KR20230135533A - Cultivating method of Haematococcus pluvialis using solar cells and light guide panel - Google Patents

Abstract

The present invention relates to a method for cultivating Haematococcus pluvialis using a solar cell and a light guide panel. The method for cultivating the Haematococcus pluvialis, according to an embodiment of the present invention, comprises the steps of: supplying carbon dioxide (CO_2) to a first culture medium in a first reactor to produce hydrogen carbonate ions (HCO_3^(-)); inoculating Haematococcus pluvialis into the first culture medium supplied with carbon dioxide; and adding calcium ions (Ca^(2+)) to the first culture inoculated with Haematococcus pluvialis, to produce calcium carbonate (CaCO_3) and photoculture the same using a light source operated by power generated from solar cells. One aspect of the present invention is to provide a method for cultivating Haematococcus pluvialis to produce calcium-mediated biomineralization and biomass containing high amounts of astaxanthin and omega-3 by introducing a solar cell system combined with a light guide panel.

Description

Cultivating method of Haematococcus pluvialis using solar cells and light guide panel}

The present invention relates to a method for cultivating Haematococcus pluvialis using a solar cell and a light guide plate, which fixes carbon dioxide (CO ₂ ) and extracts omega-3 fatty acid (ω-3 FA) and astaxanthin through calcium-mediated biomineralization. It is about technology that simultaneously improves productivity.

Large-scale greenhouse gases emitted from the use of fossil fuels are causing global warming. Accordingly, the development of carbon dioxide capture and storage technology (CCS, Carbon Capture & Sequestration) to reduce carbon dioxide is actively underway around the world. CCS technology is a method of capturing large amounts of carbon dioxide emitted from various carbon emission sources, including thermal power plants, at high concentrations and injecting them into the ground or seafloor to isolate them from the atmosphere. Although this CCS technology has the effect of reducing large amounts of carbon dioxide in a short period of time, it is difficult to realize actual CCS due to problems with stable storage, location selection, and high installation costs. Therefore, unlike CCS technology, CCU (Carbon Capture & Utilization) technology, which directly utilizes carbon dioxide for industrial purposes rather than storing it or converts it into high-value-added materials, is receiving attention, and attempts are being made in Korea to develop processes to convert carbon dioxide into various high-value-added materials. It is becoming. Among them, the biological conversion process of carbon dioxide using microalgae, a photosynthetic microorganism, is attracting attention as an economical carbon dioxide reduction technology that can simultaneously reduce carbon dioxide and produce various high value-added materials such as biofuel, bioplastics, and pharmaceuticals. Microalgae, called phytoplankton, are single-celled underwater organisms that photosynthesize and are attracting attention as a biomass resource with great potential because they can produce energy and industrial materials and reduce greenhouse gases at the same time, focusing on the fields of energy, chemistry, and the environment. Its use value is expected to expand in the future.

In the energy field, microalgae have the highest oil productivity among all biodiesel producing crops. While soybeans, oil, sunflower, and oil palm have a cultivation cycle of 4 to 8 months, microalgae multiply several times every day and can be cultivated on a daily basis. The fat content per unit weight is also high, so the annual oil production is 100% that of soybeans. It's more than double. In addition, it is possible to produce biofuel with physical properties similar to petroleum diesel as a living resource free from criticism of turning food resources into energy.

In the chemical field, microalgae have the advantage of being able to produce a variety of useful substances, and are currently industrialized mainly in the food field, and industrialization is expected to expand to the biochemical and bioplastic fields in the future.

In the environmental field, microalgae are receiving the greatest attention in terms of their ability to reduce carbon dioxide, as described above, and related research is continuously being conducted around the world. Microalgae can absorb about twice the weight of carbon dioxide as biomass, which corresponds to an absorption efficiency that is 10 to 50 times higher than that of terrestrial plants. Additionally, microalgae can be cultured regardless of specific soil or water quality. Accordingly, related companies are expanding their attempts to use microalgae in carbon dioxide reduction and factory wastewater purification projects.

Among various microalgae, Haematococcus pluvialis produces astaxanthin, a high value - added product. pluvialis ) has recently been in the spotlight. Astaxanthin, a carotenoid that can be extracted from Haematococcus pluvialis, has an excellent antioxidant effect, can be used in the human body as it is in the bioactive 3S and 3S' forms, and has excellent economic effectiveness. Accordingly, as disclosed in the patent document of the prior art document, much research is being conducted on the biological characteristics and culture process of Haematococcus pluvialis and the downstream process for astaxanthin production in order to maximize astaxanthin production. there is.

However, the overall photoculture process of Haematococcus is divided into a growth stage (growth stage, green stage) and an induction stage (induction stage, red stage) in which high value-added substances are accumulated by applying stress (salt, light, temperature, etc.). However, since the induction stage takes at least 1.5 months, overall astaxanthin productivity is low. In addition, when cultivating in an outdoor mass culture device to produce biomass on a commercial scale, there is a difference in the amount of light irradiated to each reactor due to structural limitations even during the day when light supply is smooth, resulting in differences in growth performance for each reactor. Haematococcus generally must be cultured under continuous light at the optimal amount of light (growth stage: 50 to 100 μ E/m2/s, induction stage: 150 to 300 μ E/m2/s), but due to the characteristics of the outdoor system, There is a problem of not being able to utilize light at night.

