CN106701585B - Culture method of haematococcus pluvialis - Google Patents
Culture method of haematococcus pluvialis Download PDFInfo
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- CN106701585B CN106701585B CN201510437152.2A CN201510437152A CN106701585B CN 106701585 B CN106701585 B CN 106701585B CN 201510437152 A CN201510437152 A CN 201510437152A CN 106701585 B CN106701585 B CN 106701585B
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Abstract
The invention relates to the field of microalgae biology, and discloses a method for culturing haematococcus pluvialis, which comprises the following steps: (1) culturing in the motile cell stage; (2) when the culture is carried out until the OD of the algae liquid is reached680When the value is 0.01-10, filtering and separating or performing gravity settling separation on at least part of the algae liquid to obtain a separation clear liquid I and motile cells, and returning the separation clear liquid I to the step (1) for recycling; (3) culturing the zooblast obtained in the step (2) in an immobile spore stage; (4) after the culture is completely turned to red, separating to obtain a separated clear liquid II and the immobile spores, and returning the separated clear liquid II to the step (3) for recycling; (5) and drying the acinetospore to obtain the haematococcus pluvialis product. The method for culturing haematococcus pluvialis has the advantages of stable operation in the culturing process, easy quality control, low culturing cost and no problems of waste liquid discharge and environmental pollution, and all culture solutions can be recycled in the culturing process.
Description
Technical Field
The invention relates to the field of microalgae biology, in particular to a method for culturing haematococcus pluvialis.
Background
Haematococcus pluvialis (Haematococcus pluvialis) is a unicellular green alga, the proliferation of which depends on the division of cells. When the environment is proper, the biomass of the haematococcus increases continuously, and belongs to the stage of green swimming cells; when the algae is stressed by the outside, such as illumination, temperature, pH value change and the like, spores are formed, oil and astaxanthin are accumulated, algae cells turn red and belong to the stage of the acinetospore.
The haematococcus pluvialis is rich in oil and astaxanthin, the oil content can account for 30-40% of the dry weight of cells, and the haematococcus pluvialis can be used as a potential source of biological energy; astaxanthin content can be 1.5-3.0% of the dry cell weight, and is the most important source of natural astaxanthin.
However, the existing large-scale culture and production of haematococcus pluvialis still has great difficulties, and the main reasons include: (1) the haematococcus pluvialis has low yield per unit area, is easy to be polluted by microorganisms, and has unstable contents of oil and fat and astaxanthin; (2) the haematococcus pluvialis has different growth conditions in the green swimming cell stage and the growth stage of the acinetospore, and the production cost is high.
CN102766578A discloses a method for culturing and producing haematococcus pluvialis, and specifically discloses: in a sealed tank body, the haematococcus pluvialis green swimming cells are inoculated into a culture medium, and the artificial LED light source is adopted for irradiation culture, so that the culture period can be shortened, and the concentration of the haematococcus pluvialis cells in the algae liquid can be increased. However, the process of Haematococcus conversion to acinetospores, astaxanthin accumulation and oil and fat accumulation is not involved.
CN102827766A discloses a photobioreactor for large-scale haematococcus pluvialis cultivation to produce astaxanthin, but the photobioreactor is only used in a haematococcus pluvialis 'red transfer stage' and does not comprise a haematococcus pluvialis green swimming cell growth cultivation stage.
At present, haematococcus pluvialis is generally cultivated by a one-step method at home, namely a green swimming cell stage and a fixed spore stage are carried out in the same reactor, the main purpose is to obtain high-content astaxanthin, the production of oil is rarely related, and the biggest problem of cultivation in the same reactor is that collected culture solution cannot be recycled and is discharged as waste water, so that the cultivation cost is high, the resource is wasted and the environment is polluted. For example, CN1966660A discloses an apparatus and a method for large-scale cultivation of astaxanthin by haematococcus pluvialis, wherein the whole cultivation apparatus is composed of a photobioreactor system, an aeration device, a culture solution infusion device and a static cell collection device. The growth and division of the haematococcus pluvialis are basically synchronized by the mutual flowing of culture solutions and continuous aeration; the floating dust control device arranged on the fixing frame and the natural water body are utilized to carry out large-scale haematococcus culture, so that the photobioreactor system is lowered to the required water layer depth, and the contradiction of different temperature, illumination and nutrition conditions in two stages of haematococcus culture and astaxanthin conversion is solved.
CN1680539A discloses an airlift photobioreactor for high-density culture of haematococcus pluvialis, which comprises a culture tank, a water treatment device, a lighting device and a gas supply device, and the cultivation of the haematococcus pluvialis and the accumulation of astaxanthin are completed in the same reactor mainly by controlling conditions such as illumination intensity and the like.
CN1392244A discloses a method for producing astaxanthin by cultivating haematococcus, which comprises a culture medium formula, a one-step production process, the recycling of the culture medium and a method for inducing the formation of haematococcus spores and the accumulation of astaxanthin by adjusting the pH value with carbon dioxide.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a haematococcus pluvialis culture method, wherein the swimming cell stage culture and the immobile spore stage culture are carried out in different reactors, the culture process is stable in operation and easy to control quality, all culture solution can be recycled in the culture process, the culture cost is low, and the problems of waste liquid discharge and environmental pollution are avoided.
