CN1295323C - High density culturing method of alexandrium tamarense - Google Patents

High density culturing method of alexandrium tamarense Download PDF

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CN1295323C
CN1295323C CNB2004100093591A CN200410009359A CN1295323C CN 1295323 C CN1295323 C CN 1295323C CN B2004100093591 A CNB2004100093591 A CN B2004100093591A CN 200410009359 A CN200410009359 A CN 200410009359A CN 1295323 C CN1295323 C CN 1295323C
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alexandrium tamarense
nano
cultivation
nutrient solution
nitrogen
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CN1724636A (en
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胡晗华
石岩峻
丛威
康瑞娟
蔡昭铃
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Institute of Process Engineering of CAS
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Abstract

The present invention belongs to the technical field of cultivation of microalgae, particularly to a method for high-density cultivation of alexandrium tamarense. In the initial stage of cultivation, firstly, a static or moderate stirring mode is adopted to cause the alexandrium tamarense and other nutrient components to be proliferated for a certain time by using carbon sources in a culture medium; after the cell density reaches certain degree, air is led into the culture medium; thus, the damage of the cells of the alexandrium tamarense caused by cutting caused by agitation or ventilation in the initial stage of cultivation can be avoided. Certain proportion of carbon dioxide can be mixed in the air which is led into the culture medium to provide carbon sources needed by the growth of the cells of the alexandrium tamarense; meanwhile, a certain pH value is maintained. In the initial stage of cultivation, only nitrogen and phosphorus of proper concentration are supplied to cause the alexandrium tamarense to be quickly proliferated in the initial stage of cultivation; in the later stage of cultivation, when nitrogen and phosphorus become growth limiting factors, nitrogen and phosphorus are replenished to relieve the condition of lack of nitrogen and phosphorus in the later stage. Thus, the alexandrium tamarense can be continuously proliferated to reach high cell density.

