CN103352006A - Culture method for promoting autotrophy microalgae neutral lipid accumulation - Google Patents
Culture method for promoting autotrophy microalgae neutral lipid accumulation Download PDFInfo
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Abstract
The present invention provides a culture method for promoting autotrophy microalgae neutral lipid accumulation, wherein microalgae having neutral lipid accumulation metabolism capability at a proliferation later stage is subjected to neutral lipid accumulation culture under a 400-550 nm light wave condition. According to the method, under the premise of proliferation culture of the obtained first step cells to achieve the maximum biomass, microalgae neutral lipid metabolism rapidly responses in a short time through temperature reduction on the microalgae liquid and a synergism effect of the illumination condition so as to achieve a purpose of promotion and increase of the neutral lipid accumulation efficiency and the neutral lipid accumulation amount. The blue filter membrane is a plastic film, and can be subjected to large-scale application and modification. The nitrogen limitation stress condition can be achieved through N/P ratio inversion regulation without a microalgae and media separation process. In addition, the process in the large-scale production can be simplified.
Description
Technical field
The invention belongs to oil-rich microalgae culture technique field, be specifically related to a kind of cultural method that promotes the little algae neutral fat accumulation of autotrophy.
Background technology
Under the impact of global energy crisis, the human research of seeking environmental ecology close friend's novel renewable energy is extensively carried out.The unicellular microalgae of autotrophy becomes the important materials of new forms of energy with its fast, characteristics such as biomass is high, collaborative ecologic effect excellence of growing.Wherein, in the microalgae cell, neutral fat storage total amount surpasses little algae of dry weight 20%, is the oil-producing microalgae with potentiality to be exploited.
The neutral fat of little algae accumulation metabolism is affected by coercing of multiple Factors and accumulate in the algae proliferation later stage, and therefore, the cell proliferation cultivation is conflicting often with the condition that neutral fat is accumulated.The productive culture process adopts respectively the two-step approach of vegetative cell multiplication culture and neutral fat accumulative culture to carry out usually at present, and the optimal condition of little algae propagation is provided in the first step, guarantees to obtain the most efficient maximum biomass of little algae; Second step then provides inhibition to nourish and grow but promotes the condition that neutral fat is accumulated, and keeps in optimization under the condition of high-biomass, further improves the accumulation of unit cell neutral fat.
Research discovery various environmental factors can be regulated and control the increase of energy micro algae biomass and the accumulation of grease, comprises nutrition salinity and temperature, CO
2Concentration, illumination etc.Wherein, illumination is one of most important factor, intensity of illumination, cycle and effectively radiation etc. the aspects such as the efficient of algae photosynthesis, Growth of Cells, substance metabolism approach are had significant regulating and controlling effect.In order to realize that little algae carries out the development and use of new forms of energy, various countries have carried out many-sided research to huge little algae resource, comprise condition, collection method and device, extracting method and device of germplasm screening and detection method, a large amount of culture condition, a large amount of enrichment greases etc.For example patent publication No. is the patent of invention " a kind of high-throughput screening method of oil-rich microalgae " of CN103146582A, discloses a kind of screening method to microalge.Patent publication No. is the patent of invention " a kind of method of ultrasonic secondary fluorescence staining examine microalgae grease content " of CN103163113A, discloses a kind of measuring method to microalgae grease content.Patent publication No. is that the patent of invention of CN103052706A " is produced the method as the oil-rich microalgae of biodiesel raw material ", discloses a kind ofly to produce the cultural method of the little algae of rich oil fat and induce the neutral fat accumulation method.Patent publication No. is that the patent of invention of CN102268377A " is supported the method that improves oil-producing microalgae biomass and grease accumulation with rich two stage of nitrogen-nitrogen stress training strategy " with holding concurrently, and discloses a kind of induction method that carries out the neutral fat accumulation by regulating nutritive salt and nutritional mode.But up to now, still do not utilize the filter coating filter light to promote little algae to improve the method for neutral fat accumulation.
