Method for culturing microalgae using air

Abstract

The present invention relates to a method for culturing microalgae by using air. The method for culturing microalgae according to an embodiment of the present invention, in which microalgae is cultured inside a reactor, comprises: a step for adding a growth medium and limestone to the inside of the reactor; a step for supplying air to the inside of the reactor to which the growth medium and limestone were added, the air being supplied using an air pump; and a step for inoculating microalgae inside the reactor supplied with air, and growing the microalgae by performing a first photo-culture.

Description

Microalgae cultivation method using air

[0001] The present invention relates to a method for cultivating microalgae using air, and more specifically, to a method for cultivating microalgae by supplying ambient air without a separate supply of carbon dioxide in situations where artificial carbon dioxide supply is impossible or has been interrupted.

[0002]

[0003] Massive greenhouse gas emissions from fossil fuel use are contributing to global warming. Consequently, carbon capture and storage (CCS) technology is actively being developed worldwide to reduce carbon dioxide emissions. CCS technology captures large quantities of carbon dioxide emitted from various carbon sources, including thermal power plants, at high concentrations and then injects them underground or under the seabed to isolate them from the atmosphere. While this CCS technology can effectively reduce large amounts of carbon dioxide in a short period of time, practical implementation remains challenging due to issues such as stable storage, location selection, and high installation costs. Therefore, Carbon Capture & Utilization (CCU) technology, which, unlike CCS, utilizes carbon dioxide directly for industrial purposes or converts it into high-value-added substances rather than simply storing it, is gaining attention. Furthermore, domestic efforts are underway to develop processes that convert carbon dioxide into various high-value-added substances. Among these, the biological conversion process using photosynthetic microorganisms, microalgae, is attracting attention as an economical carbon dioxide reduction technology that simultaneously reduces carbon dioxide and produces various high-value-added substances, such as biofuels, bioplastics, and pharmaceuticals. Microalgae, also known as phytoplankton, are photosynthetic, single-celled aquatic organisms. They are attracting attention as a biomass resource with significant potential for producing energy and industrial materials while simultaneously reducing greenhouse gases. Their utility is expected to expand in the future, particularly in the energy, chemical, and environmental sectors. Microalgae can play a more effective role in mitigating global warming because they consume carbon dioxide while also removing nitrogen oxides and sulfur oxides, which are causes of fine dust. Furthermore, microalgae can produce a variety of useful substances as end products.In addition to microalgae with high protein content such as chlorella, spirulina, and chlamydomonas, Haematococcus, which produces a high value-added substance called astaxanthin, is being used in health functional foods to supplement amino acids, antioxidants, and fatty acids.

[0004] For commercially viable mass cultivation of microalgae, efficient supply of carbon dioxide, the carbon source, is crucial. Optimal pH control is crucial for microalgae cultivation, as the carbon dioxide supplied during the cultivation period maintains a constant pH. Therefore, if carbon dioxide is not supplied, the pH rises, leading to the death of the microalgae.

[0005] Conventionally, carbon dioxide was captured from flue gases emitted from facilities such as power plants and incinerators, and a separate carbon dioxide supply device was used to supply the carbon dioxide to the culture medium. However, facilities such as power plants require maintenance periods, such as overhauls, which can interrupt the carbon dioxide supply. Furthermore, if there is no carbon dioxide source nearby, the outdoor culture facility must be equipped with a separate carbon dioxide transport system, resulting in significant operating costs.

[0006] Accordingly, there is an urgent need for a solution to the problems of the conventional microalgae cultivation method that uses a separate carbon dioxide supply device to artificially supply carbon dioxide.

[0007]

[0008] The present invention is intended to solve the problems of the above-described prior art, and the purpose of the present invention is to provide a method for cultivating microalgae by utilizing carbon dioxide in the air by using inexpensive limestone without a separate artificial supply of carbon dioxide.