Description

The high-density cultivation method of Alexandrium tamarense
Technical field
The invention belongs to the cultivation field of little algae, particularly relate to the high-density cultivation method of Alexandrium tamarense.
Background technology
Alexandrium tamarense (Alexandrium tamarense) is a kind of ocean dinoflagellate that produces neural paralysis toxin (PSP), is common red tide algae.It is coastal that the poisonous red tide that is caused by its often betides Japan, Europe and northeast, North America.In China Jiaozhou Bay and marine site, the South Sea once found Alexandrium tamarense kind or its sporangiocyst, once reached red tide density on the Xiamen area shrimp pool.Both at home and abroad investigators are to the environment and the nutritional factor that cause this algae red tide and produce malicious rule and carried out big quantity research, as document:
1.Parker?NS，Negri?AP，Frampton?DMF，Rodolfi?L，Tredici?MR，Blackburn?SI.Growth?of?thetoxic?dinoflagellate?Alexandrium?minutum(Dinophyceae)using?high?biomass?culturessystems.J.Appl.Phycol.，2002，14：313-324。
2.Wang?D，Ho?AYT，Hsieh?DPH.Production?of?C2?toxin?by?Alexandrium?tamarense?CI01using?different?culture?methods.J.Appl.Phycol.，2002，14：461-468。
3.Kevin?JF.Toxin?production?in?migrating?dinoflagellates：a?modeling?study?of?PSP?producingAlexandrium.Harmful?Algae，2002，1：147-155。
4.Hamasaki?K，Horie?M，Tokimitsu?S，Toda?T，Taguchi?S.Variability?in?toxicity?of?thedinoflagellate?Alexandrium?tamanrense?isolated?from?Hiroshima?Bay，western?Japan，as?areflection?of?changing?environmental?conditions.J.Plank.Res.，2001，23(3)：271-278。
Alexandrium tamarense institute toxin producing shows the intensive biological activity.Its toxin composition is wider, is mainly N-sulfo group formyl toxoid (C1, C2), Gonyaulax Diesing toxoid (GTX) and carbamate toxoid (STX), and very high pharmaceutical use is arranged.But because Alexandrium tamarense has flagellum, to shear-sensitive, growth is subjected to the inhibition of water movement easily, and the indoor cultivation difficulty is big, and culture density is low, as document:
1.Estrada?M，Berdalet?E.Effects?of?turbulence?on?phytoplankton.In：Anderson?DM，CermbellaAD，Hallegraeff?GM?eds.Physiological?Ecology?of?Harmful?Algal?Blooms.Berlin：SpringerVerlag，1998，601-618。
2.Andrew?RI，Michael?IL.Mechanisms?of?fluid?shear-induced?inhibition?of?population?growthin?a?red-tide?dinoflagellate.J.Phycol.，2002，38：683-694。
3.Juhl?AR，Latz?MI.Mechanisms?of?fuid?shear-induced?inhibition?of?population?growth?in?ared-tide?dinoflagellate.J.Phycol.，2002，38：689-694。
4.Sullivan?JM，Swift?E.Effects?of?small-scale?turbulence?on?net?growth?rate?and?size?of?tenspecies?of?marine?dinoflagellates.J.Phycol.，2003，39：83-94。
5.Sullivan?JM，Swift?E，Donaghay?PL，Rines?JEB.Small-scale?turbulence?affects?the?divisionrate?and?morphology?of?two?red-tide?dinoflagellates.Harmful?Algae，2003，2：183-199。
On the other hand, the nitrogen of culture system (preferred NaNO 3), phosphorus (preferred NaH 2PO 4) growth of frustule is also had a significant impact.Cross the propagation that low nitrogen, phosphorus concentration are unfavorable for frustule, too high nitrogen, phosphorus concentration pair cell again constitute and suppress.Present cultural method is got moderate nitrogen, phosphorus concentration, makes late stage of culture because the consumption of nutrition such as nitrogen, phosphorus makes that cell is difficult to continue to breed, thereby influences the final densities of cell cultures.The culture density of the Alexandrium tamarense of report is generally 10 at present 3The order of magnitude of individual cell/ml is no more than 10 4Individual cell/ml.
Summary of the invention
The method that the purpose of this invention is to provide a kind of high-density culture Alexandrium tamarense is to solve a large amount of needs of people to Alexandrium tamarense.
Design of the present invention is such:
At Alexandrium tamarense have flagellum, to the characteristics of shearing force sensitivity, take to leave standstill or the mode of mild stirring at the cultivation initial stage, make it to utilize carbon source and other nutrition in the substratum to be bred for some time earlier, treat to implement ventilation again after cell density acquires a certain degree, thereby make frustule avoid the cultivation initial stage by the cell injury that shearing caused that stirs or ventilation causes.Be furnished with a certain proportion of carbonic acid gas in the gas that feeds to provide the frustule growth required carbon source, keep certain pH value simultaneously.After cell reaches certain density, consumption along with the carbon source in the substratum (preferred sodium bicarbonate), the pH value of system raises, carbon source becomes limiting factor, feed carbonated air this moment, not only replenished the required carbon source of frustule growth, and made pH no longer raise, guarantee the stable of culture environment, help the propagation of frustule.
The present invention is at nitrogen, the phosphorus concentration of a cultivation initial stage moderate supply, making it can fast breeding at the cultivation initial stage, reach higher at the late stage of culture cell density, consumption aggravation to nitrogen, phosphorus, add nitrogen, phosphorus when making nitrogen, phosphorus become the limiting factor of growth, thereby alleviated the shortage of later stage nitrogen, phosphorus, give the suitable nutrient environment of cell, enable to continue propagation, reach high cell density.
The high-density cultivation method of Alexandrium tamarense of the present invention may further comprise the steps:
(1) the Alexandrium tamarense cell with logarithmic phase inserts in the culture apparatus of printing opacity, and inoculum density is 10～500cells ml -1, culture temperature is that 14～30 ℃, light intensity are 30～300 μ mol photonm -2S -1Substratum in the device is that f/2 adds rich artificial seawater (Harrison PJ, Waters RE, TaylorFJR, A broad spectrum artificial seawater medium for coastal and open oceanphytoplankton.J.Phycol., 1980,16:28～35), change nitrogen (NaNO in the substratum 3), phosphorus (NaH 2PO 4) concentration, initial nitrogen (NaNO 3) concentration is 0.05～1.0mmol/L, phosphorus (NaH 2PO 4) concentration is 0.002～0.05mmol/L;