Summary of the invention
The purpose of this invention is to provide a kind of promote the accumulation of the little algae neutral fat of autotrophy to the light method, but be the little algae neutral fat accumulation of a kind of promotion of simple large-scale operation to the light method, thereby raise the efficiency, reduce and drop into, reduce the production run cost, increase economic efficiency.
The present invention promotes the cultural method of the little algae neutral fat accumulation of autotrophy, is to be in the propagation later stage, and the little algae with neutral fat accumulation metabolic capacity is under the 400-550nm light wave condition and carries out the neutral fat accumulative culture.
400-550nm light wave condition wherein is that light transmissive material or the container with blue material forms.
The light transmissive material of described blue material is blue filter coating.
A kind of cultural method of the present invention is to be in little algae liquid in propagation later stage, changes in the booth pond that covers with blue filter coating and carries out the neutral fat accumulative culture; Specific as follows:
At first carrying out the first step cell proliferation cultivates: algae is inoculated in the seawater nutrient solution, is 22-30 ℃ in temperature, and illumination condition is to cultivate under the condition of 2000-6000lx, when being cultured to plateau maximum density 2-3 days, carrying out second step and cultivates;
Second step neutral fat accumulative culture: the algae liquid that will be cultured to the plateau maximum density is transferred to temperature to be 10-20 ℃, to carry out the neutral fat accumulative culture take blue filter coating optical filtering acquisition wavelength under the condition of 400-550nm, 2000-6000lx illumination, Light To Dark Ratio 12:12, in nutrient solution, add concentration as the sodium dihydrogen phosphate of 5g/L take the ratio of 1:1000 simultaneously, continue to cultivate and gathered in 5-7 days.
Described seawater nutrient solution composed as follows: KNO
3100 mg/L, K
2HPO
410 mg/L, MnSO
40.25 mg/L, FeC
6H
5O
72.5 mg/L, Na
2EDTA 20 mg/L, V
B120.5 mg/L, V
B15 mg/L.
The concrete cultural method of another kind of the present invention, bioreactor to be cultivated breed the little algae liquid that is in maximum biomass to the later stage, change the container that covers with blue filter coating over to, perhaps change blue plastics bag over to or change over to making through light in the bioreactor that the material of 400-550nm wave band is made, culture temperature is reduced carry out the neutral fat accumulative culture.
Method of the present invention is cultured under the prerequisite of maximum biomass in the cell proliferation of the acquisition the first step, by to the cooling of algae liquid and the synergy of illumination condition, little algae neutral fat metabolism short-term is responded fast, realize promoting and improving the purpose of neutral fat cumulative efficiency and semi-invariant.Described blue filter coating is plastic film, but mass-producing is used and repacking.Described nitrogen restriction stress conditions is to reverse by N/P ratio to regulate, and does not need the sepn process of little algae and medium to realize.Can simplify the technical process in the large-scale production.
Description of drawings
Fig. 1: the growth curve of Thalassiosira pseudonana rich oil strain NMBguh005, wherein TNL changes for the neutral fat accumulative culture phase cell number that adds stress conditions, and control is contrast;
Fig. 2: the neutral fat accumulative culture figure under two conditions of Thalassiosira pseudonana rich oil strain NMBguh005; Wherein TNL is for coercing group, and Control is control group.
Embodiment
Below in conjunction with specific embodiment method of the present invention is described in detail.
Embodiment 1: NMBguh005 carries out the neutral fat accumulative culture to Thalassiosira pseudonana rich oil strain
1, cell proliferation is cultivated:
Take from right sea water filter sterilization, add respectively KNO
3100 mg/L, K
2HPO
410 mg/L, MnSO
40.25 mg/L, FeC
6H
5O
72.5 mg/L, Na
2EDTA 20 mg/L, V
B120.5 mg/L, V
B1Each material of 5 mg/L forms micro algae culturing liquid.The inoculation Thalassiosira pseudonana carries out the cell proliferation cultivation in above-mentioned nutrient solution.Nutrient solution salinity 23 ‰, 25 ℃ of culture temperature, illumination 4000lx(D:L=12:12), culture environment is obtained by the illumination box of fluorescent light source.Cultivate water body volume 500mL, 6 is parallel.The 2mL 3 that takes a sample every day repeats microscopic count and follows the tracks of growth cycle.During proliferation process figure is shown in Fig. 1 (0-12d).Parallelly divide each 3 bottles equally with 6 when cultivating 12d, carry out respectively the contrast culture of neutral fat accumulative culture.