[0009]

[0010] A microalgae cultivation method according to an embodiment of the present invention is a microalgae cultivation method for culturing microalgae inside a reactor, comprising: (a) a step of adding a growth medium and limestone inside the reactor; (b) a step of supplying air to the inside of the reactor to which the growth medium and limestone have been added using an air pump; and (c) a step of inoculating microalgae inside the reactor to which the air has been supplied and performing a first light culture to grow the microalgae.

[0011] Additionally, in the microalgae cultivation method according to an embodiment of the present invention, the microalgae may be Chlorella sorokiniana or Haematococcus pluvialis.

[0012] Additionally, in the microalgae cultivation method according to an embodiment of the present invention, when the microalgae is Chlorella sorokiniana, the concentration of the limestone may be 80 to 100 mM.

[0013] Additionally, in the microalgae culturing method according to an embodiment of the present invention, when the microalgae is Haematococcus pluvialis, the concentration of the limestone may be 100 to 120 mM.

[0014] In addition, in the microalgae cultivation method according to an embodiment of the present invention, the microalgae cultivation method can be performed in an environment in which no carbon dioxide is supplied other than the air.

[0015] In addition, in the microalgae cultivation method according to an embodiment of the present invention, (d) the step of replacing the growth phase medium with an induction phase medium and performing a second light culture to induce accumulation of useful substances in the microalgae may be further included.

[0016] In addition, in the microalgae culturing method according to an embodiment of the present invention, the step (d) may be performed when the cell density of the microalgae reaches 1.0 g / L or more through the first light culture.

[0017]

[0018] The features and advantages of the present invention will become more apparent from the following detailed description based on the attached drawings.

[0019] Prior to this, the terms and words used in this specification and claims should not be interpreted in their usual or dictionary meanings, but should be interpreted in the sense and concept that conforms to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0020]

[0021] According to the present invention, by adding limestone to a microalgae culture medium and supplying air to culture microalgae, biomass containing a high content of lipids and astaxanthin can be mass-produced.

[0022] Additionally, biomass can be produced without loss even when the supply of artificial carbon dioxide through exhaust gas emitting facilities such as power plants is interrupted.

[0023]

[0024] Figure 1 is a schematic diagram schematically illustrating a microalgae cultivation method according to an embodiment of the present invention.

[0025] Figure 2 is a graph evaluating the potential of limestone as a source of bicarbonate ions.

[0026] Figure 3 shows the biomass concentration (g L) according to limestone concentration produced through mass cylinder culture of wild Chlorella sorokinia. -1 ) is a graph representing the

[0027] Figure 4 is a graph showing the results of an experiment to confirm whether the effect of limestone on biomass production is due to its pH control function.

[0028] Figure 5 is a graph showing the change in the weight of limestone over time in the cultivation of Chlorella sorokiniana using air and limestone.

[0029] Figure 6 is a graph showing the lipid content and lipid productivity of Chlorella sorokiniana under various conditions.

[0030] Figure 7 is a photograph showing a limestone-based chlorella sorokiniana cultivation process using a 20 L photobioreactor in an outdoor cultivation facility.

[0031] Figure 8 is a photograph showing a limestone-based chlorella sorokiniana cultivation process using a 100 L photobioreactor in an outdoor cultivation facility.

[0032] Figure 9 is a graph showing the change in biomass concentration in a limestone-based chlorella sorokiniana cultivation process using a 100 L photobioreactor in an outdoor cultivation facility.

[0033] Figure 10 is a graph showing changes in biomass concentration of Haematococcus pluvialis depending on the presence or absence of limestone under various conditions.

[0034] Figure 11 is a graph showing the astaxanthin concentration of Haematococcus pluvialis depending on the presence or absence of limestone under various conditions.

[0035] Figure 12 is a graph showing the astaxanthin productivity of Haematococcus pluvialis depending on the presence or absence of limestone under various conditions.

[0036] Figure 13 is a graph showing the change in the weight of limestone over time in the culture of Haematococcus pluvialis using air and limestone.

[0037] Figure 14 is a graph showing the fatty acid and omega-3 content of Haematococcus pluvialis under various conditions.

