音生物科技股份有限公司 | Method for cultivating navicula tenera to control gymnodinium catenatum explosion in grouper fry growing pond |
| CN105349428A (en) * | 2015-12-22 | 2016-02-24 | 中国水产科学研究院长江水产研究所 | Culture solution for fresh water navicula and culture method |
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| CN105368714A (en) * | 2015-12-22 | 2016-03-02 | 中国水产科学研究院长江水产研究所 | Culture solution and culture method for fresh water Synedra |
| CN105543096A (en) * | 2015-12-30 | 2016-05-04 | 中国水产科学研究院长江水产研究所 | Directive breeding method for culturing pond diatom in fresh water |
| CN109971648A (en) * | 2019-04-16 | 2019-07-05 | 梁严 | It is a kind of to improve the active method of tropical boat-shaped algae with hydroxyl surface material |
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| JP2001151788A (en) * | 1999-09-13 | 2001-06-05 | Toyama Prefecture | Method for culturing marine microalga, hyaluronidase inhibitor, medicinal agent, cosmetic and food comprising its extract as active ingredient |
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| CN102388126A (en) * | 2009-04-10 | 2012-03-21 | 电源开发株式会社 | Micro-alga belonging to genus navicula, process for production of oil by culture of the micro-alga, and oil collected from the micro-alga |
| CN102388126B (en) * | 2009-04-10 | 2013-10-23 | 电源开发株式会社 | Micro-alga belonging to genus navicula, process for production of oil by culture of micro-alga, and oil collected from micro-alga |
| WO2012028040A1 (en) * | 2010-11-03 | 2012-03-08 | 深圳市兆凯生物工程研发中心有限公司 | Method for open diatom cultivation |
| CN103408338A (en) * | 2013-07-23 | 2013-11-27 | 大连海洋大学 | Nutrition formula used for promoting rapid growth of benthic naviculaceae |
| CN105039166A (en) * | 2015-08-28 | 2015-11-11 | 天津海友佳音生物科技股份有限公司 | Culture solution for promoting growth of navicula tenera in open pond and culture method |
| CN105130119A (en) * | 2015-08-28 | 2015-12-09 | 天津海友佳音生物科技股份有限公司 | Method for cultivating navicula tenera to control gymnodinium catenatum explosion in grouper fry growing pond |
| CN105349428A (en) * | 2015-12-22 | 2016-02-24 | 中国水产科学研究院长江水产研究所 | Culture solution for fresh water navicula and culture method |
| CN105368713A (en) * | 2015-12-22 | 2016-03-02 | 中国水产科学研究院长江水产研究所 | Culture solution and culture method for fresh water Cyclotella |
| CN105368714A (en) * | 2015-12-22 | 2016-03-02 | 中国水产科学研究院长江水产研究所 | Culture solution and culture method for fresh water Synedra |
| CN105543096A (en) * | 2015-12-30 | 2016-05-04 | 中国水产科学研究院长江水产研究所 | Directive breeding method for culturing pond diatom in fresh water |
| CN105543096B (en) * | 2015-12-30 | 2020-02-18 | 中国水产科学研究院长江水产研究所 | Directional cultivation method of diatom in freshwater aquaculture pond |
| CN109971648A (en) * | 2019-04-16 | 2019-07-05 | 梁严 | It is a kind of to improve the active method of tropical boat-shaped algae with hydroxyl surface material |
| CN114621874A (en) * | 2021-12-28 | 2022-06-14 | 宁波浮田生物技术有限公司 | Microalgae culture medium and application |
| CN114621874B (en) * | 2021-12-28 | 2023-09-29 | 宁波浮田生物技术有限公司 | Microalgae culture medium and application thereof |
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|---|---|
| CN100392061C (en) | 2008-06-04 |
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