Accordingly, there is an urgent need for a method to solve the problems of conventional Haematococcus pluvialis culture technology.

KR 10-2019-0120876 A

The present invention is intended to solve the problems of the prior art described above. One aspect of the present invention is to introduce a solar cell system combined with calcium-mediated biomineralization and a light guide panel to produce a high content of astaxanthin and The aim is to provide a method for cultivating Haematococcus pluvialis to produce biomass containing omega-3.

In addition, another aspect of the present invention is Haematococcus pluvialis cultivation, which maximizes biomass and astaxanthin productivity by utilizing calcium carbonate produced through biomineralization under high-light Hematococcus culture conditions from the growth stage. We would like to provide a method.

The Haematococcus pluvialis culturing method according to an embodiment of the present invention includes the steps of (a) supplying carbon dioxide (CO ₂ ) to the first culture medium in a first reactor to generate hydrogen carbonate ions (HCO ₃ ^- ); (b) inoculating Haematococcus pluvialis into the first culture medium supplied with carbon dioxide; and (c) adding calcium ions (Ca ²⁺ ) to ^the first culture medium inoculated with Haematococcus pluvialis to generate calcium carbonate (CaCO ₃ ) and operating by power generated from a solar cell. It includes the step of photoculturing using a light source.

Additionally, in the H. pluvialis culturing method according to an embodiment of the present invention, in step (b), the inoculum amount of H. pluvialis may be 0.2 g/L or more.

Additionally, in the Haematococcus pluvialis culturing method according to an embodiment of the present invention, the concentration of the calcium ion added in step (c) may be 0.5 to 3.5 mM.

Additionally, in the Haematococcus pluvialis culturing method according to an embodiment of the present invention, the light source includes: an LED that generates light using power generated from the solar cell; and a light guide panel that diffuses the light generated by the LED.

In addition, in the Haematococcus pluvialis culturing method according to an embodiment of the present invention, the first reactors are plural, arranged to be spaced apart from each other, and one of the first reactors and the other one of the first reactors are arranged close to each other. The light source may be disposed between each first reactor.

In addition, in the Haematococcus pluvialis culture method according to an embodiment of the present invention, step (c) includes adding the calcium ions and performing first photoculture at a light intensity of 20 to 60 μE/m2/s. steps; And after the first photo-cultivation step, a second photo-cultivation step at a light intensity of 100 to 150 μE/m2/s may be included.

In addition, in the Hematococcus pluvialis culture method according to an embodiment of the present invention, when the cell density of the Hematococcus pluvialis reaches 1.0 g/L or more in the first photoculture, the first photoculture 2 Photoculture steps can be performed.

Additionally, in the Haematococcus pluvialis culturing method according to an embodiment of the present invention, the calcium ions in step (c) may be added by supplying calcium chloride (CaCl ₂ ) to the first culture medium.

Additionally, in the method for cultivating Haematococcus pluvialis according to an embodiment of the present invention, (d) recovering the calcium carbonate produced in step (c); and (e) adding the recovered calcium carbonate to the second culture medium in the second reactor and photoculturing the Haematococcus pluvialis.

Additionally, in the Haematococcus pluvialis culture method according to an embodiment of the present invention, step (e) may be photocultured at a light intensity of 100 μE/m2/s or more.

The features and advantages of the present invention will become more apparent from the following detailed description based on the accompanying drawings.

Prior to this, terms or words used in this specification and claims should not be construed in their usual, dictionary meaning, and the inventor may appropriately define the concept of the term in order to explain his or her invention in the best way. It must be interpreted with meaning and concept consistent with the technical idea of the present invention based on the principle that it is.

According to the present invention, astaxanthin and omega-3, which are high value-added useful substances, can be produced simultaneously from microalgae through calcium-mediated biomineralization technology, and electrical energy produced by solar cells can be used to produce biomineralization under conditions without light. Productivity can also be maximized in outdoor culture systems.

In addition, by using a light guide plate, the light emitted from the LED can be converted into flat light and diffused uniformly with high brightness, thereby overcoming the blocking phenomenon between reactors due to changes in the position of the sun when expanding the culture scale per unit area. Thus, differences in productivity between reactors can be resolved, and there is no need to install a large amount of solar panels, which is advantageous in terms of economic efficiency.

Ultimately, a system that combines bio-mineralization with solar cells using a light guide plate can be expected to have an economic effect that is incomparable to the existing one in that it can simultaneously produce high value-added substances astaxanthin and omega-3, and can be used in the same area. It is also possible to achieve a secondary effect of reducing a greater amount of carbon dioxide than was previously reduced through photoculture.

Meanwhile, it produces not only biomass but also calcium carbonate at the same time, and can be used for high-light cultivation of Haematococcus as an efficient culture strategy for Haematococcus in the summer when light is strong.

