CN108410939B - Method for increasing astaxanthin content in haematococcus pluvialis - Google Patents
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Abstract
The invention discloses a method for improving the content of astaxanthin in haematococcus pluvialis, which comprises the step of adding ethanolamine (Monoethanomine, MEA for short) into a culture solution at the motionless stage of haematococcus pluvialis culture until the final concentration is 50-400 mg/L. The method for improving the astaxanthin content in haematococcus pluvialis provided by the invention is a new technical route for improving the astaxanthin content in haematococcus pluvialis, obviously improves the astaxanthin content in cells, shortens the culture time and improves the culture efficiency.
Description
Technical Field
The invention belongs to the technical field of microalgae biology, and relates to a method for increasing the content of astaxanthin in haematococcus pluvialis, namely a method for increasing the content of astaxanthin in haematococcus pluvialis by adding ethanolamine.
Background
Astaxanthin (3, 3 ' -dihydroxy- β ' -carotene-4, 4 ' -dione) belongs to carotenoid, is a main pigment of crustacean such as shrimp, crab and salmon, has strong antioxidant function, can scavenge free radicals generated by ultraviolet irradiation in vivo, regulate and reduce such damages caused by photochemistry, and has excellent therapeutic effect on ultraviolet-induced skin cancerHaematococcus pluvialis) The content of astaxanthin in haematococcus pluvialis is the highest. Therefore, Haematococcus pluvialis has a wide prospect as a source of astaxanthin production industry.
The haematococcus pluvialis has from wide oval shape to wide oval shape, width of 19-51 microns, length of 28-63 microns, and has three nutritional types of photoautotrophic type, heterotrophic growth type and mixed nutritional type, and uses single-cell spore or zygote to make reproduction.
Aflalo et al found that Glufosinate-ammonium can influence The growth of H.pluvialis by inhibiting glutaminase activity, thereby inducing Astaxanthin Accumulation (Aflalo C, Wang B, Zarka A, et al, The Effect of The Herbicide Glufosinate (BASTA) on Astaxanthin Accumulation in The Green AlgaHaematococcus pluvialis[J]Zeitschrift Fur Naturforschung C A Journal of biosciences, 1999, 54(2): 49-54.). Patent CN104232720B discloses a method for producing astaxanthin by inducing haematococcus pluvialis by exogenously adding ethanol and seawater crystal, and the astaxanthin is accumulated. Patent CN101974599B discloses a method for stimulating haematococcus pluvialis to rapidly produce astaxanthin by using brassinolide, and salicylic acid, jasmonic acid and gibberellin are sequentially reported to have the effect of increasing the content of astaxanthin in haematococcus pluvialis.
Ethanolamine is a common organic chemical reagent, is cheap and easily available, and is industrially used for rapidly absorbing carbon dioxide gas in a closed space. Patent CN102492744B discloses the use of ethanolamine to increase the oil content of Chlorella or Scenedesmus (Sun Z, Zhang D, Yan C, et al, the motion of microbial biological production and efficacy use of CO)2from flue gas by monoethanolamine[J]. Journal ofChemical Technology&Biotechnology 2015, 90(4):730-2The effect of fixed efficiency.
Disclosure of Invention
The invention aims to provide a method for improving the content of astaxanthin in haematococcus pluvialis, obtain a new technical route for improving the content of astaxanthin in haematococcus pluvialis, obviously improve the content of astaxanthin in cells, shorten the culture time and improve the culture efficiency.
The method for improving the astaxanthin content in haematococcus pluvialis is characterized in that ethanolamine (Monoethanomine, MEA for short) is added into a culture solution at the motionless stage of haematococcus pluvialis culture to the final concentration of 50-400 mg/L.
