CN1446904A - Method for cultivating green Dunaliella salina of liking for salt extremely - Google Patents

Method for cultivating green Dunaliella salina of liking for salt extremely Download PDF

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Publication number
CN1446904A
CN1446904A CN 03109642 CN03109642A CN1446904A CN 1446904 A CN1446904 A CN 1446904A CN 03109642 CN03109642 CN 03109642 CN 03109642 A CN03109642 A CN 03109642A CN 1446904 A CN1446904 A CN 1446904A
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Prior art keywords
algae kind
bittern
nutrient solution
mentioned
algae
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张福
马若欣
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Tianjin University of Science and Technology
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Tianjin University of Science and Technology
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Abstract

A process for culturing the extremely halophilous green Du's algae used for health-care, medicinal and gene engineering purposes includes preparing culture liquid, regulating sanility and pH value, disinfecting, removing unuse algae, preparing alga seeds, inoculating, and controlling the environment of large-area culturing.

Description

The green Dunaliella salina cultural method of extreme halotolerant
Technical field
The present invention relates to the cultural method of a kind of algae, more particularly, relate to the cultural method of a kind of Dunaliella salina (Dunaliella).
Background technology
The green Dunaliella salina of (salinity is greater than 15 ° of Be ') growth has the unique biological characteristic under the extreme halotolerant bittern environment, and frond contains multiple biologically active substance.Can be applicable to fields such as body-care, bio-pharmaceuticals, genetically engineered, exploitation natural health-care products or biologics and resistant gene of salt applied research have certain application value, have a extensive future.Relate generally to for the research of Dunaliella salina (Dunaliella.salina) at present by retrieval: analyze Dunaliella salina and belong to the culture technique of having a liking for genetic diversity, Dunaliella salina between the salt kind and application, Dunaliella salina aspects such as physiological response, all do not relate to the research of extreme halotolerant green Dunaliella salina (Dunaliella.viridis) culture technique salinity.
Summary of the invention
The objective of the invention is to fill up the blank that prior art exists, provide a kind of extreme halotolerant green Dunaliella salina cultural method.
The green Dunaliella salina cultural method of extreme halotolerant of the present invention may further comprise the steps:
(1) preparation of nutrient solution, the preparation of described nutrient solution comprises the steps:
1. get the bittern that saltiness is 15～25 ° of Be ';
2. in above-mentioned bittern, add nutritive salt NaH 2PO 410g/m 3And CH 4N 2O is urea 110g/m 3
3. regulating above-mentioned solution, to make its pH value be 6～8;
4. working concentration is that the 1ppm disinfection agent of chlorine dioxide carries out the sterilizing and algae-removing processing to above-mentioned solution;
(2) preparation of algae kind, the preparation of described algae kind comprises the steps:
1. getting saltiness is 25～27 ° of Be ' bittern;
2. in above-mentioned bittern, add halogen worm dry powder 0.8～1.2g/L;
3. above-mentioned solution is placed in the illumination box and cultivates, culture condition is: illumination 10000～15000LX, and 30～40 ℃ of temperature cultivated for 1～2 week;
4. use the water droplet partition method from above-mentioned solution, to separate the algae kind;
5. it is that 15～25 ° of Be ' have also added in the aseptic bittern nutrient solution of nutritive salt that the algae kind that separation is obtained is transferred to salinity, cultivates under above-mentioned culture condition and obtains algae kind suspension 2～3 weeks;