Figures 1 and 2 are schematic diagrams showing a method for cultivating Haematococcus pluvialis according to an embodiment of the present invention.
Figure 3a is a graph showing biomass concentration (g/L) for each CaCl ₂ concentration injected in the growth stage.
Figure 3b is a graph showing astaxanthin content (%) according to CaCl ₂ concentration injected in the growth stage.
Figure 4 is a graph showing the concentration (mg L ^-1 ) of calcium carbonate produced through biomineralization according to the injection of 3mM CaCl ₂ .
Figure 5 is a graph showing the change in calcium concentration (mM) present in the culture medium on the starting and final days of culture according to the injection of 3mM CaCl ₂ .
Figure 6 is a graph showing the activity (unit 10 ⁶ cells ^-1 ) of extracellular carbonic anhydrase enzyme according to injection of 3mM CaCl ₂ .
Figure 7 is a graph showing the amount of carbon dioxide (g L ^-1 day ^-1 ) reduced in the Haematococcus pluvialis culture method incorporating biomineralization.
Figure 8a shows the effect of calcium carbonate accumulated in each cycle on biomass production (g L ^{- 1} ) when four semi-continuous cultures were performed for 60 days using the Haematococcus pluvialis culture method incorporating biomineralization. These are the analyzed experimental results.
Figure 8b is an analysis of the effect of calcium carbonate accumulated in each cycle on astaxanthin content (%) when four semi-continuous cultures were performed for 60 days using the Haematococcus pluvialis culture method incorporating biomineralization. This is the result of the experiment.
Figure 9 is a schematic diagram showing a culture method using solar cells and a light guide plate additionally in the Haematococcus pluvialis culture process incorporating biomineralization.
Figure 10a is a graph showing the amount of power while using the LED for 24 hours after converting solar energy into electric energy during the day using a solar cell and charging it in a storage battery.
Figure 10b is a graph showing the current of solar energy while the LED is used for 24 hours.
Figure 10c is a graph showing solar cell voltage, charging voltage, and load voltage while the LED is used for 24 hours.
Figure 10d is a graph showing the storage battery capacity while the LED is used for 24 hours.
Figure 11a is a photograph of a light guide plate arranged between reactors in the Haematococcus pluvialis culture method.
Figure 11b is a graph showing biomass production (g L ^-1 ) depending on biomineralization, LED operation, and light guide plate installation.
Figure 11c is a graph showing astaxanthin content (%) depending on biomineralization, LED operation, and light guide plate installation.
Figure 11d is a graph showing omega-3 content (%) depending on biomineralization, LED operation, and light guide plate installation.
Figure 12 is a graph showing the concentration (mg L ^-1 ) of calcium carbonate produced in outdoor culture of Haematococcus pluvialis combining biomineralization, LED, and light guide plate.
Figure 13 is a graph showing the omega-3 content (%) produced in outdoor culture of Haematococcus pluvialis combining biomineralization, LED, and light guide plate.
Figure 14 is a table showing fatty acids produced in outdoor culture of Haematococcus pluvialis combined with biomineralization, LED, and light guide plate.
Figure 15 is a graph showing the astaxanthin content (%) produced in outdoor culture of Haematococcus pluvialis combining biomineralization, LED, and light guide plate.
Figure 16a is a table showing carbon dioxide reduction, biomass productivity, and astaxanthin productivity according to light supply time.
Figure 16b shows the electric energy consumed and carbon dioxide emissions when only solar light is used, when solar light is used during the day and LED is used in the evening, and when solar light and light guide plate are used during the day and LED and light guide plate are used together in the evening. and a table showing the amount of carbon dioxide reduction.
Figure 17a shows the biomass production (g L ^- ) when various concentrations of calcium carbonate were injected while irradiating light of medium intensity (40 μE m ^-2 s ^{- 1} ) when culturing Haematococcus pluvialis in the growth stage. This is a graph showing ¹ ).
Figure 17b shows the biomass production (g) when various concentrations of calcium carbonate were injected while irradiating strong light (100 to 150 μE m ^-2 s ^-1 ) when culturing ^{Haematococcus} pluvialis in the growth stage. This is a graph showing L ^-1 ).
Figure 17c shows the concentration of astaxanthin when various concentrations of calcium carbonate were injected while irradiating strong light (100 to 150 μE m ^-2 s ^-1 ) when culturing ^{Haematococcus} pluvialis in the growth stage ( This is a graph showing mg L ^-1 ).
Figure 18a is a graph showing biomass production (g L ^-1 ) when cultured outdoors for 28 days with the PPPA system and the PLPA hybrid system, respectively.
Figure 18b is a graph showing astaxanthin content (%) and photosynthetic efficiency on days 0, 10, and 28 of outdoor culture of the PPPA system and the PLPA hybrid system.
Figure 18c shows the production of total omega-3 and fatty acids (mg L ^-1 ), and the productivity of total omega-3 and fatty acids (mg L ^-1 day -1) when cultured outdoors for 28 days with the PPPA system and the PLPA hybrid system, respectively ^. This is a graph showing ¹ ).

The objectives, specific advantages and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments taken in conjunction with the accompanying drawings. In this specification, when adding reference numbers to components in each drawing, it should be noted that identical components are given the same number as much as possible even if they are shown in different drawings. Additionally, terms such as “first” and “second” are used to distinguish one component from another component, and the components are not limited by these terms. Hereinafter, in describing the present invention, detailed descriptions of related known technologies that may unnecessarily obscure the gist of the present invention will be omitted.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings.

Figures 1 and 2 are schematic diagrams showing a method for cultivating Haematococcus pluvialis according to an embodiment of the present invention.