In the existing method for culturing haematococcus pluvialis, a swimming cell stage and a fixed spore stage are carried out in the same reactor, and the haematococcus pluvialis swimming cells are not separated, which is mainly caused by that: in consideration of the morphology and survival characteristics of Haematococcus pluvialis motile cells, the skilled person generally considers that the Haematococcus pluvialis in the motile cell stage cannot be separated at all, and once the Haematococcus pluvialis in the motile cell stage is separated, the Haematococcus pluvialis motile cells are extremely likely to be injured and even influence the survival of the Haematococcus pluvialis motile cells.
Therefore, in order to achieve the above object, the present invention provides a method for cultivating haematococcus pluvialis, comprising:
(1) culturing haematococcus pluvialis in a swimming cell stage;
(2) when the culture is carried out until the OD of the algae liquid is reached680When the value is 0.01-10, separating at least part of algae liquid to obtain a separation clear liquid I and haematococcus pluvialis swimming cells, and returning the separation clear liquid I to the step (1) for recycling;
(3) culturing the zooblast cells of haematococcus pluvialis obtained in the step (2) at an acinetospore stage;
(4) after the culture is completely red, separating to obtain a separated clear liquid II and haematococcus pluvialis acinetospore, and returning the separated clear liquid II to the step (3) for recycling;
(5) drying the haematococcus pluvialis acinetospore to obtain a haematococcus pluvialis product;
in the step (2), the separation mode is filtration separation or gravity settling separation.
The method can realize the continuous culture of the haematococcus pluvialis motile cells and further realize the semi-continuous culture of the haematococcus pluvialis acinetospore, the culture process is stable in operation and easy in quality control, all culture solution can be recycled in the culture process, the culture cost is low, and the problems of waste liquid discharge and environmental pollution cannot be caused.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The invention provides a method for culturing haematococcus pluvialis, which comprises the following steps:
(1) culturing haematococcus pluvialis in a swimming cell stage;
(2) when the culture is carried out until the OD of the algae liquid is reached680When the value is 0.01-10, separating at least part of algae liquid to obtain a separation clear liquid I and haematococcus pluvialis swimming cells, and returning the separation clear liquid I to the step (1) for recycling;
(3) culturing the zooblast cells of haematococcus pluvialis obtained in the step (2) at an acinetospore stage;
(4) after the culture is completely red, separating to obtain a separated clear liquid II and haematococcus pluvialis acinetospore, and returning the separated clear liquid II to the step (3) for recycling;
(5) drying the haematococcus pluvialis acinetospore to obtain a haematococcus pluvialis product;
in the step (2), the separation mode is filtration separation or gravity settling separation.
In the method of the present invention, preferably, the step (1) is performed in a swimming cell culture vessel, which is an open photobioreactor or a closed photobioreactor. The open photobioreactor and the closed photobioreactor are not particularly limited, and may be various open photobioreactors and closed photobioreactors commonly used in the art, respectively. For example, the open photobioreactor may be a racetrack photobioreactor or a box-type photobioreactor; the material of the open type photobioreactor can be metal, cement, inorganic glass, organic glass or plastic, and the plastic can comprise polyethylene, polypropylene, polyvinyl chloride, polycarbonate and the like. Preferably, the open photobioreactor is a transparent polypropylene tank or cement pond. The closed type photobioreactor can be a bubbling type photobioreactor, an airlift type photobioreactor, a stirring type photobioreactor and the like; the closed photobioreactor can be made of inorganic glass, organic glass or transparent plastic, and the transparent plastic can comprise polyethylene, polypropylene, polyvinyl chloride, polycarbonate and the like. Preferably, the closed photobioreactor is a self-cleaning bubble-type polyvinyl chloride photobioreactor.
In the method of the present invention, the Haematococcus pluvialis strain and the culture solution are not particularly limited, and may be various Haematococcus pluvialis strains and culture solutions commonly used in the art, which are well known to those skilled in the art and will not be described herein again.
In the method of the present invention, it will be understood by those skilled in the art that the culture temperature, the light intensity and the pH of the culture solution are not always controlled to an accurate point value but fluctuate within a range during the actual cultivation of Haematococcus pluvialis. Preferably, in step (1), the culture conditions include: the temperature is 15-25 deg.C, the illumination intensity is 1000-5000Lux, and the pH value is 6-8. Wherein the culture conditions further comprise: the carbon source introduced during the culture is a carbon dioxide-containing gas, and the carbon dioxide content in the gas is 1 to 20% by weight, preferably 2 to 10% by weight, and more preferably 3 to 8% by weight. When light is irradiated, the light source may be natural light, or may be an artificial light source (such as an LED light source), and is preferably natural light. The carbon dioxide containing gas may be high purity carbon dioxide or carbon dioxide containing gas emitted from an industrial process, which may include carbon dioxide containing gas emitted from power plants and refineries. Preferably, the carbon dioxide containing gas is high purity carbon dioxide or refinery hydrogen production tail gas. It will be appreciated by those skilled in the art that the carbon dioxide containing gas is cleaned of solid particles and cooled before entering the swimming cell culture device.
