KR101517838B1 - Method for cultivating photosynthetic microalgae by co-cultivation - Google Patents

Abstract

There is provided a method of cultivating a photosynthetic microalgae capable of converting nitrogen (N ₂ ) in air or water into nitrate or ammonium which can be utilized by photosynthetic microalgae by co-culturing the nitrogen-fixing cyanobacteria. According to the culturing method of the present invention, it is possible to efficiently cultivate photosynthetic microalgae in the ocean or fresh water without supplying external energy and additional nutrients.

Description

TECHNICAL FIELD The present invention relates to a method for culturing photosynthesis microalgae by co-

The present invention relates to a method for culturing photosynthetic microalgae by co-culture.

Recently, functional diversity has raised interest in photosynthetic microorganisms and microalgae, and the scope of the research is expanding in various fields. Microalgae have been actively used for research on carbon dioxide reduction, which has recently become a subject of great interest due to environmental problems such as global warming due to its ability to maintain photosynthesis, and it is noted as a sustainable energy source against depletion of fossil fuels It has also been used in research related to the production of bio-energy such as biodiesel, bio-ethanol and hydrogen gas.

However, in order to quantitatively remove carbon dioxide using microalgae or to mass-produce useful products such as bioenergy, microalgae cultivation must be carried out at a large scale and at a high concentration. Therefore, there is a need for technologies related to the construction of large-scale culture facilities. Conventionally, various types of photobioreactors installed in a room are used as a culture facility for culturing microalgae. Such a photobioreactor is mostly made of glass such as expensive pyrex which can increase the transparency of light and can be sterilized, and it has to be equipped with artificial lighting means, And technology must be invested, and even after production, it is costly to maintain and operate. Such a photobioreactor not only requires a large expense to enlarge the scale, but also involves a spatial restriction as it requires a large space. Further, it is necessary to prepare and supply a medium for microalgae cultivation and to periodically exchange microalgae. In addition, the microalgae should remove the excretion discharged from the microalgae and the metabolites obstructing growth. That is, there is a problem that manpower, equipment, and cost are required for management and operation in addition to manufacturing cost and space restriction.

Therefore, considering that economical efficiency is an important prerequisite for commercial mass cultivation, it takes a lot of cost to manufacture and maintain in the enlargement of the scale, requires a large space, Cultivation of microalgae using a photobioreactor according to the prior art, accompanied by operational costs associated with the preparation of a microorganism and the exchange of media, may be suitable for small-scale cultivation purposes for research purposes, It would not be a viable option for large-scale cultivation.

As such, costs and space-related problems have become a major obstacle to the production of useful products, including bio-energy, or the difficulty of utilizing microalgae to remove carbon dioxide, thus making microalgae available in large quantities It is necessary to develop cultivation technology which can cultivate the cells.

In this connection, the inventors of the present invention have focused on a marine which has no spatial restriction and has minimal conditions such as nutrients, carbon dioxide, water and temperature for culturing microalgae, and a method for mass production of microalgae at low cost in the ocean As a result of intensive efforts, a submerged photobioreactor using a semi-permeable membrane has been developed (Korean Patent Publication No. 2010-63260).

However, seawater is a natural culture tank in which microalgae are actually being cultured. However, as compared with a photobioreactor requiring culture and power using a large aquatic tank on the land, There is a disadvantage that the culture efficiency is lowered due to the lack of the components and the local deficiency of carbon dioxide, and further studies are needed to increase the culture efficiency.

It is an object of the present invention to provide a method for culturing a photosynthetic microalgae capable of increasing the culture efficiency in culture in a floating-type photobioreactor.

The present inventors produced the above-described floating type photobioreactor (Korean Patent Laid-Open Publication No. 2010-63260) and cultured the photosynthetic microalgae using actual seawater or fresh water. However, after a certain period of time And found it to be in a stagnant state that no longer proliferates. During the analysis of the cause, it was found that the concentration of nitrate and ammonium in the seawater or fresh water which is a culture medium gradually decreased and reached to a very low level after the stagnation period. Actual nitrogen is rich in elements, which account for 79% of the air, or some of them are dissolved in seawater or fresh water in the form of N ₂ . However, microalgae do not utilize this type of nitrogen and require nitrogen compounds such as NO ₃ ^- , NH ₂ CONH _2, NH ₃ or NH ₄ ^+. The concentrations of these nitrogen compounds in the surface waters It is not enough for ocean cultivation. The present inventors have made intensive efforts to develop a method for increasing the concentration of nitrate and ammonium in seawater or fresh water. As a result, when the nitrogen-fixing cyanobacteria were co-cultured with the photosynthetic microalga of interest, The present invention has been completed. The entire contents of Korean Patent Publication No. 2010-63260 are incorporated herein by reference.