(2) leave standstill cultivation in the past at 4～10 days that cultivate beginning, and perhaps adopted rotating speed to be no more than 60 rev/mins mild stirring, and perhaps adopted 0.5～2 hour timing at interval to stir; After cultivating 4～10 days that begin, be mixed with 0%～5%CO with the flow feeding that per minute feeds 20～200ml according to every liter of nutrient solution volume 2Air, as logical CO 2The time keep the pH value of nutrient solution in 7.6～8.8 scopes;
(3) in 4～12 days after cultivating beginning, NaNO in nutrient solution 3Add NaNO to culture system when being reduced to 0.02～0.04mmol/L 3, NaNO 3Additional amount be 0.2～2.0mmol/L nutrient solution volume;
Perhaps in 8～18 days after cultivating beginning, NaNO in nutrient solution 3Be reduced to 0.002～0.004mmol/L, or NaH in the nutrient solution 2PO 4Add NaNO to culture system when being reduced to 0.005～0.02mmol/L 3And NaH 2PO 4, NaNO 3Additional amount be 0.2～2.0mmol/L nutrient solution volume, NaH 2PO 4Additional amount be 0.02～0.2mmol/L nutrient solution volume;
(4) be cultured to algae cell density or concentration reaches preset value, perhaps regular hour, perhaps other can stop culture condition and occurs.
The present invention is owing to make the Alexandrium tamarense cell avoid the cultivation initial stage by the cell injury that shearing caused that stirs or ventilation causes in culturing process; Owing to be furnished with a certain proportion of carbonic acid gas in the gas that feeds the required carbon source of frustule growth to be provided and to keep certain pH value; Because only provide an amount of nitrogen, phosphorus concentration at the cultivation initial stage, having avoided concentration to suppress to make it can fast breeding at the cultivation initial stage, and add nitrogen, phosphorus in late stage of culture, thereby alleviated the shortage of later stage nitrogen, phosphorus, make frustule can continue propagation.Make the algae cell density that finally obtains reach 10 4Individual cell/ml～10 5Individual cell/ml is far above the general culture density of report.
Embodiment
Comparative example 1
Add 1.5 liters of substratum in 2 liters of Erlenmeyer flasks, substratum is that f/2 adds rich artificial seawater.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid, inoculum density is 500cells ml -1, culture temperature is 22 ℃, provides light source with white fluorescent lamp, intensity of illumination is 60 μ mol photonm -2S -1, light dark period is 12 hours: 12 hours.Left standstill cultivation in 0～8 day, begin to feed sterile air in the later stage (the 8th day) of logarithmic growth, air flow is 30ml/ minute.Reach 10200cells ml to the 12nd day cell density -1Reach 11200cells ml to the 18th day cell density -1, toxin output is 4.5 μ g L -1
Comparative example 2
Culture apparatus, substratum, culture temperature, intensity of illumination, periodicity of illumination, inoculum density are with comparative example 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.All the time leave standstill cultivation, stuffiness.Maximum cell density is 6600cells ml during to the 22nd day -1, toxin output is 1.8 μ g L -1
Comparative example 3
Culture apparatus, substratum, culture temperature, intensity of illumination, periodicity of illumination, inoculum density are with comparative example 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.All the time feed sterile air continuously and cultivate, air flow quantity is 80ml minute -1Maximum cell density is 5500cellsml during to the 22nd day -1, toxin output is 1.2 μ g L -1
Comparative example 4
Culture apparatus, substratum, culture temperature, intensity of illumination, periodicity of illumination, inoculum density are with comparative example 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.Remain 1 hour logical sterile air cultivation at interval, air flow is 30ml/ minute.Maximum cell density is 3500cells ml during to the 22nd day -1, toxin output is 1.9 μ g L -1
Comparative example 5
Culture apparatus, substratum, culture temperature, intensity of illumination, periodicity of illumination, inoculum density are with comparative example 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.Continuous magnetic agitation was cultivated in 0～8 day, 30 rev/mins of stirring velocitys.Later stage (the 8th day) at logarithmic growth begins to feed sterile air, and air flow is 30ml/ minute, and stirring remains unchanged.To the 14th day, cell density reached 7080cellsml -1To the 18th day, cell density reached 9310cells ml -1, toxin output is 4.6 μ g L -1
Comparative example 6
Culture apparatus, substratum, culture temperature, intensity of illumination, periodicity of illumination, inoculum density are with comparative example 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.Remain 1 hour timing at interval and stir, stirring velocity is 30 rev/mins, stuffiness.Maximum cell density is 5300cells ml during to the 22nd day -1, toxin output is 2.1 μ g L -1
Comparative example 7
Culture apparatus, substratum, culture temperature, intensity of illumination, periodicity of illumination, inoculum density are with comparative example 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.Remain continuous magnetic agitation, stirring velocity is 30 rev/mins, stuffiness.Maximum cell density is 5200cellsml during to the 22nd day -1, toxin output is 2.0 μ g L -1
Comparative example 8
Add 2.2 liters of substratum in 3 liters of airlift photobioreactors, substratum, culture temperature, intensity of illumination, inoculum density are with comparative example 1, and illumination is continuous illumination.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.Leave standstill after the inoculation and cultivated 4 days, feed sterile air then.Air flow quantity is 100ml minute -1To the 12nd day, high-cell density reached 17190cell ml -1, toxin output is 21.7 μ g L -1
Comparative example 9