In the process of the invention, preference is given toIn the step (2), the culture is carried out until the OD of the algal solution is reached680The value is 0.05 to 5, more preferably 0.1 to 1.
In the method of the present invention, it is preferable that in the step (2), at least a part of the algal solution accounts for 20 to 50% by volume of the whole algal solution.
In the method of the present invention, preferably, in the step (2), at least a part of the algae liquid is transferred to a separation and storage device for separation. The transfer method is not particularly limited, and various transfer methods commonly used in the art may be used. The transfer method is preferably gravity transfer, overflow transfer, or siphon transfer, and more preferably overflow transfer.
In the method of the present invention, preferably, in the step (2), the separation method is filtration separation. The filtration and separation method may be normal pressure filtration and separation, and vacuum filtration and separation, and is more preferably normal pressure filtration and separation.
In the method of the present invention, preferably, the separation and storage and transportation device is a cylindrical separation empty column, the height-to-diameter ratio of the cylindrical separation empty column is 1 to 10:1, and more preferably 1.5 to 5:1, and the bottom of the cylindrical separation empty column is provided with a filter medium. The filter medium is not particularly limited, and may be various filter media commonly used in the art, and preferably, the filter medium is glass sand, filter cloth, or filter paper, and more preferably, glass sand; the pore size of the filter medium is 1 to 60 μm, and more preferably 10 to 30 μm.
In the process of the present invention, the cylindrical separation hollow column is preferably provided with a stirrer, further preferably the stirrer is an anchor stirrer, a gate stirrer or a ribbon stirrer, still further preferably a gate stirrer.
In the method of the present invention, it should be understood by those skilled in the art that the number of the separation and storage devices depends on the volume of the zoospore culture device and the immobile spore culture device, and may be 1 or more, and may be operated in parallel.
In the method of the present invention, preferably, the method of returning the separated clear solution I to the step (1) for reuse comprises: the centrate I is mixed with make-up (fresh) culture broth in a media tank and returned to the motile cell culture.
In the method of the present invention, preferably, the step (3) is performed in an acinetospore culture device, wherein the acinetospore culture device is an open photobioreactor or a closed photobioreactor, preferably an open photobioreactor. The open photobioreactor and the closed photobioreactor are not particularly limited, and may be various open photobioreactors and closed photobioreactors commonly used in the art, respectively. For example, the open photobioreactor may be a racetrack photobioreactor or a box-type photobioreactor; the material of the open type photobioreactor can be metal, cement, inorganic glass, organic glass or plastic, and the plastic can comprise polyethylene, polypropylene, polyvinyl chloride, polycarbonate and the like. Preferably, the open photobioreactor is a transparent polypropylene tank or cement pond. The closed type photobioreactor can be a bubbling type photobioreactor, an airlift type photobioreactor, a stirring type photobioreactor and the like; the closed photobioreactor can be made of inorganic glass, organic glass or transparent plastic, and the transparent plastic can comprise polyethylene, polypropylene, polyvinyl chloride, polycarbonate and the like.
In the method of the present invention, preferably, in the step (3), the haematococcus pluvialis motile cells and the separated clear solution II are mixed in a separation and storage device and then transferred to an acinetospore culture device for culturing in an acinetospore stage. Further preferably, the means of transfer into the acinetospore culture is siphon transfer or vacuum transfer, even more preferably vacuum transfer.
In the method of the present invention, the manner in which the stressed haematococcus pluvialis accumulates lipids and astaxanthin is not particularly limited, and various manners commonly used in the art may be used. For example, in step (3), the culture mode may be temperature stress culture, light stress culture or stress agent stress culture. Preferably, the culture mode is stress culture of a stress agent. Further preferably, the conditions under which the stress agent stresses the culture include: the pH value is 7-9, the temperature is 24-30 ℃, the illumination intensity is 5000-15000Lux, the stress agent is sodium salt, and the addition amount of the stress agent is 0.005-0.05mol per liter of the culture solution, more preferably 0.02-0.05mol per liter of the mixed algae solution; still more preferably, the stress agent is sodium bicarbonate.
In the method of the present invention, it will be understood by those skilled in the art that the number of the acinetospore cultivators should match the growth cycle, the harvest quantity, the growth cycle of the haematococcus pluvialis accumulated oil and astaxanthin in the zoospore cultivators, and may be 1, or a plurality of parallel operations, preferably 2-5.
In the method of the present invention, preferably, in the step (3), after the transfer is finished, the separation and storage device is washed with deionized water to be neutral for the next separation and transfer, and the washing liquid is stored for standby. For example, the cleaning solution can be sent to a cleaning solution tank for storage.
In the method of the present invention, preferably, the cleaning solution is recycled to the step (3), and further preferably, the recycling manner includes: mixing the cleaning solution and haematococcus pluvialis motile cells in a separation and storage and transportation device, and transferring the mixture to an acinetospore culture device for culturing in an acinetospore stage; or the recycling mode comprises the following steps: in the culture at the acinetospore stage, the wash was transferred to an acinetospore incubator for water replenishment.