As shown in Figures 1 and 2, the Haematococcus pluvialis culture method according to an embodiment of the present invention supplies carbon dioxide (CO ₂ ) to the first culture medium in the first reactor to produce hydrogen carbonate ions ( Step of producing HCO ₃ ^- ), in the first culture medium supplied with carbon dioxide, Haematococcus pluvialis ( Haematococcus pluvialis ), and adding calcium ions (Ca ²⁺ ) to the first culture medium ^inoculated with Haematococcus pluvialis to generate calcium carbonate (CaCO ₃ ), and using the power produced from the solar cell. It includes the step of photoculturing using a light source operated by.

The present invention relates to the microalgae Haematococcus pluvialis ( Haematococcus pluvialis ) to simultaneously produce omega-3 fatty acids and astaxanthin and improve their productivity. Previously, the method of producing astaxanthin and omega-3 from Haematococcus pluvialis was different, and the productivity was very low, so the present invention was developed as a solution to this problem.

Specifically, the H. pluvialis culturing method according to an embodiment of the present invention includes a carbon dioxide supply step, a H. pluvialis inoculation step, and calcium ion addition and photoculture steps.

The carbon dioxide supply step is a process of supplying carbon dioxide to the first culture medium to photoculture the microalgae Haematococcus pluvialis. Here, the first culture medium may be filled into a predetermined reactor. In addition, the first culture medium is not particularly limited as long as it enables the growth of Haematococcus pluvialis, and for example, a carbon-deficient autotrophic medium can be used. Carbon dioxide can be supplied by supplying air containing 4 to 6% (v/v) carbon dioxide, or by using a CO ₂ incubator. Additionally, carbon dioxide-containing exhaust gas emitted from industrial facilities such as power plants can be supplied to the culture medium. When carbon dioxide is supplied to the culture medium, water and carbon dioxide react to produce hydrogen ions and hydrogen carbonate ions (HCO ₃ ^- ). The supply of carbon dioxide can be temporary or continuous. Therefore, carbon dioxide can be continuously supplied until the calcium ion addition and photoculture step (S400), which will be described later.

In the Haematococcus pluvialis inoculation step, Haematococcus pluvialis is inoculated into the first culture medium supplied with carbon dioxide. Haematococcus pluvialis is a microalgae that has excellent antioxidant effects and produces astaxanthin as a bioactive product. Haematococcus pluvialis cells are rich in nutrients and cell division mainly occurs under low light conditions, and when nutrients are depleted and high light stress occurs, astaxanthin, a carotenoid with high antioxidant properties, is produced as a secondary metabolite inside the cells. It is accumulated as. Carotenoids are usually at a low level in the growth stage, but their content is known to gradually increase in the induction stage.

Here, the appropriate inoculation amount of Haematococcus pluvialis is 0.2 g/L or more. This is because, when the inoculum amount is less than 0.2 g/L, growth potential may be impaired when calcium ions are injected for biomineralization, which will be described later.

In the calcium ion addition and photoculture step ^, biomineralization is induced through the addition of calcium ions (Ca ²⁺ ) and photoculture is performed. Haematococcus pluvialis has Carbon Anhydrase (CA), which acts as a catalyst for the reaction where water and carbon dioxide react to produce hydrogen ions and hydrogen carbonate ions. Here, the hydrogen carbonate ions generated in the carbon dioxide supply step and the calcium ions added to the first culture medium react to produce calcium carbonate (CaCO ₃ ).

Calcium ions to induce biomineralization can be injected by supplying calcium chloride (CaCl ₂ ). However, it is not necessary to supply calcium chloride to inject calcium ions, and Ca(OH) ₂ , Ca(NO ₃ ) ₂ ·4H ₂ O, Ca(CH ₃ COO) ₂ ·H ₂ O, CaSO ₄ ·2H All known calcium salts that can supply calcium ions by dissolving in the culture medium, such as _2O , can be used. At this time, since the concentration of added calcium ions affects the productivity of biomass, astaxanthin, and omega-3, the appropriate concentration is 0.5 to 3.5 mM, preferably 2.5 to 3.5 mM.

Meanwhile, the photoculture process may be performed under different light intensity conditions depending on the growth stage and induction stage. In the growth stage, when cell division mainly occurs, the first photoculture is performed at a light intensity of 20 to 60 μE/m2/s, and in the induction stage when astaxanthin accumulates, the second photoculture is performed at a light intensity of 100 to 150 μE/m2/s. You can. Here, calcium ions can be injected in the growth stage (first photoculture stage). Entry from the growth stage to the induction stage can be judged by morphological changes in Haematococcus pluvialis or depletion of nitrogen sources in the culture medium. In addition, the induction step can be performed when the cell density of Haematococcus pluvialis in the culture medium is 1.0 g/L or more, so calcium ions are added and the cell density of Haematococcus pluvialis is 1.0 through the first photoculture. When reaching g/L or more, a second photoculture step can be performed at the above light intensity.

When calcium ions are added, calcium carbonate and water are produced, so the pH of the culture medium decreases. Carbon dioxide is dissolved and hydrogen carbonate ions and hydrogen ions are continuously produced, so the pH decreases to about 3 to 4.5. Since biomineralization proceeds at pH 7.5 to 8.5, preferably pH 7.7 to 8.0, the pH of the culture medium can be maintained at 7.5 to 8.5 by adding a basic solution to the culture medium. Here, the basic solution is selected from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), ammonia (NH ₄ OH), calcium hydroxide (Ca(OH) ₂ ), and magnesium hydroxide (Mg(OH) ₂ ). Any one or more may be dissolved in a solution.