A method for increasing the astaxanthin content in Haematococcus pluvialis comprises the following steps:
1) obtaining a haematococcus pluvialis culture solution in a swimming growth stage:
autotrophic culturing haematococcus pluvialis in a closed photobioreactor, wherein the selected culture medium is BG11 or BM culture medium or other artificial culture medium suitable for growth of haematococcus pluvialis cells, the culture temperature is controlled to be 25-30 ℃, and the light intensity is 100 mu mol/(m) mol2S) introduction of sterile gas mixture (CO)2The volume concentration of the culture medium is 2-10%, and the balance is air), culturing haematococcus pluvialis to the late logarithmic phase, and obtaining an autotrophic haematococcus pluvialis culture solution;
or mixedly culturing Haematococcus pluvialis in a closed columnar photoreactor, wherein the culture medium is BG11 or BM culture medium supplemented with 1g/L sodium acetate, the culture temperature is controlled at 25-30 deg.C, and the light intensity is 100 μmol/(m)2S) introduction of sterile gas mixture (CO)2The volume concentration of the culture medium is 2-10%, and the balance is air), culturing haematococcus pluvialis to the late logarithmic phase, and obtaining a polycultured haematococcus pluvialis culture solution;
or in a closed stirring tank, heterotrophically culturing haematococcus pluvialis, wherein the culture medium is prepared by adding 1g/L of sodium acetate on the basis of BG11 or BM culture medium, the culture temperature is controlled to be 25-30 ℃, the stirring speed is 50-150 rpm, and the haematococcus pluvialis is cultured until the end of logarithmic growth phase of the haematococcus pluvialis to obtain a heterotrophic haematococcus pluvialis culture solution;
2) accumulation of astaxanthin in Haematococcus pluvialis in the immotile growth stage
Diluting the haematococcus pluvialis culture solution in the swimming stage obtained in the step 1) by using sterilized/disinfected deionized water or a nitrogen-deficient culture medium (BG 11 or BM culture medium) as a diluting solution until the cell concentration is 0.1-0.5 g/L, adding ethanolamine until the final concentration is 50-400 mg/L, then starting induction culture, and giving illumination and mixing in the culture process, wherein the light intensity is 50-1000 mu mol/(m culture medium)2S) light quantum flux, controlling the culture temperature to be 25-35 ℃;
and after 7-14 days of culture, harvesting haematococcus pluvialis when the haematococcus pluvialis culture solution is changed from green to red and the color is not obviously changed under microscopic conditions.
Further, ethanolamine is added to a final concentration of 100-200 mg/L.
Further, in step 2), adding the sterilized MEA at one time or in batches; further, it is preferable that the sterilized MEA is sterilized by ultraviolet irradiation.
Further, in the culture process in the step 2), the illumination can be selected from artificial illumination and natural illumination; further preferably, the artificial lighting comprises using incandescent lamps and LED lamp bars.
Further, the light intensity is 150-300 [ mu ] mol/(m) in the culture process in the step 2)2S) light quantum flux.
Further, in the step 2), the method of mixing the culture solution is performed by introducing CO in a closed culture system2The culture solution is driven to mix by gas, and the culture medium is driven to mix by a stirring impeller in an open culture mode; preferably, CO is introduced2The gas volume concentration is 2-10%.
Experiments of examples 1-5 prove that when ethanolamine is added into the haematococcus pluvialis culture solution, the astaxanthin content is improved by 53-221% compared with that under normal culture conditions, the yield is improved by 52-206%, and the fact that the ethanolamine has a great application prospect in the field of astaxanthin production by using haematococcus pluvialis is fully demonstrated.
The invention has the following function mechanism and creative description:
glufosinate-ammonium induces the accumulation of astaxanthin, its mechanism is that inhibit glutaminase activity in the algal cell and then influence the growth of haematococcus pluvialis and synthesis of astaxanthin, patent CN104232720B adds ethanol and sea crystal exogenously and induces the method that haematococcus pluvialis produces astaxanthin, its mechanism of action lies in that it causes the osmotic pressure of water environment or pH value to change through adding exogenous chemical reagent, what changed finally is the external environment that the cell grows, start from the external environment and make the cell produce the hard spore wall to resist the external stress, thus turn into the stationary phase and accumulate astaxanthin; as for brassinolide, salicylic acid, jasmonic acid and gibberellin which belong to plant hormones, astaxanthin is accumulated by inhibiting cell division to induce the generation of spores, and the method is not particularly obvious in the improvement of the astaxanthin content produced by haematococcus pluvialis, the culture period is long, and in addition, the cost of the chemical reagents and hormones is far higher than that of ethanolamine.
Patent CN102492744B and related papers of the inventor disclose the use of ethanolamine in chlorella or scenedesmus, but the above technical proposal only utilizes CO of ethanolamine itself2Absorption characteristics by increasing CO2The fixation efficiency of the method enables the ethanolamine with a certain concentration to improve the concentration of the available carbon source in the culture solution, thereby increasing the carbon fixation efficiency of chlorella or scenedesmus and further influencing the biochemical composition and growth rate of chlorella or scenedesmus.