In the method of the present invention, it will be understood by those skilled in the art that, in the step (4), the reddening state of the acinetospore can be observed by an optical microscope, and the separation is performed when the acinetospore turns red from the periphery to the center.
In the method of the present invention, the separation method in step (4) is preferably centrifugal separation, filtration separation or gravity settling separation, and more preferably gravity settling separation.
In the method of the present invention, preferably, the method of returning the separated clear liquid II to the step (3) for reuse comprises: and conveying the separated clear liquid II to a separation and storage and transportation device through a pump, and uniformly mixing the separated clear liquid II with the separated haematococcus pluvialis swimming cells. Further preferably, the separated clear liquid II is delivered by a centrifugal pump. As will be appreciated by those skilled in the art, to facilitate recycling of the separated clear liquid II, the separated clear liquid II may be stored in a clear liquid tank for later use.
In the method of the invention, in the step (5), haematococcus pluvialis acinetospore can be firstly sent to a concentration tank and then dried. The drying method is not particularly limited, and various drying methods commonly used in the art may be used, and the drying method is preferably natural drying, vacuum drying or spray drying, and more preferably cyclone spray drying.
Examples
The present invention will be described in detail below by way of examples, but the present invention is not limited thereto.
Haematococcus pluvialis was purchased from the institute of Wuhan aquatic organisms, Chinese academy of sciences.
Measuring the optical density value (OD value) of the algae liquid: the optical density value is measured by a spectrophotometer, and the absorbance value of the algae liquid at the wavelength of 680nm is measured by using distilled water as a contrast and is used as an index of the concentration of the algae liquid.
BG11 medium with the composition shown in Table 1-2 was used in the culture, and NaNO was added to BG11 medium3Is a nitrogen source.
TABLE 1BG11 culture Medium
| Components | Content, mg/L |
| K2HPO4·3H2O | 40 |
| NaNO3 | 1500 |
| Na2CO3 | 20 |
| MgSO4·7H2O | 75 |
| CaCl2·2H2O | 36 |
| Citric acid | 6 |
| Ferric ammonium citrate | 6 |
| EDTA-2Na | 1 |
| Microelement A5 | 1 |
TABLE 2 Trace element A5
| Components | Composition of mg/L |
| H3BO3 | 2860 |
| MnCl2·4H2O | 1810 |
| ZnSO4·7H2O | 222 |
| CuSO4·5H2O | 79 |
| NaMoO4·5H2O | 390 |
| Co(NO3)2·6H2O | 50 |
The refinery hydrogen production tail gas is the refinery hydrogen production tail gas of China petrochemical company Shijiazhuang refining chemical division company, the content of carbon dioxide is 20-25 wt%, and solid particles are purified and removed and cooled before use. Cooling to 25 deg.C, mixing with air to obtain mixed gas with carbon dioxide content of 6 wt%, and controlling pH value by controlling air intake flow of the mixed gas during Haematococcus pluvialis culture.
The closed type photobioreactor is a polyvinyl chloride film bag, the diameter is 220mm, the height is 1500mm, the volume is 35L, and an inner guide cylinder is not arranged.
The open type photobioreactor is a transparent polypropylene box body, the volume is 20L, and the light receiving area is 0.095m2。
Culture rate (g/m) of haematococcus pluvialis2And/d) is the dry weight of haematococcus pluvialis/the light receiving area of the haematococcus pluvialis swimming cell culture stage/the culture time of the haematococcus pluvialis swimming cell culture stage.
The separation and storage device is a 30L cylindrical organic glass separation empty tower with a glass sand filter medium at the bottom, the height-diameter ratio is 2:1, the frame type stirring is carried out, the conveying pipe is inserted into the bottom from the top and is connected with the immobile spore culture device, the diameter of the conveying pipe is 10mm, the specification of the glass sand is G3, and the pore diameter is 15-30 mu m.
Example 1
This example illustrates a method for cultivating Haematococcus pluvialis using a closed photobioreactor.
In this embodiment, both the zoophocyte culture device and the immobile spore culture device are closed photo-bioreactors.
(1) First-stage culture of Haematococcus pluvialis
Inoculating Haematococcus pluvialis into 2000mL triangular flask containing 1000mL BG11 culture medium, inoculating the OD of culture solution after inoculating Haematococcus pluvialis680The value was 0.015. Culturing the algae solution at 20-22 deg.C and 1800-2200Lux with pH of 6-7 to OD of 0.3. The pH value is adjusted by high-purity carbon dioxide in the culture process.
(2) The culture of haematococcus pluvialis swimming cell stage was carried out in 1 closed photobioreactor: sterilizing the closed photobioreactor, adding 28L BG11 culture medium and primarily cultured Haematococcus pluvialis strain, and adding initial OD of culture solution680The value was 0.08. The mixed gas enters from the bottom of the closed type photobioreactor through the dispersion of the air stone in a bubbling mode, and stirring and mixing are generated, wherein the gas inlet flow rate is 200L/h. Culturing at 20-24 deg.C, illumination intensity of 1800-5000Lux, and pH value of 6-8, wherein the pH value is controlled by gas inlet flow of mixed gas.