(3) salinity of the above-mentioned algae kind suspension of raising makes saltiness reach 20～25 ° of Be ', at illumination 2300～2600LX, carries out guarantor's kind of algae kind under the condition that temperature is 18～22 ℃;
(4) in nutrient solution according to 8 * 10 3Cells/ml～12 * 10 3Cells/ml density inoculation algae kind;
(5) be that 20～35 ℃, the pH value of nutrient solution are to cultivate under 6～8 the condition in illumination, temperature, threw in one time of nutrition salt NaH in per 7～10 days to nutrient solution 2PO 410g/m 3And CH 4N 2O110g/m 3
The best inoculum density of described step (4) is 10 * 10 3Cells/ml.The optimum temps of described step (5) is 25～30 ℃.
The green Dunaliella salina cultural method of extreme halotolerant of the present invention is cultured this algae for the big area industrialization provides the necessary technology approach, for this algae of application and development provides possibility.The green Dunaliella salina of extreme halotolerant has the value of research, exploitation.
Embodiment
Below in conjunction with embodiment the present invention is described further.
Embodiment 1:
1. preparation nutrient solution: buy salinity from senior system halogen district, long reed Yan Qu saltern and be 100 liters in the bittern of 15 ° of Be ', at first use absorbent cotton elimination foul; Add nutritive salt NaH then 2PO 41 gram and urea are CH 4N 2O 11 grams; Then test pH value, if pH value in 6～8 these scopes, need not to regulate, if exceed this scope, using rare 10%HCl or 10%NaOH to regulate pH value is 6～8; Use the disinfection agent of chlorine dioxide (CLO of market sale at last 2Content is 2%) 5 grams carry out sterilizing and algae-removing to bittern and handle, and promptly make nutrient solution.Place inoculation use after 48 hours.
2. the algae kind prepares: 1 liter in bittern fetching salinity from senior system halogen district, beach long reed Yan Qu saltern, Chinese Bohai Sea Gulf and be 25 ° of Be '.At first be pre-cultivation, halogen worm dry powder 1 gram that adds in bittern then uses Erlenmeyer flask to cultivate in illumination box, and culture condition is: illumination 10000LX, 40 ℃ of temperature; Cultivated for 1 week, after microscopy finds that green Dunaliella salina takes place, use the water droplet partition method to separate the algae kind, the algae kind that separation is obtained is transferred to salinity rapidly to be 15 ° of Be ' and to have added in the aseptic bittern nutrient solution of nutritive salt, cultivates according to above-mentioned culture condition and can make algae kind suspension 2 weeks.
3. the algae kind is protected and planted: the nutrient solution salinity is brought up to 20 ° of Be ' gradually, and culture condition is regulated to: illumination 2500LX, 20 ℃ of temperature.
4. inoculation: before the inoculation, use blood counting chamber method test algae kind density of suspending liquid, long-pending according to algae kind density and inoculation culture liquid, according to 1 * 10 4Cells/ml density inoculation algae kind.Recording algae kind density of suspending liquid is 200 * 10 4Cells/ml picks and places 100 liters of the nutrient solutions put after 48 hours, throws in algae kind suspension 500ml;
5. regulation and control culture environment: above-mentioned postvaccinal 100 liters of nutrient solution pH values are controlled between 6～8, throw in nutritive salt NaH 2PO 41 gram and urea 11 grams added once in per 7 days; Cultivate under 25 ℃ of room temperatures, indoor common fluorescent lamp illuminate condition, cultivated 12 days, the green Dunaliella salina of extreme halotolerant can take place in a large number in the water body.
Embodiment 2:
1. preparation nutrient solution: buy salinity from senior system halogen district, long reed Yan Qu saltern and be 100 liters in the bittern of 18 ° of Be ', at first use absorbent cotton elimination foul; Add nutritive salt NaH then 2PO 41 gram and urea are CH 4N 2O 11 grams; Then test pH value, if pH value in 6～8 these scopes, need not to regulate, if exceed this scope, using rare 10%HCl or 10%NaOH to regulate pH value is 6～8; Use the disinfection agent of chlorine dioxide (CLO of market sale at last 2Content is 2%) 5 grams carry out sterilizing and algae-removing to bittern and handle, and promptly make nutrient solution.Place inoculation use after 48 hours.