Ethanolamine is known to be used to rapidly absorb gases such as carbon dioxide, and the role of ethanolamine in the culture process of Haematococcus pluvialis has not been reported, but the inventors unexpectedly increased the astaxanthin content of Haematococcus pluvialis rapidly by adding ethanolamine to the culture broth at the motionless stage of the Haematococcus pluvialis culture. Unlike the exogenous inducer inducing the accumulation of astaxanthin and its mechanism of action reported in the above-mentioned document, the ethanolamine of the present invention is different from the above-mentioned chemical agents and hormones, and the inventors have studied the action of ethanolamine as a small molecular substance in the transition of different growth stages of Haematococcus pluvialis carefully and found that, unlike the mechanism of ethanolamine increasing the supply of carbon source to increase the growth rate of Chlorella vulgaris and Scenedesmus, the present invention inhibits photosynthesis of the cells of Haematococcus pluvialis by the addition of ethanolamine, and unlike the species of Chlorella vulgaris and Scenedesmus which do not accumulate astaxanthin, the inhibition rapidly develops Haematococcus pluvialis into chlamydospores and starts to accumulate astaxanthin.
Drawings
FIG. 1 is a graph showing the effect of ethanolamine in a closed photobioreactor on astaxanthin content in Haematococcus pluvialis.
FIG. 2 is a graph showing the effect of ethanolamine addition on astaxanthin content in Haematococcus pluvialis.
FIG. 3 is a graph showing the effect of ethanolamine in raceway ponds on astaxanthin content in Haematococcus pluvialis.
Detailed Description
Example 1
A columnar glass photobioreactor (with the height of 50 cm and the inner diameter of 6 cm) and BG11 culture medium are adopted to carry out autotrophic culture in a room to obtain the haematococcus pluvialis culture solution in a swimming stage. The algae is Haematococcus pluvialis, and is from freshwater algae seed bank of aquatic organisms of Chinese academy of sciences, and is numbered 712. The induction of astaxanthin was carried out in the same reactor and the culture broth was diluted with BG11 medium deficient in nitrogen so that the initial cell concentration during the induction phase was 0.2 g (dry weight)/L. When preparing the medium, MEA was added to a final concentration of 100 mg/L, and the medium was sterilized at 121 ℃ and 0.1MPa for 20 minutes, cooled and diluted. Aerating and mixing by using aeration stones (with the aperture of 30-60 mu m) commonly used in the field, wherein 8 LED light bars provide light with the intensity of 200 mu mol/(m)2S) white light illumination. Controlling the room temperature to be 25 ℃ by using an air conditioner, and continuously introducing CO for 24 h2The mixed gas drives the culture solution to mix, the flow rate of the mixed gas is 100 mL/min, and the composition is 2% of CO by volume content298% by volume of air. After 8 days of batch induction culture, harvesting by using a centrifugal mode under the conditions that the rotating speed is 5000rpm and the time is 5 min, and measuring the content of astaxanthin in the obtained algae powder.
Adding MEA with final concentration of 100 mg/L into Haematococcus pluvialis culture solution, and performing batch induction culture for 8 days to obtain astaxanthin content of 1.67% of cell dry weight; while the same reactor and the same culture conditions were used to culture the same microalgae, except that no MEA was added, 12 days were required for the cells to become completely red, and the astaxanthin content accounted for 0.52% of the cell dry weight. In comparison, the astaxanthin content increased by 221% after 100 mg/LMEA was added. As shown in fig. 1.
Adding MEA with final concentration of 100 mg/L into Haematococcus pluvialis culture solution, and performing induction culture in batch mode for 8 days to obtain cell concentration of 0.168 g (dry weight)/L; the same microalgae were cultured in the same reactor under the same culture conditions except that MEA was not added and the culture was carried out in batch for 12 days at a cell concentration of 0.175 g (dry weight)/L. In comparison, the astaxanthin yield is improved by 206% after 100 mg/LMEA is added.
Example 2
The same as example 1, except that MEA was added to the Haematococcus pluvialis culture solution to a final concentration of 200 mg/L.
Adding MEA with final concentration of 200mg/L into Haematococcus pluvialis culture solution, and performing batch induction culture for 8 days to obtain astaxanthin content of 0.85% of cell dry weight; while the same reactor and the same culture conditions were used to culture the same microalgae, except that no MEA was added, 12 days were required for the cells to become completely red, and the astaxanthin content accounted for 0.52% of the cell dry weight. In comparison, the astaxanthin content is increased by 63% after 200 mg/LMEA is added. As shown in fig. 1.