Add the 100mL substratum in the triangular flask of 250mL, culture temperature, intensity of illumination are with comparative example 1, and illumination is continuous illumination.In the substratum except nitrogen (NaNO 3) concentration and phosphorus (NaH 2PO 4) concentration is outward all with comparative example 1.Initial nitrogen (NaNO 3) concentration is 0.0882mmol/L, phosphorus (NaH 2PO 4) concentration is 0.036mmol/L.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid, inoculum density is 60cells.ml -1Culturing process leaves standstill all the time.The 10th day (nitrogen (NaNO of this moment in inoculation back 3) concentration is 0.03mmol/L, phosphorus (NaH 2PO 4) concentration is 0.02mmol/L) add the NaNO of 0.80mmol/L 3, can make cell density reach 19950cells ml at the 20th day -1Reached high-density 43540cellsml on the 24th day -1
Comparative example 10
Culture apparatus, initial substratum, culture temperature, intensity of illumination, light dark period, inoculum density are with comparative example 9.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.Culturing process leaves standstill all the time.The 14th day (nitrogen concentration of this moment is that 0.003mmol/L, phosphorus concentration are 0.01mmol/L) adds the NaNO of 0.80mmol/L simultaneously after inoculation 3NaH with 0.072mmol/L 2PO 4, can make cell density reach 17780cells ml at the 20th day -1Reached high-density 52300cells ml on the 24th day -1
Embodiment 1
In 3 liters of airlift photobioreactors, add 2.2 liters of substratum.In the substratum except nitrogen (NaNO 3) concentration and phosphorus (NaH 2PO 4) concentration all adds rich artificial seawater, initial nitrogen (NaNO with f/2 outward 3) concentration is 0.0882mmol/L, phosphorus (NaH 2PO 4) concentration is 0.036mmol/L.Culture temperature is 22 ℃, provides light source with white fluorescent lamp, and intensity of illumination is 60 μ molphotonm -2S -1, illumination is continuous illumination.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid, inoculum density is 500cells ml -1Leave standstill after the inoculation and cultivated 4 days, feed sterile air then, air flow quantity is 150ml minute -1The 6th day (nitrogen concentration of this moment is that 0.028mmol/L, phosphorus concentration are 0.021mmol/L) adds the NaNO of 0.80mmol/L after the inoculation 3, can make cell density reach 20850cells ml at the 14th day -1Reached high-density 46630cellsml on the 18th day -1
Embodiment 2
Add 2.2 liters of substratum in 3 liters of airlift photobioreactors, culture temperature, periodicity of illumination, inoculum density are with embodiment 1.Intensity of illumination is 100 μ molphotonm -2S -1, initial substratum is with embodiment 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.The inoculation back keeps 1 hour logical sterile air cultivation at interval, and air flow is 30ml/ minute.Cultivate and change the sterile air flow into 150ml minute after 4 days -1The 6th day (nitrogen concentration of this moment is that 0.031mmol/L, phosphorus concentration are 0.023mmol/L) adds the NaNO of 0.80mmol/L after the inoculation 3, can make cell density reach 22850cells ml at the 14th day -1Reached high-density 44530cellsml on the 18th day -1
Embodiment 3
Add 2.2 liters of substratum in 3 liters of airlift photobioreactors, culture temperature, intensity of illumination, periodicity of illumination, inoculum density are with embodiment 1.Initial substratum is with embodiment 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.Leave standstill after the inoculation and cultivated 5 days, feed sterile air then, air flow quantity is 100ml minute -1The 10th day (nitrogen concentration of this moment is that 0.0026mmol/L, phosphorus concentration are 0.009mmol/L) adds the NaNO of 0.80mmol/L simultaneously after inoculation 3NaH with 0.072mmol/L 2PO 4, can make cell density reach 18860cells ml at the 15th day -1Reached high-density 55600cells ml on the 17th day -1
Embodiment 4
Add 1.5 liters of substratum in 2 liters of Erlenmeyer flasks, culture temperature, periodicity of illumination, inoculum density are with embodiment 1.Intensity of illumination is 100 μ molphotonm -2S -1, initial substratum is with embodiment 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.Remain continuous magnetic agitation after the inoculation and cultivate 30 rev/mins of stirring velocitys.Cultivate and feed sterile air after 4 days, the sterile air flow is 80ml minute -1The 10th day (nitrogen concentration of this moment is that 0.0023mmol/L, phosphorus concentration are 0.0075mmol/L) adds the NaNO of 1.0mmol/L simultaneously after inoculation 3NaH with 0.08mmol/L 2PO 4, can make cell density reach 19760cells ml at the 14th day -1Reached high-density 58500cells ml on the 17th day -1
Embodiment 5
Add 2.2 liters of substratum in 3 liters of airlift photobioreactors, culture temperature, intensity of illumination, periodicity of illumination are with embodiment 1.Initial substratum is with embodiment 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid, inoculum density is 300cells ml -1Leave standstill after the inoculation and cultivated 5 days, feed then and be mixed with 1%CO 2Sterile air, and the pH value of keeping nutrient solution is in 8.2～8.6 scopes, air flow quantity is 100ml minute -1The 7th day (nitrogen concentration of this moment is that 0.03mmol/L, phosphorus concentration are 0.02mmol/L) adds the NaNO of 0.80mmol/L after the inoculation 3, can make cell density reach 24650cells ml at the 14th day -1Reached density 48730cellsml on the 18th day -1Reached high-density 76500cells ml on the 22nd day -1
Embodiment 6
Add 2.2 liters of substratum in 3 liters of airlift photobioreactors, culture temperature, intensity of illumination, periodicity of illumination, inoculum density are with embodiment 1.Initial substratum is with embodiment 1.Insert Alexandrium tamarense (Alexandrium tamarense) and plant liquid.The inoculation back keeps 1 hour logical sterile air cultivation at interval, and air flow is 30ml/ minute.Cultivate to change into after 6 days feeding and be mixed with 5%CO 2Sterile air, and the pH value of keeping nutrient solution is in 7.8～8.2 scopes, air flow quantity is 50ml minute -1The 12nd day (nitrogen concentration of this moment is that 0.0022mmol/L, phosphorus concentration are 0.008mmol/L) adds the NaNO of 0.80mmol/L simultaneously after inoculation 3NaH with 0.070mmol/L 2PO 4, can make cell density reach 24860cells ml at the 16th day -1Reached density 61600cells ml on the 19th day -1Reached high-density 77800cells ml on the 23rd day -1