2. the algae kind prepares: fetch salinity from senior system halogen district, beach long reed Yan Qu saltern, Chinese Bohai Sea Gulf and be 1 liter in 26% bittern.At first be pre-cultivation, add halogen worm dry powder 0.8 gram in bittern, then use Erlenmeyer flask to cultivate in illumination box, culture condition is: illumination 12000LX, 40 ℃ of temperature; Cultivated for 1.5 weeks, after microscopy finds that green Dunaliella salina takes place, use the water droplet partition method to separate the algae kind, the algae kind that separation is obtained is transferred to salinity rapidly to be 18 ° of Be ' and to have added in the aseptic bittern nutrient solution of nutritive salt, cultivates according to above-mentioned culture condition and can make algae kind suspension 2～3 weeks.
3. the algae kind is protected and planted: the nutrient solution salinity is brought up to 22 ° of Be ' gradually, and culture condition is regulated to: illumination 2300LX, 18 ℃ of temperature.
4. inoculation: before the inoculation, use blood counting chamber method test algae kind density of suspending liquid, long-pending according to algae kind density and inoculation culture liquid, according to 8 * 10 3Cells/ml density inoculation algae kind.Recording algae kind density of suspending liquid is 160 * 10 4Cells/ml picks and places 100 liters of nutrient solutions putting 48 hours, throws in algae kind suspension 500ml;
5. regulation and control culture environment: above-mentioned postvaccinal 100 liters of nutrient solution pH values are controlled between 6～8, throw in nutritive salt NaH 2PO 41 gram and urea 11 grams added once in per 9 days; Cultivate under 20 ℃ of room temperatures, indoor common fluorescent lamp illuminate condition, cultivated 13 days, the green Dunaliella salina of extreme halotolerant can take place in a large number in the water body.
Embodiment 3:
1. preparation nutrient solution: buy salinity from senior system halogen district, long reed Yan Qu saltern and be 100 liters in the bittern of 22 ° of Be ', at first use absorbent cotton elimination foul; Add nutritive salt NaH then 2PO 41 gram and urea are CH 4N 2O 11 grams; Then test pH value, if pH value in 6～8 these scopes, need not to regulate, if exceed this scope, using rare 10%HCl or 10%NaOH to regulate pH value is 6～8; Use the disinfection agent of chlorine dioxide (CLO of market sale at last 2Content is 2%) 5 grams carry out sterilizing and algae-removing to bittern and handle, and promptly make nutrient solution.Place inoculation use after 48 hours.
2. the algae kind prepares: 1 liter in bittern fetching salinity from senior system halogen district, beach long reed Yan Qu saltern, Chinese Bohai Sea Gulf and be 26 ° of Be '.At first be pre-cultivation, add halogen worm dry powder 1.2 grams in bittern, then use Erlenmeyer flask to cultivate in illumination box, culture condition is: illumination 15000LX, 30 ℃ of temperature; Cultivated for 2 weeks, after microscopy finds that green Dunaliella salina takes place, use the water droplet partition method to separate the algae kind, the algae kind that separation is obtained is transferred to salinity rapidly to be 22 ° of Be ' and to have added in the aseptic bittern nutrient solution of nutritive salt, cultivates according to above-mentioned culture condition and can make algae kind suspension 3 weeks.
3. the algae kind is protected and planted: the nutrient solution salinity is brought up to 25 ° of Be ' gradually, and culture condition is regulated to: illumination 2600LX, 22 ℃ of temperature.