Adding MEA with final concentration of 200mg/L into Haematococcus pluvialis culture solution, and performing batch induction culture for 8 days with cell concentration of 0.170 g (dry weight)/L; the same microalgae were cultured in the same reactor under the same culture conditions except that MEA was not added and the culture was carried out in batch for 12 days at a cell concentration of 0.175 g (dry weight)/L. In comparison, the astaxanthin yield increased 59% after the addition of 200 mg/LMEA.
Example 3
The other examples are the same as example 1 except that the culture medium of Haematococcus pluvialis was derived from the induction stage and ethanolamine was added. Specifically, a closed cylindrical glass photobioreactor (with the height of 50 cm and the inner diameter of 6 cm) and BG11 culture medium with 1g/L sodium formate added are adopted indoors to carry out mixed culture to obtain a haematococcus pluvialis culture solution in a swimming stage; astaxanthin induction was carried out in the same reactor, the culture broth was diluted with BG11 medium deficient in nitrogen so that the initial cell concentration in the induction phase was 0.2 g (dry weight)/L, 50 mg/L of MEA was added to the culture broth to a final concentration of 100 mg/L on each of days 1 and 4 of the induction culture, and the added MEA solution was sterilized by UV sterilization.
Adding 50 mg/L twice to 100 mg/L MEA in Haematococcus pluvialis culture solution, and performing induction culture in batch mode for 8 days to obtain astaxanthin content of 1.48% of cell dry weight; the same microalgae were cultured in the same reactor under the same culture conditions, except that no MEA was added, and the batch culture was carried out for 8 days, and the astaxanthin content was 0.55% based on the dry weight of the cells. In comparison, the astaxanthin content increased 169% after two additions of 50 mg/L MEA to a final concentration of 100 mg/L. As shown in fig. 2.
Adding 50 mg/L twice to 100 mg/L MEA in Haematococcus pluvialis culture solution, and performing induction culture in batch for 8 days at cell concentration of 0.177 g (dry weight)/L; the same microalgae were cultured in the same reactor under the same culture conditions except that no MEA was added and the culture was performed in batch for 8 days, and the cell concentration was 0.180 g (dry weight)/L. In comparison, astaxanthin production was increased by 164% after two additions of 50 mg/L MEA to a final concentration of 100 mg/L.
Example 4
The other example was the same as example 3 except that ethanolamine was added. Specifically, astaxanthin induction was performed in a closed cylindrical glass photobioreactor (height 50 cm, inner diameter 6 cm), the culture broth was diluted with nitrogen-deficient BG11 medium so that the initial cell concentration at the induction stage was 0.2 g (dry weight)/L, 25 mg/L of MEA was added to a final concentration of 100 mg/L on each of days 1, 2, 4, 6, and 8 of induction culture, and the added MEA solution was sterilized by uv sterilization.
Adding 25 mg/L of MEA with final concentration of 100 mg/L into Haematococcus pluvialis culture solution four times, and performing batch induction culture for 8 days, wherein the astaxanthin content accounts for 0.84% of cell dry weight; the same microalgae were cultured in the same reactor under the same culture conditions, except that no MEA was added, and the batch culture was carried out for 8 days, and the astaxanthin content was 0.55% based on the dry weight of the cells. In comparison, the astaxanthin content increased by 53% after four additions of 25 mg/L to a final concentration of 100 mg/L MEA. As shown in fig. 2.
Adding 25 mg/L of MEA with final concentration of 100 mg/L into Haematococcus pluvialis culture solution four times, and performing induction culture in batch mode for 8 days at cell concentration of 0.180 g (dry weight)/L; the same microalgae were cultured in the same reactor under the same culture conditions except that no MEA was added and the culture was performed in batch for 8 days, and the cell concentration was 0.180 g (dry weight)/L. In comparison, astaxanthin production increased by 52% after four additions of 25 mg/L to a final concentration of 100 mg/L MEA.