(3) Separating and transferring haematococcus pluvialis swimming cells: OD of culture solution after 7 days of culture680And (3) when the value reaches 0.3, transferring 8.4L of haematococcus pluvialis algae liquid from the closed photobioreactor in the step (2) to a separation, storage and transportation device in an overflow transfer mode. And (3) filtering the mixture by using glass sand to obtain a separated clear liquid I and haematococcus pluvialis swimming cells, mixing the separated clear liquid I with a supplemented culture medium in a culture medium tank, pumping the mixture into the closed photobioreactor in the step (2) by using a pump to ensure that the volume of the culture liquid in the closed photobioreactor is 28L, and continuing culturing the haematococcus pluvialis swimming cells. Pumping 8.4L liquid from clear liquid tank into separation and storage device, and filtering to obtain Haematococcus pluvialis swimming liquidAnd (4) mixing the cells. After being mixed evenly, the mixture is vacuumized, and the mixed liquid enters the immobile spore culturing device through a conveying pipe.
(4) And (3) washing of a separation and storage and transportation device: and after the vacuum transferring is finished, adding deionized water to wash the separation and storage and transportation device to be neutral, and feeding the cleaning liquid into a clear liquid tank for later use.
(5) Haematococcus pluvialis acinetospore conversion, accumulation of lipids and astaxanthin were carried out in 2 parallel acinetospore cultivators, i.e. closed photobioreactors: and (3) after the mixed solution is transferred to a 1 st closed photobioreactor in vacuum, adding 0.02mol of stress agent sodium bicarbonate in terms of per liter of mixed algal solution to perform conversion of the acinetospore and accumulation of the oil and fat and the astaxanthin. The culture temperature is 28-32 deg.C, the illumination intensity is 5000-15000Lux, and the pH value is 7-9. The mixed gas enters from the bottom of the closed type photobioreactor through the dispersion of the air stone in a bubbling mode, stirring and mixing are carried out, the air input of the mixed gas is 60L/h, and the pH value is controlled through the air input flow of the mixed gas.
Observing under an optical microscope after culturing for 10 days, wherein the acinetospore turns red from the periphery to the center, stopping air intake, standing for separation to obtain a separated clear liquid II and haematococcus pluvialis acinetospore, allowing the separated clear liquid II to enter a clear liquid tank, allowing the haematococcus pluvialis acinetospore to enter a concentration tank, and performing cyclone spray drying to obtain haematococcus pluvialis algae powder.
OD of culture solution in the closed photobioreactor for haematococcus pluvialis swimming cell stage culture in step (3)680When the value reaches 0.3, the step (3) is repeated to transfer the mixed solution to the 2 nd immovable spore culture device, namely the 2 nd closed type photobioreactor. And circulating in sequence.
The culture speed of the haematococcus pluvialis is 3g/m according to the total volume of the culture solution in the swimming cell culture stage of the haematococcus pluvialis2The content of oil and fat in the obtained haematococcus pluvialis is 36 percent, and the content of astaxanthin is 1.5 percent.
Example 2
This example illustrates a method for cultivating Haematococcus pluvialis using an open photobioreactor.
In this embodiment, the zoophocyte culture device and the immobile spore culture device are both open photobioreactors.
(1) First-stage culture of Haematococcus pluvialis
Inoculating Haematococcus pluvialis into 2000mL triangular flask containing 1000mL BG11 culture medium, inoculating the OD of culture solution after inoculating Haematococcus pluvialis680The value was 0.015. Culturing the algae solution at 20-22 deg.C and 1800-2200Lux with pH of 6-7 to OD of 0.3. The pH value is adjusted by high-purity carbon dioxide in the culture process.
(2) The culture of haematococcus pluvialis swimming cell stage was carried out in 1 open photobioreactor: adding 16L BG11 culture medium and Haematococcus pluvialis strain of primary culture into open photobioreactor, and collecting the initial OD of culture solution680The value was 0.03. The mixed gas enters the photobioreactor in a bubbling mode through the dispersion of the air stone, and is stirred and mixed, and the gas inflow rate is 80L/h. Culturing at 22-25 deg.C, illumination intensity of 1800-5000Lux, and pH value of 6-8, wherein the pH value is controlled by gas inlet flow of mixed gas.
(3) Separating and transferring haematococcus pluvialis swimming cells: OD of culture solution after 8 days of culture680And (3) when the value reaches 0.28, transferring 4.8L of haematococcus pluvialis algae liquid from the open type photobioreactor in the step (2) to a separation, storage and transportation device in an overflow transfer mode. And (3) filtering the mixture by using glass sand to obtain a separated clear liquid I and haematococcus pluvialis swimming cells, mixing the separated clear liquid I with a supplemented culture medium in a culture medium tank, pumping the mixture into the open photobioreactor in the step (2) by using a pump to enable the volume of the culture liquid in the open photobioreactor to be 16L, and continuing culturing the haematococcus pluvialis swimming cells. 4.8L of liquid is pumped into a separation and storage device from a clear liquid tank by a pump and is mixed with the haematococcus pluvialis swimming cells obtained by filtering. After being mixed evenly, the mixed solution enters the immobile spore culturing device through the siphon of the conveying pipe.