4. inoculation: before the inoculation, use blood counting chamber method test algae kind density of suspending liquid, long-pending according to algae kind density and inoculation culture liquid, according to 1.2 * 10 4Cells/ml density inoculation algae kind.Recording algae kind density of suspending liquid is 240 * 10 4Cells/ml picks and places 100 liters of nutrient solutions putting 48 hours, throws in algae kind suspension 500ml;
5. regulation and control culture environment: above-mentioned postvaccinal 100 liters of nutrient solution pH values are controlled between 6～8, throw in nutritive salt NaH 2PO 41 gram and urea 11 grams added once in per 10 days; Cultivate under 20 ℃ of room temperatures, indoor common fluorescent lamp illuminate condition, cultivated 15 days, the green Dunaliella salina of extreme halotolerant can take place in a large number in the water body.
Embodiment 4:
1. preparation nutrient solution: buy salinity from senior system halogen district, long reed Yan Qu saltern and be 100 liters in the bittern of 25 ° of Be ', at first use absorbent cotton elimination foul; Add nutritive salt NaH then 2PO 41 gram and urea are CH 4N 2O 11 grams; Then test pH value, if pH value in 6～8 these scopes, need not to regulate, if exceed this scope, using rare 10%HCl or 10%NaOH to regulate pH value is 6～8; Use the disinfection agent of chlorine dioxide (CLO of market sale at last 2Content is 2%) 5 grams carry out sterilizing and algae-removing to bittern and handle, and promptly make nutrient solution.Place inoculation use after 48 hours.
2. the algae kind prepares: 1 liter in bittern fetching salinity from senior system halogen district, beach long reed Yan Qu saltern, Chinese Bohai Sea Gulf and be 27 ° of Be '.At first be pre-cultivation, add halogen worm dry powder 1 gram in bittern, then use Erlenmeyer flask to cultivate in illumination box, culture condition is: illumination 10000LX, 40 ℃ of temperature; Cultivated for 1 week, after microscopy finds that green Dunaliella salina takes place, use the water droplet partition method to separate the algae kind, the algae kind that separation is obtained is transferred to salinity rapidly to be 25 ° of Be ' and to have added in the aseptic bittern nutrient solution of nutritive salt, cultivates according to above-mentioned culture condition and can make algae kind suspension 3 weeks.
3. the algae kind is protected and planted: the nutrient solution salinity is controlled at 25 ° of Be ', and culture condition is regulated to: illumination 2300LX, 18 ℃ of temperature.
4. inoculation: before the inoculation, use blood counting chamber method test algae kind density of suspending liquid, long-pending according to algae kind density and inoculation culture liquid, according to 1 * 10 4Cells/ml density inoculation algae kind.Recording algae kind density of suspending liquid is 200 * 10 4Cells/ml picks and places 100 liters of the nutrient solutions put after 48 hours, throws in algae kind suspension 500ml;
5. regulation and control culture environment: above-mentioned postvaccinal 100 liters of nutrient solution pH values are controlled between 6～8, throw in nutritive salt NaH 2PO 41 gram and urea 11 grams added once in per 7 days; Cultivate under 26 ℃ of room temperatures, outdoor natural solar light irradiation condition, cultivated 15 days, the green Dunaliella salina of extreme halotolerant can take place in a large number in the water body.
The resistant gene of salt of the green Dunaliella salina of extreme halotolerant is the best gene of finding in the present vegitabilia of salt tolerance, can extract, and is applied to the genetically engineered field; The glycerine composition utilizes the ability of the green Dunaliella salina synthesis of natural of extreme halotolerant plant glycerine can produce natural phant glycerine up to more than 60% in the Dunaliella salina cell paste that the extreme halotolerant environment is grown down; The green Dunaliella salina cell paste of extreme halotolerant contains vegetable-protein, chlorophyll, glycerine and other kind biologically active substances, can be applicable to fields such as body-care, bio-pharmaceuticals, exploitation natural health-care products or biologics.