In order to accomplish the above object, the present invention provides a method for culturing a biological culture system, comprising the steps of: injecting seawater or fresh water into a culture container of a floating type photobioreactor including a culture container and a lifting means for fixing the culture container; Inoculating the seawater or fresh water with nitrogen-fixing cyanobacteria and photosynthetic microalgae to be cultivated; Sealing the culture vessel and fixing it to the floating means, and then introducing the vessel into the ocean or fresh water; And co-culturing the nitrogen-fixing cyanobacteria and photosynthetic microalgae. The present invention also provides a method for culturing a photosynthetic microalgae.

The culture container may be any light permeable material capable of transmitting light, such as a glass, a plastic, a semi-permeable membrane, a material having a specific wavelength filter function, a gas capable of transmitting and blocking a nutrient and a nutrient, A permeable material may be used, preferably a flexible material. Optionally, the culture vessel may have at least one window made of a semi-permeable membrane at a portion of the frame made of impermeable plastic, which has the advantage of structural stability and cost savings. The semi-permeable membrane may be made of cellulose, acetyl cellulose (CA), cellulose 2.5 acetate (cellulose acetate), or the like. Cellulose triacetate (CTA), modified cellulose, curophan, polyvinyl alcohol (PVA), poly (methyl methacrylate), poly (methyl methacrylate) Ethylene-vinyl alcohol copolymer (EVA), polyacrylonitrile (PAN), poly (lactic-co-glycolic acid), polylactic acid (PLA ), Polypropylene (PP), polyacrylamide, polysulfone (PSF) or poly (ethylene-co-vinyl acetate) can be used. As the impermeable film, conventionally used polyethylene, polystyrene (PS) polypropylene, polyethylene terephthalate (PET), hidensity polyethylene (HDPE), polycarbonate and the like can be used.

The nitrogen-fixing blue-green algae are not intended to genus Nostoc, Anabaena genus, genus Crocosphaera, Cyanothece in, Trichormus in, or in Richella Calothrix preferably a limited lay.

Preferably, the photosynthetic microalgae to be cultivated are not limited to, but are not limited to, Botryococcus, Chlorella, Crypthecodinium, Cylindrotheca, Dunaliella, Isochrysis, Monallanthus, Nannochloris, Nannochloropsis, Neochloris, Nitzschia, Phaeodactylum, Schizochytrium, Tetraselmis or Haematococcus .

Meanwhile, the culture container may further include a separate isolation barrier for spatially separating the nitrogen-fixing blue algae and the photosynthetic microalgae to be cultured. This is because if the nitrogen-fixing cyanobacteria directly contact with the photosynthetic microalgae to be cultured, they can secrete a substance that inhibits the growth of the photosynthetic microalgae to be cultured, so they are cultured in a separated state for a certain period of time, , Nitrogen compounds immobilized by nitrogen-fixing cyanobacteria can be absorbed by the photosynthetic microalgae to be cultured. Alternatively, the nitrogen-fixing cyanobacteria may first be destroyed prior to removal of the separation barrier, and then the isolation barrier may be removed. It is preferred that the isolation barrier is a semi-permeable membrane or a biodegradable polymer and that the semi-permeable membrane protects the gas, water and ions, particularly nitrogen fixation, while preventing the passage of microalgae and alleopathic chemicals The nitrogen compound produced by the cyanobacterium can be passed through any material as long as it passes freely. However, it is possible to use cellulose acetate, CA, cellulose 2.5 acetate, cellulose triacetate (CTA), cellulose triacetate Modified cellulose, curophan, polyvinylalcohol (PVA), poly (methyl methacrylate), PMMA, ethylene-vinyl alcohol copolymers, vinyl alcohol copolymer (EVA), polyacrylonitrile (PAN), poly (lactic-co-glycolic acid), polylactic acid (PLA) Polypropylene (PP), polyacrylamide, polysulfone (PSF) or poly (ethylene-co-vinyl acetate) can be used. The biodegradable polymer includes, but is not limited to, polyglycolic acid, PGA, poly-L-lactic acid, PLLA, poly-DL- 3-hydroxybutylate, poly (3HV-co-3HB)}, polycaprolactone {Poly (3-hydroxyvalerate-co- (ε-caprolactone, PCL) poly (ester-amide), poly (ester-uretane), or polybutylene succinic acid-polybutylene succinate-cobutylene adipate, PBSA}. The biodegradable polymer is decomposed by seawater or fresh water after a lapse of a certain period of time, so that the nitrogen-fixing cyanobacteria isolated inside can be exposed to the photosynthetic microalgae to be cultured.