Meanwhile, photoculture is performed using a light source operated by power generated from solar cells. A solar cell is a device that converts solar energy into electrical energy and is used for solar power generation by forming a solar panel. In particular, in the case of outdoor culture, sunlight cannot be used at night, so light culture can be performed using the electrical energy produced by solar cells. The light source according to the present invention can use an LED that generates light using power generated from a solar cell. At this time, the electric energy produced from the solar cell is stored in the capacitor, and the LED can use the electric energy stored in the capacitor as a power source. However, in order to produce electrical energy from solar cells, high costs are required to build the initial system and there are limitations in operating the entire outdoor culture, so the light source according to the present invention uses a light guide panel (LGP) in addition to LED. You can. The light guide plate is a member that diffuses the light generated by the LED, and has a fine pattern formed inside it through special processing to induce scattering of light. In the case of large-scale outdoor culture with multiple first reactors arranged, a solar power generation system capable of producing large-scale electrical energy must be built in order to irradiate light using LEDs for each reactor, but the light generated from LEDs must be transmitted through a light guide plate. Since the reactor can be irradiated uniformly, the cost required to build a solar power generation system can be reduced, and differences in productivity between reactors due to the blocking phenomenon between reactors due to changes in the sun's position can be prevented or minimized. there is.

Referring to FIG. 2, during large-scale outdoor culture in which multiple reactors (PBRs) are arranged together, a shading effect occurs in which one reactor obscures another reactor in the reactor array. To solve this problem, the present invention As another embodiment (PLPA system), a plurality of first reactors are arranged spaced apart from each other, and a light source composed of an LED and a light guide plate can be placed between one first reactor and the other first reactor arranged close to each other. there is. Here, the distance between one first reactor and the other first reactor arranged close to each other includes between the row of one first reactor and the row of the other second reactor arranged close to each other.

On the other hand, when a high content of astaxanthin and omega-3 accumulate in H. pluvialis cells through the addition of calcium ions and the photoculture step, H. pluvialis can be separated from calcium carbonate and recovered. there is. Here, Haematococcus pluvialis and calcium carbonate can be separated by density difference. For example, Haematococcus pluvialis and calcium carbonate can be separated using a centrifuge. At this time, since calcium carbonate exists in a crystallized state, it can be easily separated without the injection of additional chemicals.

When calcium carbonate is recovered, Haematococcus pluvialis can be photo-cultured by adding the recovered calcium carbonate to the second culture medium in the second reactor. Here, calcium carbonate can be added at the growth stage and photocultured at high light intensity of 100 μE/m2/s or more. Because calcium carbonate has anisotropic properties, it scatters strong light, allowing strong light that was only applied to a specific area to be evenly irradiated to cells in all areas. This high-light culture using calcium carbonate can be used as a culture strategy for Haematococcus pluvialis, especially in summer when light is strong.

Hereinafter, the present invention will be described in more detail through experimental examples.

Experimental Example 1. Manufacturing process of components for photosynthetic biological reaction and mineralization process

To maximize the amount of carbon dioxide that can be dissolved and to supply gas containing 5% carbon dioxide directly into the culture medium for biomineralization, eight 500mL (width: 5cm, height: 60cm) mass cylinders were prepared.

Experimental Example 2. Detailed manufacturing process of components

A multi-purpose inlet/outlet made of silicone was used to block the inlet of the prepared mass cylinder and allow gas injection and sampling. To install the gas line and sampling line, a 2-3mm hole was made in the multi-purpose inlet/outlet made of silicone, and two Teflon tubes were cut to match the height of the mass cylinder and inserted into the created hole. A stone sparger was connected to the bottom of the Teflon tube through which gas was supplied, and air containing 5% carbon dioxide was supplied.

Experimental Example 3. Microalgae photoculture process

NIES-C was used as the medium, and 450 ml each was injected into the mass cylinder and aeration was performed. Afterwards, 10mM KOH was injected and the pH was set between 7.5 and 8. This is to ensure that the pH is maintained between 7.5 and 8 even when carbon dioxide is continuously supplied due to the buffering effect of KOH. Microalgae are Haematococcus pluvialis was used, and the initial inoculation amount was 0.2 g L ^{- 1} based on biomass, and in the mass cylinder, it was 40 μE m -2 s ^{- 1} in the growth stage of 6 to 8 days, and 100 to 100 μE m ^-2 s - 1 in the induction stage of 10 to 12 days. Light ^was irradiated at 150 μE m ^-2 s ^-1 . The final culture volume of the mass cylinder was 500 mL.

Experimental Example 4. Biomass process in growth stage

Two mass cylinders were used for each condition, and experiments were conducted to find the optimal CaCl ₂ concentration to induce biomineralization in a short time while inoculating cells. To find the optimal CaCl ₂ concentration, biomineralization was performed by adding 1mM to 4mM in a mass cylinder and cultured for 16 days, and the results are shown in Figures 3A to 3B. Figure 3a is a graph showing biomass concentration (g/L) by CaCl ₂ concentration injected in the growth stage, and Figure 3b is a graph showing astaxanthin content (%) by CaCl ₂ concentration injected in the growth stage.