Example 5
A sealed stirred tank reactor (the volume is 50L, the rotating speed is 36 r/min) and BG11 culture medium are adopted to carry out heterotrophic culture by adding 1g/L sodium acetate as the culture medium, and the haematococcus pluvialis culture solution in the swimming stage is obtained. The algae is Haematococcus pluvialis, and is from freshwater algae seed bank of aquatic organisms of Chinese academy of sciences, and is numbered 712. Astaxanthin induction is carried out by adopting a runway pool under the outdoor natural illumination condition, the length of the runway pool is 2.4 m, the diameter of a bend is 1.0 m, the height of the pool wall is 0.45 m, the culture depth is 10 cm, and the actual culture volume is 200L. The culture broth was diluted with deionized water so that the initial cell concentration during the induction phase was 0.2 g (dry weight)/L. Adding MEA into diluted water to final concentration of 100 mg/L, sterilizing by hypochlorous acid oxidation method, wherein the dosage of hypochlorous acid is 0.5 mL of hypochlorous acid/L culture medium, adding sodium thiosulfate after 12 h to neutralize until the color of the starch potassium iodide test paper is not changed. The illumination intensity is the outdoor actual light intensity, the culture temperature is controlled not to be higher than 30 ℃ by a method of spraying the side wall of the raceway pond in the culture process, and the power of the stirring paddle is adjusted, so that the flow rate of the culture solution is 20 cm/s. After 10 days of the induction culture in the batch, the algal powder was harvested by filtration, wherein the pore diameter of the hydrophobic membrane was 2 μm, and the astaxanthin content in the algal powder obtained was measured.
Adding MEA with final concentration of 100 mg/L into Haematococcus pluvialis culture solution, and performing batch induction culture for 10 days to obtain astaxanthin content of 1.19% of cell dry weight; while the same microalgae were cultured in the same reactor under the same culture conditions, except that no MEA was added, and the batch culture was carried out for 10 days, the astaxanthin content was 0.70% based on the dry weight of the cells. In comparison, the astaxanthin content is improved by 70 percent after 100 mg/LMEA is added. As shown in fig. 3.
Adding MEA with final concentration of 100 mg/L into Haematococcus pluvialis culture solution, and performing batch induction culture for 10 days with cell concentration of 0.152 g (dry weight)/L; the same microalgae were cultured in the same reactor under the same culture conditions except that no MEA was added and the culture was carried out in batch for 10 days, the cell concentration was 0.151 g (dry weight)/L. In comparison, the astaxanthin yield increased by 71% after 100 mg/LMEA addition.
In examples 1 to 5, the content of astaxanthin in Haematococcus pluvialis was determined according to the literature (Gunn, Zhang Huim, Yang Jian, etc.; quick determination method of astaxanthin content in Haematococcus pluvialis [ J]Food research and development, 2016, 37(12): 125-. The method specifically comprises the following steps: centrifuging 50mL of culture solution at 5000rpm for 3min, discarding supernatant, adding 5mL of aqueous solution containing 5% NaOH and 30% methanol, performing 70 deg.C water bath for 5 min, shaking frequently during heat preservation, centrifuging at 5000rpm for 3min, removing supernatant (chlorophyll is extracted into supernatant and destroyed by strong base), and collecting algae residue. 3mL of dimethyl sulfoxide containing a small amount of acetic acid (5 drops of 10 mL) is added into the centrifuge tube, the mixture is shaken up and kept at 70 ℃ for 5 min, and the centrifuge tube is shaken continuously during the heat preservation. Centrifuging at 5000rpm for 3min, transferring the supernatant into a 10 mL volumetric flask, and repeating for at least 3 times to make the algae residue at the bottom of the residual centrifuge tube colorless or light color. And (3) metering the volume of the supernatant collected for multiple times to 10 mL by using dimethyl sulfoxide, and measuring the absorbance A at the wavelength of 492nm after diluting by 10 times, wherein the dimethyl sulfoxide is used as a blank control. According to the formula C = (4.5 × A × V)a）/VbCalculating astaxanthin concentration, and calculating astaxanthin content and yield based on dry weight of algal cells, wherein VaIs the volume of dimethyl sulfoxide, VbIs the volume of the algae liquid, and A is the absorbance value at 492 nm.