Claims (2)

1. the cultural method of an Alexandrium tamarense, it is characterized in that: this method may further comprise the steps:
(1) the Alexandrium tamarense cell with logarithmic phase inserts in the culture apparatus of printing opacity, and the substratum in the device is that f/2 adds rich artificial seawater, changes NaNO in the substratum 3, NaH 2PO 4Concentration, initial NaNO 3Concentration is 0.05～1.0mmol/L, NaH 2PO 4Concentration is 0.002～0.05mmol/L;
(2) left standstill cultivation in the past at 4～10 days that cultivate beginning, perhaps adopt rotating speed to be no more than 60 rev/mins mild stirring, perhaps adopt 0.5～2 hour the timing in interval to stir, after cultivating 4～10 days that begin, be mixed with 0%～5%CO with the flow feeding that per minute feeds 20～200ml according to every liter of nutrient solution volume 2Air, as logical CO 2The time keep the pH value of nutrient solution in 7.6～8.8 scopes;
(3) in 4～12 days after cultivating beginning, NaNO in nutrient solution 3Add NaNO to culture system when being reduced to 0.02～0.04mmol/L 3, NaNO 3Additional amount be 0.2～2.0mmol/L nutrient solution volume;
Perhaps in 8～18 days after cultivating beginning, NaNO in nutrient solution 3Be reduced to 0.002～0.004mmol/L, or NaH in the nutrient solution 2PO 4Add NaNO to culture system when being reduced to 0.005～0.02mmol/L 3And NaH 2PO 4, NaNO 3Additional amount be 0.2～2.0mmol/L nutrient solution volume, NaH 2PO 4Additional amount be 0.02～0.2mmol/L nutrient solution volume;
(4) be cultured to algae cell density or concentration reaches preset value, perhaps can stop culture condition and occur.
2. method according to claim 1 is characterized in that: the inoculum density of described step (1) is 10～500cells ml -1, culture temperature is that 14～30 ℃, light intensity are 30～300 μ mol photonm -2S -1
CNB2004100093591A 2004-07-21 2004-07-21 High density culturing method of alexandrium tamarense Expired - Fee Related CN1295323C (en)