As a result of the experiment, it was confirmed that the highest biomass concentration (g L ^-1 ) was found at 3mM, and it was also confirmed that injection beyond that inhibited growth (see Figure 3a). In addition, the astaxanthin content was also highest when biomineralization was performed by injecting 3 mM CaCl ₂ (see Figure 3b). When CaCl ₂ was injected to proceed with mineralization in the growth stage, growth was inhibited even at 1mM when the initial concentration was low, showing that the initial hematococcus concentration should be at least 0.2 g L ^-1 or more. Could know.

In addition, in order to confirm whether calcium carbonate is produced from H. pluvialis, when H. pluvialis was cultured without injecting CaCl ₂ and when H. pluvialis was not inoculated, the culture medium was Calcium carbonate concentration and its change were measured when 3mM CaCl ₂ was injected into each of the inoculated cultures, and the results are shown in Figures 4 and 5. Figure 4 is a graph showing the concentration (mg L ^{- 1} ) of calcium carbonate produced through biomineralization according to the injection of 3mM CaCl ₂ , and Figure 5 is a graph showing the concentration in the culture medium on the start and last days of culture according to the injection of 3mM CaCl ₂ This is a graph showing the change in calcium concentration (mM).

When 3mM CaCl ₂ was injected and Haematococcus was cultured, the concentration of calcium carbonate produced was 20.85 mg L ^-1 . When the concentration of calcium carbonate was measured when Haematococcus culture was performed without biomineralization, it was almost non-existent at 3.4 mg L ^{- 1} . Additionally, when biomineralization was induced by injecting 3mM CaCl ₂ into a culture medium without Haematococcus inoculation, the concentration of calcium carbonate was found to be 1.0 mg L ^{- 1} . Summarizing the results, it was confirmed that biomineralization partially occurred from Haematococcus and calcium carbonate was produced (see Figure 4). Unlike the Haematococcus culture medium in which there was no change in calcium concentration, when the calcium concentration of biomineralized Haematococcus was measured, it was confirmed that the concentration of calcium carbonate produced decreased from 3.64mM to 3.39mM (Figure 5 reference).

Figure 6 is a graph showing the activity of extracellular carbonic anhydrase enzyme (unit 10 ⁶ cells ^-1 ) according to injection of 3mM CaCl ₂ , and Figure 7 is a graph showing Haematococcus pluvialis fused with biomineralization. This is a graph showing the amount of carbon dioxide (g L ^-1 day ^-1 ) reduced in the culture method.

Referring to Figure 6, biomineralization is induced by the rapid conversion of carbon dioxide into hydrogen carbonate ions by the extracellular carbonic anhydrase enzyme. As a result of checking whether biomineralization was actually high when CaCl ₂ was injected, it was found that CaCl ₂ It was confirmed that it was 137 times higher than when not injected, and the carbon dioxide fixation efficiency was 46.3% superior (see Figure 7).

Experimental Example 5. Semi-continuous process of biomineralization of Haematococcus pluvialis

Figure 8a shows the effect of calcium carbonate accumulated in each cycle on biomass production (g L ^-1 ) when four semi-continuous cultures were performed for 60 days using the Haematococcus pluvialis culture method incorporating biomineralization. This is the result of the analyzed experiment, and Figure 8b shows the astaxanthin content (%) of calcium carbonate accumulated in each cycle when four semi-continuous cultures were performed for 60 days using the Haematococcus pluvialis culture method incorporating biomineralization. This is the result of an experiment analyzing the effect on .

Most of the carbon dioxide supplied to the culture exists as hydrogen carbonate ions in the culture medium, so reusing the culture medium is essential to reduce carbon dioxide, especially in terms of scale-up. For this purpose, when the biomineralization process was performed in 4 batches, it was confirmed that biomass loss of Haematococcus occurred from the 2nd culture, and the loss occurred up to 30.1% after the 4th culture ( see Figure 8a). It was confirmed that the astaxanthin content also increased by more than 24% when calcium carbonate was removed (see Figure 8b). The produced calcium carbonate was separated after culturing and used for culturing Haematococcus under high-light conditions (Experimental Example 8).

Experimental Example 6. Biomineralization process of Haematococcus pluvialis with solar cell and light guide plate fused

When cultivating Haematococcus outdoors using a vinyl photobioreactor to mass-culture, there are limitations in supplying constant light. Because the intensity of light changes over time and the supply of light is cut off in the evening, the cultivation period is prolonged and it is difficult to continuously produce biomass containing a certain amount of astaxanthin. In addition, in the mass culture system, reactors were obscured from each other, making it difficult to irradiate the same amount of light to all reactors, making it difficult to produce the same batch. Therefore, a system was built as shown in Figure 9 that stores electricity using solar cells during the time when sunlight is stably supplied (09:00 to 16:00) and uses LEDs 24 hours a day to supply light without consuming electricity costs. did. Figure 9 is a schematic diagram showing a culture method using solar cells and a light guide plate additionally in the Haematococcus pluvialis culture process incorporating biomineralization, and Figure 10a shows solar energy converted into electrical energy during the day using solar cells. A graph showing the amount of power while the LED is used for 24 hours after conversion and charging in the storage battery. Figure 10b is a graph showing the current of solar energy while the LED is used for 24 hours. Figure 10c is a graph showing the current of solar energy while the LED is used for 24 hours. 10D, a graph showing the solar cell voltage, charging voltage, and load voltage, is a graph showing the storage battery capacity while the LED is used for 24 hours.