(4) And (3) washing of a separation and storage and transportation device: after the siphon transfer is finished, deionized water is added to wash the separation and storage and transportation device to be neutral, and the cleaning liquid enters a clear liquid tank for later use.
(5) Haematococcus pluvialis acinetospore transformation, accumulation of oil and fat and astaxanthin were carried out in 2 parallel acinetospore cultivators, open photobioreactors: after the mixed solution is siphoned and transferred to the 1 st open type photobioreactor, 0.03mol of stress agent sodium bicarbonate is added into each liter of the mixed algae solution for conversion of the acinetospore and accumulation of the oil and fat and the astaxanthin. The culture temperature is 28-32 deg.C, the illumination intensity is 5000-15000Lux, and the pH value is 7-9. The mixed gas enters the open type photobioreactor in a bubbling mode through the dispersion of the air stone, stirring and mixing are carried out, the air input of the mixed gas is 40L/h, and the pH value is controlled through the air input flow of the mixed gas.
Observing under an optical microscope after culturing for 10 days, wherein the acinetospore turns red from the periphery to the center, stopping air intake, standing and separating to obtain a separated clear liquid II and haematococcus pluvialis acinetospore, allowing the separated clear liquid II to enter a clear liquid tank, allowing the haematococcus pluvialis acinetospore to enter a concentration tank, and performing vacuum drying to obtain haematococcus pluvialis algae powder.
OD of culture solution in open photobioreactor for haematococcus pluvialis swimming cell stage culture in step (3)680When the value reaches 0.28, the step (3) is repeated to transfer the mixed solution to the 2 nd immovable spore culture device, namely the open photobioreactor. And circulating in sequence.
The culture speed of the haematococcus pluvialis is 2.7g/m according to the total volume of the culture solution in the swimming cell culture stage of the haematococcus pluvialis2The content of oil and fat in the obtained haematococcus pluvialis is 35 percent, and the content of astaxanthin in the haematococcus pluvialis is 1.6 percent.
Example 3
This example illustrates a method for cultivating Haematococcus pluvialis using a composite photobioreactor.
In this embodiment, the swimming cell culture device is a closed photobioreactor, and the immobile spore culture device is an open photobioreactor.
(1) First-stage culture of Haematococcus pluvialis
Inoculating Haematococcus pluvialis into 2000mL triangular flask containing 1000mL BG11 culture medium, inoculating the OD of culture solution after inoculating Haematococcus pluvialis680The value was 0.015. Culturing the algae solution at 20-22 deg.C and 1800-2200Lux with pH of 6-7 to OD of 0.3.The pH value is adjusted by high-purity carbon dioxide in the culture process.
(2) The culture of haematococcus pluvialis swimming cell stage was carried out in 1 closed photobioreactor: adding 28L BG11 culture medium and Haematococcus pluvialis strain of primary culture into a closed photobioreactor, and collecting the initial OD of the culture solution680The value was 0.075. The mixed gas enters the photobioreactor in a bubbling mode through the dispersion of the air stone, and is stirred and mixed, and the gas inflow rate is 200L/h. Culturing at 20-24 deg.C, illumination intensity of 1800-5000Lux, and pH value of 6-8, wherein the pH value is controlled by gas inlet flow of mixed gas.
(3) Separating and transferring haematococcus pluvialis swimming cells: OD of culture solution after 7 days of culture680And (3) when the value reaches 0.31, transferring 8.4L of haematococcus pluvialis algae liquid from the closed photobioreactor in the step (2) to a separation, storage and transportation device in an overflow transfer mode. And (3) filtering the mixture by using glass sand to obtain a separated clear liquid I and haematococcus pluvialis swimming cells, mixing the separated clear liquid I with a supplemented culture medium in a culture medium tank, pumping the mixture into the closed photobioreactor in the step (2) by using a pump to ensure that the volume of the culture liquid in the closed photobioreactor is 28L, and continuing culturing the haematococcus pluvialis swimming cells. 8.4L of liquid is pumped into a separation and storage device from a clear liquid tank by a pump and is mixed with the haematococcus pluvialis swimming cells obtained by filtering. After being mixed evenly, the mixed solution enters the immobile spore culturing device through the siphon of the conveying pipe.
(4) And (3) washing of a separation and storage and transportation device: after the siphon transfer is finished, deionized water is added to wash the separation and storage and transportation device to be neutral, and the cleaning liquid enters a clear liquid tank for later use.
(5) Haematococcus pluvialis acinetospore transformation, accumulation of oil and fat and astaxanthin were carried out in 2 parallel acinetospore cultivators, open photobioreactors: after the mixed solution is siphoned and transferred to the 1 st open type photobioreactor, 0.05mol of stress agent sodium bicarbonate is added into each liter of the mixed algae solution for conversion of the acinetospore and accumulation of the oil and fat and the astaxanthin. The culture temperature is 28-32 deg.C, the illumination intensity is 5000-15000Lux, and the pH value is 7-9. The mixed gas enters the photobioreactor in a bubbling mode through the dispersion of the air stone, and is stirred and mixed, the air input of the mixed gas is 40L/h, and the pH value is controlled through the air input flow of the mixed gas.