Claims (3)

1. the green Dunaliella salina cultural method of extreme halotolerant is characterized in that, comprises the steps:
(1) preparation of nutrient solution, the preparation of described nutrient solution comprises the steps:
1. get the bittern that saltiness is 15～25 ° of Be ';
2. in above-mentioned bittern, add nutritive salt NaH 2PO 410g/m 3And CH 4N 2O 110g/m 3
3. regulating above-mentioned solution, to make its pH value be 6～8;
4. working concentration is that the 1ppm disinfection agent of chlorine dioxide carries out the sterilizing and algae-removing processing to above-mentioned solution;
(2) preparation of algae kind, the preparation of described algae kind comprises the steps:
1. getting saltiness is 25～27 ° of Be ' bittern;
2. in above-mentioned bittern, add halogen worm dry powder 0.8～1.2g/L;
3. above-mentioned solution is placed in the illumination box and cultivates, culture condition is: illumination 10000～15000LX, and 30～40 ℃ of temperature cultivated for 1～2 week;
4. use the water droplet partition method from above-mentioned solution, to separate the algae kind;
5. it is that 15～25 ° of Be ' have also added in the aseptic bittern nutrient solution of nutritive salt that the algae kind that separation is obtained is transferred to salinity, cultivates under above-mentioned culture condition and obtains algae kind suspension 2～3 weeks;
(3) salinity of the above-mentioned algae kind suspension of raising makes saltiness reach 20～25 ° of Be ', at illumination 2300～2600LX, carries out guarantor's kind of algae kind under the condition that temperature is 18～22 ℃;
(4) in nutrient solution according to 8 * 10 3Cells/ml～12 * 10 3Cells/ml density inoculation algae kind;
(5) be that 10～35 ℃, the pH value of nutrient solution are to cultivate under 6～8 the condition in illumination, temperature, threw in one time of nutrition salt NaH in per 7～10 days to nutrient solution 2PO 410g/m 3And CH 4N 2O110g/m 3
2. the green Dunaliella salina cultural method of extreme halotolerant according to claim 1 is characterized in that the inoculum density of described step (4) is 10 * 10 3Cells/ml.
3. the green Dunaliella salina cultural method of extreme halotolerant according to claim 1 is characterized in that the temperature of described step (5) is 25～30 ℃.
CN 03109642 2003-04-11 2003-04-11 Method for cultivating green Dunaliella salina of liking for salt extremely Pending CN1446904A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014512194A (en) * 2011-05-12 2014-05-22 韓国海洋科学技術院 Method for producing salt containing useful components of microalgae and produced salt
CN106830490A (en) * 2017-03-28 2017-06-13 精河县精河盐化有限责任公司 A kind of processing method of the sodium sulfate type salt lake water for the cultivation of salt algae
CN108085258A (en) * 2018-01-03 2018-05-29 山东华维藻业有限公司 A kind of utilize puies forward the method that bromine bittern cultivates Dunaliella salina in banked track pond
CN108308591A (en) * 2017-12-18 2018-07-24 潍坊友容实业有限公司 The method for manufacturing plant salt using Dunaliella salina
CN108641964A (en) * 2018-03-29 2018-10-12 天津科技大学 A method of utilizing the broken breeding green Du Shi algaes of osmotic pressure
WO2019238914A1 (en) 2018-06-15 2019-12-19 Isp Investments Llc Method for obtaining an aqueous extract of dunaliella salina and cosmetic uses of same

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014512194A (en) * 2011-05-12 2014-05-22 韓国海洋科学技術院 Method for producing salt containing useful components of microalgae and produced salt
CN106830490A (en) * 2017-03-28 2017-06-13 精河县精河盐化有限责任公司 A kind of processing method of the sodium sulfate type salt lake water for the cultivation of salt algae
CN108308591A (en) * 2017-12-18 2018-07-24 潍坊友容实业有限公司 The method for manufacturing plant salt using Dunaliella salina
CN108085258A (en) * 2018-01-03 2018-05-29 山东华维藻业有限公司 A kind of utilize puies forward the method that bromine bittern cultivates Dunaliella salina in banked track pond
CN108641964A (en) * 2018-03-29 2018-10-12 天津科技大学 A method of utilizing the broken breeding green Du Shi algaes of osmotic pressure
WO2019238914A1 (en) 2018-06-15 2019-12-19 Isp Investments Llc Method for obtaining an aqueous extract of dunaliella salina and cosmetic uses of same

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