CN103352006B - 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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- CN103352006B CN103352006B CN201310331939.1A CN201310331939A CN103352006B CN 103352006 B CN103352006 B CN 103352006B CN 201310331939 A CN201310331939 A CN 201310331939A CN 103352006 B CN103352006 B CN 103352006B
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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 promoting autotrophy microalgae neutral lipid accumulation.
Background technology
Under the impact of global energy crisis, the research that the mankind find the novel renewable energy of environmental ecology close friend is extensively carried out.The features such as the unicellular microalgae of autotrophy is fast with its growth, biomass is high, collaborative ecologic effect is excellent, become the important materials of new forms of energy.Wherein, in microalgae cell, neutral fat storage total amount exceedes micro-algae of dry weight 20%, is the oil-producing microalgae with potentiality to be exploited.
The neutral fat accumulation metabolism of micro-algae affects in the algae proliferation later stage by coercing of multiple Factors and carry out accumulating, and therefore, the condition that cell proliferation cultivation and neutral fat are accumulated is conflicting often.Current productive culture process adopts the two-step approach of vegetative cell multiplication culture and neutral fat accumulative culture to carry out usually respectively, and the optimal condition providing micro-algae to breed in the first step, guarantees to obtain the most efficient maximum biomass of micro-algae; Second step then provides and suppresses to nourish and grow but the condition promoting neutral fat to accumulate, and under maintaining the condition of high-biomass, improves the accumulation of unit cell neutral fat further in optimization.
Research finds the increase of various environmental factors controllable energy microalgae biomass and the accumulation of grease, comprises nutrition salinity and temperature, CO
2concentration, illumination etc.Wherein, illumination is one of most important factor, and the aspect such as efficiency, Growth of Cells, substance metabolism approach to algae photosynthesis such as intensity of illumination, cycle and Net long wave radiation has significant regulating and controlling effect.The exploitation of new forms of energy are carried out in order to realize micro-algae, various countries have carried out many-sided research to huge micro-algae resource, comprise germplasm screening and detection method, mass propgation condition, the condition of a large amount of enrichment grease, collection method and device, extracting method and device etc.Such as 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 ultrasonic wave added fluorescent dye detects the method for 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 " produces the method for the oil-rich microalgae as biodiesel raw material ", discloses a kind of cultural method and the induction neutral fat accumulation method of producing the micro-algae of rich oil fat.Patent publication No. is that the patent of invention of CN102268377A " supports the method improving oil-producing microalgae biomass and oil accumulation with rich nitrogen-nitrogen stress two benches training strategy " with holding concurrently, and discloses a kind of induction method by regulating nutritive salt and nutritional mode to carry out neutral fat accumulation.But up to now, still do not utilize filter coating filter light to carry out promoting that micro-algae improves the method for neutral fat accumulation.
Summary of the invention
The object of this invention is to provide a kind of promote autotrophy microalgae neutral lipid accumulation give light method, namely simple, can the micro-algae neutral fat accumulation of the promotion of large-scale operation give a light method, thus to raise the efficiency, reduce and drop into, reduce production run cost, increase economic efficiency.
The present invention promotes the cultural method of autotrophy microalgae neutral lipid accumulation, is to be in the propagation later stage, carries out neutral fat accumulative culture under micro-algae with neutral fat accumulation metabolic capacity is in 400-550nm light wave condition.
Wherein 400-550nm light wave condition is formed with the light transmissive material of blue material or container.
The light transmissive material of described blue material is blue filter film.
A kind of cultural method of the present invention is the micro-algae liquid by being in the propagation later stage, proceeds to carry out neutral fat accumulative culture in the booth pond of blue filter film covering; Specific as follows:
First carrying out the first step cell proliferation cultivation: be inoculated in by algae in cultivation in sea water liquid, is 22-30 DEG C in temperature, and illumination condition is cultivate under the condition of 2000-6000lx, when being cultured to plateau maximum density 2-3 days, carries out second step cultivation;
Second step neutral fat accumulative culture: the algae liquid being cultured to plateau maximum density is transferred to temperature and is 10-20 DEG C, obtains that wavelength is 400-550nm, 2000-6000lx illumination with blue filter membrane filtration light, carries out neutral fat accumulative culture under the condition of Light To Dark Ratio 12:12, in nutrient solution, add with the ratio of 1:1000 the sodium dihydrogen phosphate that concentration is 5g/L simultaneously, continue to cultivate and gather for 5-7 days.
Described cultivation in sea water liquid 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, that bioreactor is cultivated micro-algae liquid that propagation to the later stage is in maximum biomass, proceed to the container covered with blue filter film, or proceed to blue plastics bag or proceed to in the bioreactor making to make at the material of 400-550nm wave band through light, culture temperature being reduced and carries out neutral fat accumulative culture.
Method of the present invention is under the cell proliferation of the acquisition the first step is cultured to the prerequisite of maximum biomass, by to the cooling of algae liquid and the synergy of illumination condition, micro-algae neutral fat metabolism short-term is responded fast, realizes the object promoting and improve neutral fat cumulative efficiency and semi-invariant.Described blue filter film is plastic film, can mass-producing application and repacking.Described nitrogen restriction stress conditions is reversed by N/P ratio to regulate, and do not need the sepn process of micro-algae and medium to realize.The technical process in large-scale production can be simplified.