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CN100375783C (en) * 2006-03-21 2008-03-19 中盐制盐工程技术研究院 Method for culturing 'Yuanshizao' alga in large scale outdoor, and its processing method
CN100441227C (en) * 2006-07-24 2008-12-10 厦门大学 Atexandrium tamarense culture liquid sterilizing process
CN102134553B (en) * 2010-01-27 2013-11-06 中国科学院过程工程研究所 Tubular photobioreactor and system and method for culturing microalgae cells
CN102758027B (en) * 2011-04-28 2013-12-25 杭州汉徽光电科技有限公司 Generation environment control method and system for microalgae cultivation
CN105237543A (en) * 2015-11-19 2016-01-13 上海市农业科学院 Method for purification preparation of N-sulfocarbamoyl toxins

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CN1218831A (en) * 1998-07-02 1999-06-09 中国科学院武汉植物研究所 Regulating and controlling method for carbon source and PH value in cultivating spirulina
CN1376777A (en) * 2001-03-26 2002-10-30 中国科学院化工冶金研究所 Process for culturing microalga cells by combination of supplementing carbon by solvent with collecting them by air floating

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Publication number Priority date Publication date Assignee Title
CN85103319A (en) * 1985-05-03 1986-11-05 日清制油株式会社 Method for culturing marine chlorella
CN1218831A (en) * 1998-07-02 1999-06-09 中国科学院武汉植物研究所 Regulating and controlling method for carbon source and PH value in cultivating spirulina
CN1376777A (en) * 2001-03-26 2002-10-30 中国科学院化工冶金研究所 Process for culturing microalga cells by combination of supplementing carbon by solvent with collecting them by air floating

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