For composition and the proportioning of nutrient solution, be with follow-up neutral fat accumulative culture in the wavelength of illumination induce mutually collaborative, thereby guarantee to obtain in little algae maximum neutral fat cumulative amount.
2, neutral fat accumulative culture:
After cell proliferation was cultured to plateau maximum biomass 2-3 days, changing 3 culturing bottles over to temperature was that the culture temperature that guarantees algae in 20 ℃ the incubator reduces, and other 3 culturing bottles are maintained at and continue in the original incubator to cultivate.Newly change the incubator fluorescent light source over to and all isolate with blue plastic film, capable of regulating illumination intensity reaches 4000lx(D:L=12:12).Adding concentration according to the ratio of 1:1000 simultaneously is the sodium dihydrogen phosphate of 5g/L.Continue to cultivate 5-7 days.Same track cells variable density in the neutral fat accumulative culture process, the 1mL that takes a sample simultaneously detects the quantitative variation of neutral fat with the Nile red staining.Neutral fat accumulative culture 5 days.
Cultivate after 5 days, in the cooling, nitrogen is coerced and filter coating to the light culture condition under, cell count is coerced average decline 7%.The neutral fat semi-invariant of unit cell continues to keep significantly improving under the former culture condition (p＜0.05) than control group, and maximum reaches 40%(Fig. 2).
The above results shows, method of the present invention can obviously improve the content of the neutral fat in little algae.
Claims (6)
1. a cultural method that promotes the little algae neutral fat accumulation of autotrophy is characterized in that, is to be in the propagation later stage, and the little algae with neutral fat accumulation metabolic capacity is under the 400-550nm light wave condition and carries out the neutral fat accumulative culture.
2. cultural method as claimed in claim 1 is characterized in that, described 400-550nm light wave condition is to form with the light transmissive material of blue material or container.
3. cultural method as claimed in claim 2, the light transmissive material that it is characterized in that described blue material is blue filter coating.
4. cultural method claimed in claim 1 it is characterized in that and will be in little algae liquid in propagation later stage, changes in the booth pond that covers with blue filter coating and carries out the neutral fat accumulative culture; Concrete steps are as follows:
At first carrying out the first step cell proliferation cultivates: algae is inoculated in the seawater nutrient solution, is 22-30 ℃ in temperature, and illumination condition is to cultivate under the condition of 2000-6000lx, when being cultured to plateau maximum density 2-3 days, carrying out second step and cultivates;
Second step neutral fat accumulative culture: the algae liquid that will be cultured to the plateau maximum density is transferred to temperature to be 10-20 ℃, to carry out the neutral fat accumulative culture take blue filter coating optical filtering acquisition wavelength under the condition of 400-550nm, 2000-6000lx illumination, Light To Dark Ratio 12:12, in nutrient solution, add concentration as the sodium dihydrogen phosphate of 5g/L take the ratio of 1:1000 simultaneously, continue to cultivate and gathered in 5-7 days.
5. cultural method as claimed in claim 4 is characterized in that the composed as follows of described seawater nutrient solution: KNO
3100 mg/L, K
2HPO
410 mg/L, MnSO
40.25 mg/L, FeC
6H
5O
72.5 mg/L, Na
2EDTA 20 mg/L, V
B120.5 mg/L, V
B15 mg/L.
6. cultural method claimed in claim 1, it is characterized in that the little algae liquid that is in maximum biomass to the later stage is bred in the bioreactor cultivation, change the container that covers with blue filter coating over to, perhaps change blue plastics bag over to or change over to making through light in the bioreactor that the material of 400-550nm wave band is made, culture temperature is reduced carry out the neutral fat accumulative culture.