Accompanying drawing explanation
Fig. 1: the growth curve of Thalassiosira pseudonana rich oil strain NMBguh005, wherein TNL is the neutral fat accumulative culture phase cell number change adding stress conditions, and control is contrast;
Neutral fat accumulative culture figure under Fig. 2: Thalassiosira pseudonana rich oil strain NMBguh005 two conditions; 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: neutral fat accumulative culture is carried out to Thalassiosira pseudonana rich oil strain NMBguh005
1, cell proliferation is cultivated:
Take from the sterilization of right sea water filter, add KNO respectively
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
b1the each material of 5 mg/L forms micro algae culturing liquid.Cell proliferation cultivation is carried out in inoculation Thalassiosira pseudonana to above-mentioned nutrient solution.Nutrient solution salinity 23 ‰, culture temperature 25 DEG C, 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.Sample 2mL 3 every day and repeat microscopic count tracking growth cycle.Proliferation process figure is as shown in Fig. 1 (0-12d).Parallelly divide each 3 bottles equally by 6 when cultivating 12d, the contrast carrying out neutral fat accumulative culture is respectively cultivated.
For composition and the proportioning of nutrient solution, be induce with the wavelength of illumination in follow-up neutral fat accumulative culture mutually to work in coordination with, thus ensure in micro-algae, to obtain maximum neutral fat cumulative amount.
2, neutral fat accumulative culture:
After cell proliferation is cultured to plateau maximum biomass 2-3 days, 3 culturing bottles being proceeded to temperature is ensure in the incubator of 20 DEG C that the culture temperature of algae reduces, and other 3 culturing bottles are maintained in original incubator and continue to cultivate.Newly proceed to incubator fluorescent light source all to isolate with blue plastic film, regulating illumination intensity reaches 4000lx(D:L=12:12).Add concentration according to the ratio of 1:1000 is the sodium dihydrogen phosphate of 5g/L simultaneously.Continue to cultivate 5-7 days.Same track cells variable density in neutral fat accumulative culture process, samples 1mL detects neutral fat quantitative change with Nile red staining simultaneously.Neutral fat accumulative culture 5 days.
Cultivate after 5 days, in cooling, N stress and filter coating under light culture condition, cell count is coerced average decline 7%.The neutral fat semi-invariant of unit cell, continues to maintain significantly improving (p < 0.05) under former culture condition than control group, maximumly reaches 40%(Fig. 2).
The above results shows, method of the present invention can significantly improve the content of the neutral fat in micro-algae.
Claims (3)
1. promote a cultural method for autotrophy microalgae neutral lipid accumulation, it is characterized in that, be to be in the propagation later stage, under the Thalassiosira pseudonana with neutral fat accumulation metabolic capacity is in 400-550nm light wave condition, carry out neutral fat accumulative culture; Its concrete steps are as follows:
First carrying out the first step cell proliferation cultivation: be inoculated in by algae in cultivation in sea water liquid, is 22-30 DEG C in temperature, and illumination condition is cultivate under the condition of 2000-6000lx, when being cultured to plateau maximum density 2-3 days, carries out second step cultivation;
Second step neutral fat accumulative culture: the algae liquid being cultured to plateau maximum density is transferred to temperature and is 10-20 DEG C, obtains that wavelength is 400-550nm, 2000-6000lx illumination with blue filter membrane filtration light, carries out neutral fat accumulative culture under the condition of Light To Dark Ratio 12:12, in nutrient solution, add with the ratio of 1:1000 the sodium dihydrogen phosphate that concentration is 5g/L simultaneously, continue to cultivate and gather for 5-7 days; Described cultivation in sea water liquid composed as follows: KNO
3100mg/L, K
2hPO
410mg/L, MnSO
40.25mg/L, FeC
6h
5o
72.5mg/L, Na
2eDTA 20mg/L, VB
120.5mg/L, VB
15mg/L.
2. cultural method as claimed in claim 1, is characterized in that, described 400-550nm light wave condition, is to be formed with the light transmissive material of blue material or container.
3. cultural method as claimed in claim 2, is characterized in that the light transmissive material of described blue material is blue filter film.
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| FR3025214A1 (en) * | 2014-08-26 | 2016-03-04 | Fermentalg | NEW PROCESS FOR CULTIVATION OF ALGAE, PARTICULARLY MICROALGUES |
| PT3018198T (en) * | 2014-11-07 | 2018-11-05 | Neste Oyj | Method of 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. |
| CN106635810B (en) * | 2016-12-06 | 2020-02-14 | 沈阳化工研究院有限公司 | Microalgae and culture method of 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 |
| CN113502226A (en) * | 2021-08-31 | 2021-10-15 | 西藏大学 | Culture medium for separating, purifying and amplifying culture of plateau microalgae |
| 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 |
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| CN102443562A (en) * | 2010-10-12 | 2012-05-09 | 中国石油化工股份有限公司 | Culture medium and culture method for promoting fast propagation of microalgae |
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