As shown in Figures 10a to 10d, there is no solar energy during the evening, so the charging power and charging current reach '0', and the storage battery capacity is suitable for 24-hour use as the energy stored during the day is sufficient, and solar energy is drawn during the day. reached the maximum. Additionally, the load power, load current, solar cell voltage, charging voltage, and load voltage did not drop and were constant as it was continuously operated. Also, during the day, solar energy is converted into electric energy through solar cells and the energy is stored in the storage battery. When the LED is turned on 24 hours a day, the energy is used by the LED, so the capacity drops significantly during the evening. At this time, you can see that the LED does not reach 0 but reaches about 10 to 15 mAh, which shows that the electrical energy stored in the storage battery is sufficient. And since there is a lot of solar energy that can be stored during the day, the electric energy is stored in the storage battery again, reaching the maximum capacity (100 mAh).

Since the initial cost to build a solar cell is significant, as the scale of cultivation increases, there is a limit to cultivating all reactors using solar cells. Therefore, LEDs are supplied using an excellent light guide plate that can effectively disperse the supplied light. By developing technology, we have developed an economical process system that produces biomass containing a high concentration of astaxanthin at high speed by consistently supplying optimal light for cultivation. Figure 11a is a photograph of a light guide plate placed between reactors in the Haematococcus pluvialis culture method, and Figure 11b shows biomass production (g L ^-1 ) depending on biomineralization, LED operation, and light guide plate installation. 11C is a graph showing astaxanthin content (%) depending on biomineralization, LED operation, and light guide plate installation, and FIG. 11D is a graph showing omega-3 content (%) depending on biomineralization, LED operation, and light guide plate installation. This is a graph representing .

As shown in Figure 11a, a light guide plate (LGP) is installed between reactors (mass cylinders) that are placed close to each other and are optimized to irradiate light to the facing reactors at once (biomineralization, LED, LGP) ) A process system was established, and Haematococcus culture was first performed in a mass cylinder. Here, solar cells were charged and used outdoors, and the electric energy generated by the solar cells operated the LED. When cultivating, both the amount of biomass and the astaxanthin content were excellent in a hybrid system that supplied light for 24 hours using LED and LGP. Biomass increased by 52.5% compared to the control (see Figure 11b), and astaxanthin content increased by 74.34% (see Figure 11c). The most important part here is that the omega-3 content increased by 24.26%, and among them, the EPA content increased as well as 1% of DHA, which was not accumulated in the general culture system, producing the best substance among high value-added useful substances (Figure 11c). Omega-3 with high EPA and DHA content has a recent market price of $12,540,000/ton, which is superior to that of astaxanthin ($7,000,000/ton).

Experimental Example 7. Hybrid process in outdoor culture using photosynthetic bioreactor

A hybrid system was constructed and cultured at the Korea District Heating Corporation's outdoor culture facility in Pangyo. Cultivation was carried out in a 100 liter reactor, and exhaust gas containing 3 to 5% carbon dioxide supplied by Korea District Heating Corporation was injected into the reactor. Figure 12 is a graph showing the concentration (mg L ^-1 ) of calcium carbonate produced in outdoor culture of Haematococcus pluvialis combined with biomineralization, LED, and light guide plate, and Figure 13 is a graph showing the concentration of calcium carbonate (mg L -1 ) produced in outdoor culture of Haematococcus pluvialis combined with biomineralization, LED, and light guide plate. A graph showing the omega-3 content (%) produced in outdoor culture of Haematococcus pluvialis combined, Figure 14 is a graph showing the omega-3 content (%) produced in outdoor culture of Haematococcus pluvialis combined with biomineralization, LED, and light guide plate. A table showing fatty acids, Figure 15 is a graph showing the astaxanthin content (%) produced in outdoor culture of Haematococcus pluvialis combined with biomineralization, LED and light guide plate, Figure 16a is a graph showing carbon dioxide according to light supply time A table showing reduction amount, biomass productivity, and astaxanthin productivity, Figure 16b shows the case of using only solar light, using solar light during the day and using LED in the evening, using solar light and light guide plate during the day, and using LED and light guide in the evening. This table shows the electrical energy consumed, carbon dioxide emissions, and carbon dioxide reduction when light guide plates are used together.

As a result of the culture, it was confirmed that the calcium carbonate concentration was about 5.6 times higher (see Figure 12) and the omega-3 content was also 2.67 times higher (see Figure 13). Among omega-3s, the EPA content was 2.7 times higher than under normal culture conditions, and DHA, which had not been accumulated, was also accumulated to 1.14% (see Figure 14). Astaxanthin content increased to 54.7% compared to the control (see Figure 15). In conclusion, it was confirmed that when Haematococcus was exposed to constant light for 24 hours, the carbon dioxide reduction increased by 105.4% compared to when only sunlight was used, and biomass and astaxanthin productivity increased by 130.4% and 82.3%, respectively. (See Figure 16a). Additionally, using LED with solar power increases electrical energy and carbon dioxide emissions, so there are limits as scale increases. Therefore, when used with a light guide plate, electrical energy and carbon dioxide emissions could be reduced by 6.51 times, and even the electric energy used in the light guide plate could be used as solar energy due to the solar cell (see Figure 16b).