In addition, the isolation barrier may be installed not only perpendicularly to the water surface but also horizontally. In this case, fine algae favoring high light intensity in the upper layer and fine algae preferred in the lower layer are disposed, It can be optimized. For example, if the microalgae to be cultured favor high light intensity, and the nitrogen-fixing cyanobacteria prefer low light intensity, the microalgae to be cultured can be arranged in the upper layer and the nitrogen-fixed blue algae can be arranged in the lower layer. On the contrary, if the microalgae to be cultured prefer low light intensity and the nitrogen-fixed cyanobacteria prefer high light intensity, the microalgae to be cultured can be placed in the upper layer and the nitrogen-fixed blue algae can be arranged in the lower layer.

On the other hand, seawater or fresh water may be natural sea water or fresh water, but it is preferable to use a filter that is filtered with a filter to prevent contamination of other microorganisms.

According to the present invention, the nitrogen gas in the air is fixed in the form of ammonium and nitrate using nitrogen-fixed cyanobacteria to form carbon dioxide and nitrogen components necessary for microalgae growth, , And it is expected that the pH, which can be increased by microalgae growth, can be controlled.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram for explaining the concept of co-culture of nitrogen-fixed blue-green algae and microalgae to be cultured using the floating type bioreactor of the present invention. FIG.
FIG. 2 is a view showing an embodiment of a floating type photobioreactor capable of co-culturing nitrogen-fixed blue algae and microalgae to be cultured, the left and right being divided by a vertical isolation barrier.
FIG. 3 is a view showing still another embodiment of a floating type photobioreactor capable of co-culturing nitrogen-fixed blue algae and microalgae to be cultured, the upper and lower layers being divided by a horizontal isolation barrier.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be described in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram for explaining the concept of a co-cultivation method of a nitrogen-fixing blue-green algae and a photosynthetic microalgae to be cultured using a floating biological reactor according to an embodiment of the present invention. The culture container 101 is made of a light-transmitting material, and has a structure capable of containing seawater therein, but any shape can be used, but a pillow type is most preferable. The light transmissive material is a light transmissive material such as glass or impermeable plastic, a semitransmissive film, a material having a specific wavelength filter function, a light permeable material capable of transmitting or shielding gases and nutrients, or a composite material. In this case, the system is closed system completely closed with seawater or fresh water. In this case, the inside is not filled with seawater, and the air such as CO ₂ is injected into the upper part to be used for photosynthesis. So that the surface 104 can be floated. It is possible to float on the sea surface by lifting means connected to be fixed to the culture vessel even if no air is injected. The semi-permeable membrane that can be used herein is not particularly limited, but includes cellulose acetate, cellulose acetate acetate, cellulose triacetate (CTA), modified cellulose Polyvinyl alcohol (PVA), poly (methyl methacrylate), PMMA, ethylene-vinyl alcohol copolymer (EVA), and the like. Polyacrylonitrile (PAN), poly lactic-co-glycolic acid (PLA), polylactic acid (PLA), polypropylene (PP), polyacrylamide , Polysulfone (PSF) or poly (ethylene-co-vinyl acetate) can be used. As the impermeable film, conventionally used polyethylene, polystyrene (PS) polypropylene, polyethylene terephthalate (PET), hidensity polyethylene (HDPE), polycarbonate and the like can be used.

Optionally, the culture vessel may have a partially semi-permeable membrane. In this case, a plurality of windows made of a semitransparent film may be provided in the structure made of the opaque film.

Wherein the nitrogen fixing blue-green algae (102) is capable of converting a nitrogen-containing compound form, such as nitrate, nitrite, ammonium to secure the air nitrogen, one possible use be any, Nostoc genus, Anabaena genus, Crocosphaera in, Cyanothece in, Trichormus It is preferred that the genus is of the genus Richella or Calothrix .

The photosynthetic microalgae 103 to be cultivated can be grown by supplying the nitrogen fixed by the nitrogen-fixing cyanobacteria 102 in the form of nitrogen compounds.