Observing under an optical microscope after culturing for 10 days, wherein the acinetospore turns red from the periphery to the center, stopping air intake, standing for separation to obtain a separated clear liquid II and haematococcus pluvialis acinetospore, allowing the separated clear liquid II to enter a clear liquid tank, allowing the haematococcus pluvialis acinetospore to enter a concentration tank, and performing cyclone spray drying to obtain haematococcus pluvialis algae powder.
OD of culture solution in the closed photobioreactor for haematococcus pluvialis swimming cell stage culture in step (3)680When the value reaches 0.31, the step (3) is repeated to transfer the mixed solution to the 2 nd immovable spore culture device, namely the open photobioreactor. And circulating in sequence.
The culture speed of the haematococcus pluvialis is 3.1g/m according to the total volume of the culture solution in the swimming cell culture stage of the haematococcus pluvialis2The content of oil and fat in the obtained haematococcus pluvialis is 39 percent, and the content of astaxanthin in the haematococcus pluvialis is 1.5 percent.
Example 4
This example illustrates a method for cultivating Haematococcus pluvialis using a composite photobioreactor.
In this embodiment, the swimming cell culture device is an open photobioreactor, and the immobile spore culture device is a closed photobioreactor.
(1) First-stage culture of Haematococcus pluvialis
Inoculating Haematococcus pluvialis into 2000mL triangular flask containing 1000mL BG11 culture medium, inoculating the OD of culture solution after inoculating Haematococcus pluvialis680The value was 0.015. Culturing the algae solution at 20-22 deg.C and 1800-2200Lux with pH of 6-7 to OD of 0.3. The pH value is adjusted by high-purity carbon dioxide in the culture process.
(2) The culture of haematococcus pluvialis swimming cell stage was carried out in 1 open photobioreactor: adding 16L BG11 culture medium and Haematococcus pluvialis strain of primary culture into open photobioreactor, and collecting the initial OD of culture solution680The value was 0.025. The mixed gas enters the photobioreactor in a bubbling mode through the dispersion of the air stone and is generatedStirring and mixing are carried out, and the air inlet flow is 80L/h. Culturing at 22-25 deg.C, illumination intensity of 1800-5000Lux, and pH value of 6-8, wherein the pH value is controlled by gas inlet flow of mixed gas.
(3) Separating and transferring haematococcus pluvialis swimming cells: OD of culture solution after 7 days of culture680And (3) when the value reaches 0.29, transferring 4.8L of haematococcus pluvialis algae liquid from the open type photobioreactor in the step (2) to a separation, storage and transportation device in an overflow transfer mode. And (3) filtering the mixture by using glass sand to obtain a separated clear liquid I and haematococcus pluvialis swimming cells, mixing the separated clear liquid I with a supplemented culture medium in a culture medium tank, pumping the mixture into the open photobioreactor in the step (2) by using a pump to enable the volume of the culture liquid in the open photobioreactor to be 16L, and continuing culturing the haematococcus pluvialis swimming cells. 4.8L of liquid is pumped into a separation and storage device from a clear liquid tank by a pump and is mixed with the haematococcus pluvialis swimming cells obtained by filtering. After being mixed evenly, the mixed solution enters the immobile spore culturing device through the conveying pipe.
(4) And (3) washing of a separation and storage and transportation device: and after the vacuum transferring is finished, adding deionized water to wash the separation and storage and transportation device to be neutral, and feeding the cleaning liquid into a clear liquid tank for later use.
(5) Haematococcus pluvialis acinetospore conversion, accumulation of lipids and astaxanthin were carried out in 2 parallel acinetospore cultivators, i.e. closed photobioreactors: and (3) after the mixed solution is transferred to a 1 st closed photobioreactor in vacuum, adding 0.02mol of stress agent sodium bicarbonate in terms of per liter of mixed algal solution to perform conversion of the acinetospore and accumulation of the oil and fat and the astaxanthin. The culture temperature is 28-32 deg.C, the illumination intensity is 5000-15000Lux, and the pH value is 7-9. The mixed gas enters from the bottom of the closed type photobioreactor through the dispersion of the air stone in a bubbling mode, stirring and mixing are carried out, the air input of the mixed gas is 60L/h, and the pH value is controlled through the air input flow of the mixed gas.
Observing under an optical microscope after culturing for 10 days, wherein the acinetospore turns red from the periphery to the center, stopping air intake, standing and separating to obtain a separated clear liquid II and haematococcus pluvialis acinetospore, allowing the separated clear liquid II to enter a clear liquid tank, allowing the haematococcus pluvialis acinetospore to enter a concentration tank, and performing vacuum drying to obtain haematococcus pluvialis algae powder.
OD of culture solution in open photobioreactor for haematococcus pluvialis swimming cell stage culture in step (3)680When the value reaches 0.29, the step (3) is repeated to transfer the mixed solution to the 2 nd immovable spore culture device, namely the closed photobioreactor. And circulating in sequence.
The culture speed of the haematococcus pluvialis is 2.6g/m according to the total volume of the culture solution in the swimming cell culture stage of the haematococcus pluvialis2The content of oil and fat in the obtained haematococcus pluvialis is 38 percent, and the content of astaxanthin in the haematococcus pluvialis is 1.8 percent.