Experimental Example 8. Haematococcus pluvialis culture process injected with calcium carbonate in the growth stage

When cultivating Haematococcus pluvialis in the growth stage, the light intensity was varied and various concentrations of calcium carbonate were injected. Calcium carbonate produced through biomineralization in Experimental Example 5 was used. Figure 17a shows the biomass production (g L ^- ) when various concentrations of calcium carbonate were injected while irradiating light of medium intensity (40 μE m ^-2 s ^{- 1} ) when culturing Haematococcus pluvialis in the growth stage. ¹ ), a graph showing FIG. 17b, when various concentrations of calcium carbonate were injected while irradiating strong light (100 to 150 μE m ^-2 s ^{- 1} ) during culturing of Haematococcus pluvialis in the growth stage. A graph showing the biomass ^production (g L ^-1 ) of FIG ^. 17c shows the ^various This is a graph showing the astaxanthin concentration (mg L ^-1 ) when calcium carbonate was injected.

Because calcium carbonate causes a double scattering phenomenon, the culture results confirmed that when light of high intensity is applied, the strong light can be dispersed into several medium intensity lights. In fact, when light was irradiated under general culture conditions, there was little change in biomass depending on the amount of calcium carbonate (see Figure 17a). However, when strong light was irradiated from the growth stage, typical Haematococcus cells died in 2 days, but biomass production and astaxanthin content were the best when 2mM calcium carbonate was injected along with cell inoculation (Figure 17b) , see 17c).

Example 9. PLPA hybrid system in outdoor culture

Referring to Figure 2, in order to increase the culture scale per area, as a result of arranging multiple photobioreactors (PBRs) at high density (PPPA system), the photobioreactors in the middle receive little light due to the blocking effect. . Therefore, a PLPA hybrid system was constructed by placing light guide plates between photobioreactors and supplying light with LEDs to induce biomineralization. Additionally, the LED's electrical energy was supplied from solar cells collected during the day, so no additional electrical energy was used. Figure 18a is a graph showing biomass production (g L ^-1 ) when outdoor cultured for 28 days with the PPPA system and the PLPA hybrid system, respectively, and Figure 18b is a graph showing the 0th and 10th days of outdoor culture of the PPPA system and the PLPA hybrid system. A graph showing the astaxanthin content (%) and photosynthetic efficiency on the 28th day, Figure 18c shows the total production of omega-3 and fatty acids (mg L ^-1 ) when cultured outdoors for 28 days with the PPPA system and the PLPA hybrid system, respectively. , and a graph showing the productivity of total omega-3 and fatty acids (mg L ^-1 day ^-1 ).

Biomass production (g/L), astaxanthin content (%), photosynthetic efficiency at 10 days (μmol/mg/Chl/min), and total omega-3 productivity (mg/L) of the PLPA hybrid system in a dense photobioreactor batch. L/day) and total fatty acid productivity (mg/L/day) were compared with the PPPA system, increasing by 3.34 times, 3.30 times, 2.17 times, 4.12 times, and 3.43 times, respectively, confirming that it was an efficient system.

Although the present invention has been described in detail through specific examples and experimental examples, this is for detailed explanation of the present invention, and the present invention is not limited thereto, and those with ordinary knowledge in the field within the technical spirit of the present invention It is clear that modifications and improvements are possible.

All simple modifications or changes of the present invention fall within the scope of the present invention, and the specific scope of protection of the present invention will be made clear by the appended claims.

Claims (10)

(a) supplying carbon dioxide (CO ₂ ) to the first culture medium in the first reactor to generate hydrogen carbonate ions (HCO ₃ ^- );
(b) inoculating Haematococcus pluvialis into the first culture medium supplied with carbon dioxide; and
(c) Calcium ions (Ca ²⁺ ) are added to the first culture medium inoculated with Haematococcus pluvialis to generate calcium carbonate (CaCO ₃ ), and operated by power generated from a solar cell. A Haematococcus pluvialis culturing method comprising the step of photoculturing using a light source.
In claim 1,
In step (b), the inoculum amount of Haematococcus pluvialis is 0.2 g/L or more.
In claim 1,
A method for cultivating Haematococcus pluvialis wherein the concentration of the calcium ion added in step (c) is 0.5 to 3.5 mM.
In claim 1,
The light source is,
LED that generates light using power generated from the solar cell; and
A Haematococcus pluvialis culture method comprising a light guide panel that diffuses the light generated by the LED.
In claim 4,
A plurality of first reactors are arranged to be spaced apart from each other,
A method of cultivating Haematococcus pluvialis wherein the light source is disposed between one of the first reactors and the other first reactor disposed close to each other.
In claim 1,
In step (c),
Adding the calcium ions and performing first photoculture at a light intensity of 20 to 60 μE/m2/s; and
After the first photoculture step, a second photoculture step at a light intensity of 100 to 150 μE/m2/s. Haematococcus pluvialis culture method comprising a.
In claim 6,
A H. pluvialis culturing method in which the second photo-culture step is performed when the cell density of H. pluvialis reaches 1.0 g/L or more through the first photo-culture.
In claim 1,
The calcium ion in step (c) is a Haematococcus pluvialis culture method in which calcium chloride (CaCl ₂ ) is supplied to the first culture medium.
In claim 1,
(d) recovering the calcium carbonate produced in step (c); and
(e) adding the recovered calcium carbonate to the second culture medium in the second reactor and photoculturing the H. pluvialis.
In claim 9,
The step (e) is a method of cultivating Haematococcus pluvialis by photoculturing at a light intensity of 100 μE/m2/s or more.