Seawater is injected through a part of the opening of the culture container 101, and then the nitrogen-fixed algae 102 and the photosynthetic microalgae 103 to be cultured are inoculated at an appropriate ratio, and then the opening is sealed. At this time, since the above-mentioned proper ratio needs to occupy a large number of microalgae to be cultivated, it can be adjusted to 1: 2 to 1: 100, and it can be adjusted according to the nitrogen concentration in seawater or fresh water. The sealed culture vessel is immersed in the floatation means and put into the ocean or fresh water. The culturing vessel floated on the water surface 104 by the floatation means has a structure in which the nitrogen-fixed cyanobacteria 102 and the photosynthetic microalgae 103 to be present inside by the wind and the waves are mixed, ₂ and the nutrients of seawater are used for photosynthesis and growth. When the cultivation is completed, the culture container is separated to recover the nitrogen-fixed cyanobacteria 102 and the photosynthetic microalgae 103 to be cultivated. Since the nitrogen-fixing cyanobacterium 102 is a kind of photosynthetic microalgae, it is not necessary to separate the nitrogen-fixing cyanobacterium 102 and the photosynthetic microalgae 103 to be cultured, It is also possible to separate by centrifugation using the difference in specific gravity.

FIG. 2 is a view showing an embodiment of a floating type photobioreactor capable of co-culturing nitrogen-fixed blue algae and microalgae to be cultured, the left and right being divided by a vertical isolation barrier.

In this case, the floating type photobioreactor has an isolation barrier 105 in the vertical direction perpendicular to the water surface in the culture container. Only the nitrogen-fixed blue algae 102 can be cultured on the boundary of the isolation barrier 105, and only the photosynthetic microalgae 103 to be cultivated on the other side. In this case, the isolation barrier 105 may be a semi-permeable membrane or a biodegradable polymer material. In the former case, nitrogen compounds produced by nitrogen-fixing cyanobacteria can be transported by osmotic pressure toward the opposite side of the isolation barrier. In this case, the size of the pores of the semipermeable membrane can be controlled to restrict the movement of growth inhibitory substances that can be secreted by the nitrogen-fixing cyanobacterium 105. In the latter case, the isolation barrier 105 is a biodegradable polymer material, which decomposes when a culture time of a predetermined period elapses, so that the nitrogen-fixing cyanobacteria 102 can be mixed with the photosynthetic microalgae 103 to be cultivated. In this case, if necessary, the nitrogen-fixing cyanobacteria 102 may be dissolved before the biodegradable polymer material is decomposed to prevent the growth delay of the photosynthetic microalgae to be cultivated by the metabolites of the nitrogen-fixing cyanobacteria 102. The biodegradable polymer includes, but is not limited to, polyglycolic acid, PGA, poly-L-lactic acid, PLLA, poly-DL- 3-hydroxybutylate, poly (3HV-co-3HB)}, polycaprolactone {Poly (3-hydroxyvalerate-co-3-hydroxybutylate) (ε-caprolactone, PCL) poly (ester-amide), poly (ester-uretane), or polybutylene succinic acid-polybutylene succinate-cobutylene adipate, PBSA}.

Likewise, the culture vessel may also have a partially semipermeable membrane, in which case one or more windows made of a semipermeable membrane may be provided in the structure made of impermeable plastic.

3 is a view showing still another embodiment of a floating type photobioreactor capable of co-culturing the nitrogen-fixing blue algae 102 and the cultured microalgae 103 by being divided into upper and lower layers by a horizontal isolation barrier 105 to be.

In this case, when the nitrogen-fixing cyanobacterium 102 is preferred to have a high light intensity, the microalgae 103 to be cultured are arranged in the upper layer portion 106, Can be maximized. In this case, the horizontal isolation barrier 105 is preferably a semi-permeable membrane, and it is possible to pass the nitrogen compound produced by the nitrogen-fixing cyanobacteria but the alleopathic chemical produced by the nitrogen-fixing cyanobacteria It is more preferable that it can not pass. In addition, the horizontal isolation barrier 104 may be a semi-permanent material, but is made of a biodegradable material. When a certain period of time elapses, the horizontal isolation barrier 104 may be dissolved by seawater or fresh water to mix limited microalgae. The cyanobacterium 102 may first be destroyed so that only the internal nitrogen compound can be used by the microalgae 103 to be cultivated.

The foregoing description of specific embodiments of the invention has been presented for purposes of illustration and description. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention as defined by the appended claims. Do.