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| CN104651235A (en) * | 2015-03-02 | 2015-05-27 | 宁波大学 | Thalassiosira pseudonana and application of thalassiosira pseudonana as mercenaria mercenaria larva breeding bait |
| CN105199959A (en) * | 2015-10-22 | 2015-12-30 | 湛江恒兴南方海洋科技有限公司 | Macro-element nutrient salt formula for large-scale culture of Thalassiossira sp. |
| WO2016030629A1 (en) * | 2014-08-26 | 2016-03-03 | Fermentalg | Method for culture of protists |
| CN106635810A (en) * | 2016-12-06 | 2017-05-10 | 沈阳化工研究院有限公司 | Microalgae and method for culturing microalgae |
| CN107208034A (en) * | 2014-11-07 | 2017-09-26 | 耐斯特公司 | The method for cultivating algae |
| CN110628645A (en) * | 2019-10-10 | 2019-12-31 | 广东海洋大学深圳研究院 | Concentration culture method of Cyclotella meini seawater strain |
| CN113502226A (en) * | 2021-08-31 | 2021-10-15 | 西藏大学 | Culture medium for separating, purifying and amplifying culture of plateau microalgae |
| CN116024093A (en) * | 2023-03-27 | 2023-04-28 | 中国科学院海洋研究所 | Light supply method in dunaliella salina culture process and application |
| CN118599932A (en) * | 2024-08-08 | 2024-09-06 | 广东海洋大学 | Method for promoting eukaryotic microalgae to synthesize lipid |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016030629A1 (en) * | 2014-08-26 | 2016-03-03 | Fermentalg | Method for culture of protists |
| WO2016030630A1 (en) * | 2014-08-26 | 2016-03-03 | Fermentalg | Novel method for culture of algae, in particular microalgae |
| FR3025217A1 (en) * | 2014-08-26 | 2016-03-04 | Fermentalg | PROCESS FOR CULTIVATION OF PROTISTS |
| FR3025214A1 (en) * | 2014-08-26 | 2016-03-04 | Fermentalg | NEW PROCESS FOR CULTIVATION OF ALGAE, PARTICULARLY MICROALGUES |
| US11667885B2 (en) | 2014-11-07 | 2023-06-06 | Neste Oyj | Method of cultivating algae |
| CN107208034A (en) * | 2014-11-07 | 2017-09-26 | 耐斯特公司 | The method for cultivating algae |
| CN104651235A (en) * | 2015-03-02 | 2015-05-27 | 宁波大学 | Thalassiosira pseudonana and application of thalassiosira pseudonana as mercenaria mercenaria larva breeding bait |
| CN105199959A (en) * | 2015-10-22 | 2015-12-30 | 湛江恒兴南方海洋科技有限公司 | Macro-element nutrient salt formula for large-scale culture of Thalassiossira sp. |
| CN106635810A (en) * | 2016-12-06 | 2017-05-10 | 沈阳化工研究院有限公司 | Microalgae and method for culturing microalgae |
| CN110628645B (en) * | 2019-10-10 | 2021-07-06 | 广东海洋大学深圳研究院 | Concentration culture method of Cyclotella meini seawater strain |
| CN110628645A (en) * | 2019-10-10 | 2019-12-31 | 广东海洋大学深圳研究院 | Concentration culture method of Cyclotella meini seawater strain |
| CN113502226A (en) * | 2021-08-31 | 2021-10-15 | 西藏大学 | Culture medium for separating, purifying and amplifying culture of plateau microalgae |
| CN116024093A (en) * | 2023-03-27 | 2023-04-28 | 中国科学院海洋研究所 | Light supply method in dunaliella salina culture process and application |
| CN116024093B (en) * | 2023-03-27 | 2023-06-13 | 中国科学院海洋研究所 | Light supply method in dunaliella salina culture process and application |
| CN118599932A (en) * | 2024-08-08 | 2024-09-06 | 广东海洋大学 | Method for promoting eukaryotic microalgae to synthesize lipid |
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