According to the method for culturing haematococcus pluvialis, the culture in the swimming cell stage and the culture in the immobile spore stage are carried out in different reactors, the culture process is stable in operation and easy to control, all culture solution can be recycled in the culture process, the culture cost is low, and the problems of waste liquid discharge and environmental pollution are avoided.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (29)
1. A method for cultivating haematococcus pluvialis, which comprises the following steps:
(1) culturing haematococcus pluvialis in a swimming cell stage;
(2) when cultivatingRaising to OD of algae liquid680When the value is 0.05-5, separating at least part of algae liquid to obtain a separation clear liquid I and haematococcus pluvialis swimming cells, and returning the separation clear liquid I to the step (1) for recycling;
(3) culturing the zooblast cells of haematococcus pluvialis obtained in the step (2) at an acinetospore stage;
(4) after the culture is completely red, separating to obtain a separated clear liquid II and haematococcus pluvialis acinetospore, and returning the separated clear liquid II to the step (3) for recycling;
(5) drying the haematococcus pluvialis acinetospore to obtain a haematococcus pluvialis product;
in the step (2), the separation mode is filtration separation, and the pore diameter of a filter medium used for filtration is 1-60 μm; the volume of at least part of the algae solution accounts for 20-50% of the total algae solution;
in the step (4), the separation mode is centrifugal separation, filtration separation or gravity settling separation.
2. The method according to claim 1, wherein in step (1), the culture conditions comprise: the temperature is 15-25 deg.C, the illumination intensity is 1000-5000Lux, and the pH value is 6-8.
3. The method according to claim 1, wherein in the step (2), the culture is performed until OD of algal solution is reached680The value is 0.1 to 1.
4. The method of claim 1, wherein in step (2), at least a portion of the algal liquor is transferred to a separation and storage facility for separation.
5. The method according to claim 4, wherein in the step (2), the transferring is performed by gravity transfer, overflow transfer or siphon transfer.
6. The method according to claim 5, wherein in the step (2), the transferring manner is overflow transferring.
7. The method according to claim 1, wherein in the step (2), the separation mode is normal pressure filtration separation.
8. The method according to claim 4, wherein the separation and storage and transportation device is a cylindrical separation void having an aspect ratio of 1-10:1 and the bottom of the cylindrical separation void is fitted with a filter medium.
9. The method according to claim 8, wherein the separation and storage means is a cylindrical separation void having an aspect ratio of 1.5-5: 1.
10. The method of claim 8, wherein the filter media is a frit, a filter cloth, or a filter paper.
11. The method of claim 10, wherein the filter media is a frit; the pore size of the filter medium is 10-30 μm.
12. The method according to claim 8, wherein the cylindrical separation void column is provided with an agitator.
13. The method of claim 12, wherein the agitator is an anchor agitator, a gate agitator, or a ribbon agitator.
14. The method of claim 13, wherein the agitator is a gate agitator.
15. The method according to claim 4, wherein in step (3), the haematococcus pluvialis motile cells and the separated clear solution II are mixed in the separation and storage device and then transferred to an acinetospore culture device for culturing in an acinetospore stage.
16. The method of claim 15, wherein the transferring into the acinetospore culture is by siphon transfer or vacuum transfer.
17. The method of claim 16, wherein the means of transferring into the acinetospore culture is vacuum transfer.
18. The method according to claim 1, wherein in the step (3), the culture is temperature stress culture, light stress culture or stress agent stress culture.
19. The method according to claim 18, wherein in the step (3), the culture manner is a stress agent stress culture.
20. The method of claim 18, wherein the conditions under which the stress agent is stress cultured comprise: the pH value is 7-9, the temperature is 24-30 ℃, the illumination intensity is 5000-15000Lux, the stress agent is sodium salt, and the addition amount of the stress agent is 0.005-0.05mol per liter of the culture solution.
21. The method of claim 20, wherein the stress agent is added in an amount of 0.02-0.05mol per liter of the mixed algal solution.
22. The method of claim 20, wherein the stress agent is sodium bicarbonate.
23. The method as claimed in claim 15, wherein in step (3), after the transfer is finished, the separation and storage device is washed to neutral with deionized water for the next separation and transfer, and the washing liquid is stored for later use.
24. The method of claim 23, wherein the cleaning solution is recycled to step (3).
25. The method of claim 24, wherein the means for recycling comprises: mixing the cleaning solution and the haematococcus pluvialis motile cells in the separation and storage and transportation device, and transferring the mixture to an acinetospore culture device for culturing in an acinetospore stage; or the recycling mode comprises the following steps: in the culture at the acinetospore stage, the wash was transferred to an acinetospore incubator for water replenishment.
26. The method according to claim 1, wherein in the step (4), the separation mode is gravity settling separation.
27. The method according to claim 1, wherein in the step (5), the drying manner is natural drying, vacuum drying or spray drying.
28. The method according to claim 27, wherein in the step (5), the drying manner is cyclone spray drying.
29. The method of claim 1, wherein step (1) is carried out in a swimming cell culture vessel, which is an open photobioreactor or a closed photobioreactor; and (3) the step (3) is carried out in an immobile spore culture device which is an open photobioreactor or a closed photobioreactor.
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