101: Culture vessel 102: Nitrogen fixed cyanobacteria
103: Microalgae to be cultured 104: Water surface
105: isolation barrier 106: upper layer
107: Lower layer

Claims (16)

Injecting seawater or fresh water into the culture container of a floating type photobioreactor including a culture container made of a light transmitting material and a floatation means for fixing the culture container;
Inoculating the seawater or fresh water with nitrogen-fixing cyanobacteria and photosynthetic microalgae to be cultivated;
Sealing the culture vessel and fixing it to the floating means, and then introducing the vessel into the ocean or fresh water; And
And co-culturing the nitrogen-fixing cyanobacteria and photosynthetic microalgae. The optical transmission material according to claim 1, wherein the light transmitting material is at least one selected from the group consisting of glass, impermeable plastic, semi-permeable film, material having a specific wavelength filter function, light permeable material capable of transmitting and blocking a gas and nutrients, A method of culturing a photosynthetic microalgae which is a material. The method of claim 1, wherein the culture vessel is provided with at least one window made of a semi-permeable membrane in a structure made of impermeable plastic. The transflective film according to claim 2 or 3, wherein the semi-permeable membrane is selected from the group consisting of cellulose, cellulose acetate (CA), cellulose 2.5 acetate, cellulose triacetate (CTA), modified cellulose, Curophan, polyvinylalcohol (PVA), poly (methyl methacrylate), PMMA, ethylene-vinyl alcohol copolymer (EVA), poly Polyacrylonitrile (PAN), poly lactic-co-glycolic acid, polylactic acid (PLA), polypropylene (PP), polyacrylamide, Wherein the photosynthetic microalgae are polysulfone (PSF) or poly (ethylene-co-vinyl acetate). The method according to claim 2 or 3, wherein the impermeable plastic is polyethylene, polystyrene (PS) polypropylene, PET (polyethylene terephthalate), HDPE (hidensity polyethylene), polycarbonate. The method of claim 1, wherein the nitrogen fixation is blue-green algae Nostoc genus, genus Anabaena, genus Crocosphaera, Cyanothece in, Trichormus in, or in Richella Calothrix layman, how cultivation of photosynthetic algae. The method according to claim 1, wherein the photosynthetic microalgae to be cultured is a culture method of photosynthetic microalgae which is Botryococcus, Chlorella, Crypthecodinium, Cylindrotheca, Dunaliella, Isochrysis, Monallanthus, Nannochloris, Nannochloropsis, Neochloris, Nitzschia, Phaeodactylum, Schizochytrium, Tetraselmis or Haematococcus . The culture method of photosynthetic microalgae according to claim 1, wherein the incubation type photobioreactor further comprises an isolation barrier for isolating the nitrogen-fixing cyanobacteria and the photosynthetic microalgae to be cultivated in the culture container. The method of cultivating a photosynthetic microalgae according to claim 8, wherein the isolation barrier is provided in a direction perpendicular or horizontal to the water surface. 10. The method of claim 8 or 9, wherein the isolation barrier comprises a semi-permeable membrane or a biodegradable polymer. 10. The method according to claim 9, wherein when the isolation barrier is installed in a horizontal direction with respect to the water surface and the microalgae to be cultured favor high light intensity, the microalgae to be cultivated are placed in the upper layer and nitrogen- And the microbial cells are placed in the lower layer portion. [10] The method according to claim 9, wherein when the isolation barrier is installed in a horizontal direction with respect to the water surface and the microalga to be cultured prefer low light intensity, the microalgae to be cultivated are arranged in the lower layer portion, And then the microbial cells are placed in the upper layer. The method of claim 10, wherein the semi-permeable membrane is selected from the group consisting of cellulose, cellulose acetate (CA), cellulose 2.5 acetate, cellulose triacetate (CTA), modified cellulose, polyvinyl alcohol (PVA), poly (methyl methacrylate), PMMA, ethylene-vinyl alcohol copolymer (EVA), polyacrylonitrile polyacrylonitrile (PAN), poly lactic-co-glycolic acid, polylactic acid (PLA), polypropylene (PP), polyacrylamide, polysulfone , PSF) or poly (ethylene-co-vinyl acetate). The biodegradable polymer according to claim 10, wherein the biodegradable polymer is selected from the group consisting of polyglycolic acid (PGA), poly (L-lactic acid), PLLA, poly- 3-hydroxybutylate, poly (3HV-co-3HB)}, polycaprolactone {Poly (3-hydroxyvalerate-co- ε-caprolactone, PCL} poly (ester-amide), poly (ester-uretane)} or polybutylene succinic acid-polybutylene succinate -cobutylene adipate, PBSA}. < / RTI > 11. The method according to claim 10, wherein the biodegradable polymer is decomposed after a lapse of a predetermined time. The method of claim 1, wherein the seawater or fresh water is filtered through a filter.

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