Claims (6)
1. A method for increasing the astaxanthin content in Haematococcus pluvialis comprises the following steps:
1) obtaining a haematococcus pluvialis culture solution in a swimming growth stage:
autotrophic culturing haematococcus pluvialis in a closed photobioreactor, wherein the selected culture medium is BG11 culture medium, the culture temperature is controlled to be 25-30 ℃, and the light intensity is 100 mu mol/(m)2S), introducing sterile mixed gas, and culturing haematococcus pluvialis to the late logarithmic phase to obtain an autotrophic haematococcus pluvialis culture solution;
or polyculture culturing Haematococcus pluvialis in a closed photobioreactor, adding 1g/L sodium acetate based on selected BG11 culture medium, controlling the culture temperature at 25-30 deg.C and light intensity at 100 μmol/(m)2S), introducing sterile mixed gas, and culturing haematococcus pluvialis to the late logarithmic phase to obtain a polycultured haematococcus pluvialis culture solution;
or heterotrophic haematococcus pluvialis culture liquid is obtained by heterotrophic culture in a closed stirring tank, 1g/L sodium acetate is added on the basis of the selected BG11 culture medium, the culture temperature is controlled to be 25-30 ℃, the stirring speed is 50-150 rpm, and the culture is carried out until the end of logarithmic growth phase of the haematococcus pluvialis;
wherein, CO in the mixed gas2The volume concentration of the air is 2-10%, and the balance is air;
it is characterized by also comprising the following steps,
2) accumulation of astaxanthin in haematococcus pluvialis in the stationary growth phase:
using sterilized/disinfected deionized water or a nitrogen-deficient culture medium as a diluting solution, wherein the nitrogen-deficient culture medium is a nitrogen-deficient BG11 culture medium, diluting the swimming-stage haematococcus pluvialis culture solution obtained in the step 1) by using the diluting solution until the cell concentration is 0.1-0.5 g/L, adding sterilized/disinfected ethanolamine until the final concentration is 100-200 mg/L, starting induction culture, and providing illumination and mixing in the culture process, wherein the light intensity is 200 [ mu ] mol/(m ] m2S) light quantum flux, controlling the culture temperature to be 25-35 ℃,
after culturing for 7-14 days, harvesting haematococcus pluvialis when the haematococcus pluvialis culture solution is changed from green to red and the color is not obviously changed under microscopic conditions;
and the haematococcus pluvialis is from a freshwater algae seed bank of aquatic organisms institute of Chinese academy of sciences, and is numbered 712.
2. The method for increasing the astaxanthin content in Haematococcus pluvialis according to claim 1, wherein the sterilized ethanolamine is added in step 2) at one time or in batches.
3. The method according to claim 2, wherein the lighting in step 2) is selected from artificial lighting and natural lighting.
4. The method of claim 3, wherein the artificial lighting comprises incandescent lighting or LED lighting.
5. The method according to claim 2, wherein the mixing method of step 2) comprises: introducing CO under closed culture mode2Mixing the haematococcus pluvialis culture solution with gas, and mixing the haematococcus pluvialis culture solution with a stirring impeller in an open culture mode.
6. The method of claim 5, wherein the CO is introduced into the haematococcus pluvialis to increase the astaxanthin content of the haematococcus pluvialis2The volume concentration of the gas is 2-10%.
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| CN109294920A (en) * | 2018-10-19 | 2019-02-01 | 清华大学深圳研究生院 | A method of addition nano material induces haematococcus pluvialis efficient accumulation astaxanthin |
| CN109679853A (en) * | 2019-01-18 | 2019-04-26 | 昆明理工大学 | The method for improving haematococcus pluvialis biomass and astaxanthin yield using fulvic acid |
| CN109609385A (en) * | 2019-02-28 | 2019-04-12 | 福建康是美生物科技有限公司 | A kind of cultural method of haematococcus pluvialis |
| WO2021057711A1 (en) * | 2019-09-23 | 2021-04-01 | 山东拜昂生物技术有限公司 | Method for producing astaxanthin by heterotrophic culture of haematococcus pluvialis |
| CN111808755A (en) * | 2020-08-03 | 2020-10-23 | 中国科学院天津工业生物技术研究所 | Method for increasing microalgae cell density |
| CN113549555B (en) * | 2021-09-08 | 2024-07-09 | 攀枝花市格萨拉生物技术有限责任公司 | Method for producing natural astaxanthin by culturing haematococcus pluvialis |
| CN114480133A (en) * | 2022-01-25 | 2022-05-13 | 山西农业大学 | Culture method and application of haematococcus pluvialis |
| CN117470823A (en) * | 2023-11-16 | 2024-01-30 | 河北科技大学 | Method for detecting toxicity of glufosinate by fluorescence of microcystis aeruginosa |
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