[0038] Figure 15 is a photograph showing a limestone-based Haematococcus pluvialis cultivation process using a 20 L photobioreactor in an outdoor cultivation facility.

[0039] Figure 16 is a graph showing biomass concentration, astaxanthin content, astaxanthin productivity, and astaxanthin concentration in a limestone-based Haematococcus pluvialis cultivation process using a 20 L photobioreactor in an outdoor cultivation facility.

[0040] Figure 17 is a photograph showing a limestone-based Haematococcus pluvialis cultivation process using a 100 L photobioreactor in an outdoor cultivation facility.

[0041] Figure 18 is a graph showing biomass concentration, astaxanthin content, and astaxanthin productivity in a limestone-based Haematococcus pluvialis cultivation process using a 100 L photobioreactor in an outdoor cultivation facility.

[0042]

[0043] The objects, 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 reference numerals are given to components in each drawing, it should be noted that, as far as possible, identical components are given the same numerals even if they are shown in different drawings. Furthermore, terms such as "first," "second," etc. are used to distinguish one component from another, and the components are not limited by these terms. In the following description of the present invention, detailed descriptions of related known technologies that may unnecessarily obscure the gist of the present invention will be omitted.

[0044] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings.

[0045]

[0046] Fig. 1 is a schematic diagram schematically illustrating a microalgae culturing method according to an embodiment of the present invention. Referring to Fig. 1, a microalgae culturing method according to an embodiment of the present invention is a microalgae culturing method for culturing microalgae inside a reactor, including a step of adding a growth medium and limestone inside the reactor, a step of supplying air using an air pump to the inside of the reactor to which the growth medium and limestone have been added, and a step of inoculating microalgae inside the reactor to which air has been supplied and performing a first light culture to grow the microalgae.

[0047] The present invention relates to a method for cultivating microalgae by supplying ambient air without a separate carbon dioxide supply in situations where artificial carbon dioxide supply is impossible or has been interrupted. Conventionally, carbon dioxide in combustion exhaust gas emitted from facilities such as power plants and incinerators has been captured and carbon dioxide has been supplied to a culture medium using a separate carbon dioxide supply device. However, in facilities such as power plants, the supply of carbon dioxide is interrupted during periods of equipment maintenance such as overhaul, and in cases where there is no carbon dioxide supply source near an outdoor cultivation facility, there is a problem in that a separate carbon dioxide transport device must be installed. The present invention has been devised as a solution to solve these problems of conventional microalgae cultivation methods.

[0048] The microalgae cultivation method according to an embodiment of the present invention is a method of culturing microalgae inside a reactor, and the microalgae to be cultivated are Dunaliella, Chlamydomonas, Scenedesmus, Chlorella, Ettlia, Micractinium, Coelastrum, Haematococcus, Cosmarium, Pediastrum, Pandorina, Eudorina, Euglena, Spirulina, Botryococcus, Tetraselmis, Nannochloropsis, Coccomyxa, Phaeodactylum, It may be at least one species selected from the group consisting of Schizochytrium, Arthrospira, Nitzschia, Isochrysis, Microcystis, Nannochloris, Synechococcus, Synechocystis, and Aurantiochytrium. Preferably, it may be Chlorella sorokiniana or Haematococcus pluvialis. Chlorella sorokiniana has excellent productivity for biofuel that can replace fossil fuels, and Haematococcus pluvialis can produce astaxanthin, a high value-added useful substance that produces antioxidants.

[0049] Specifically, the microalgae cultivation method according to an embodiment of the present invention includes a limestone addition step, an air supply step, and a first light cultivation step.

[0050] The limestone addition step involves adding growth medium and limestone to the reactor. The photo-cultivation process of microalgae is divided into a growth stage and an induction stage, which begins when stress (salt, light, temperature, etc.) is applied. The growth medium contains nutrients necessary for the growth stage of microalgae. For example, TAP-C can be used as a growth medium for Chlorella, and NIES-C can be used as a growth medium for Haematococcus. Limestone is a rock formed by the accumulation of bones, clams, and snail shells of aquatic animals, and is added to the growth medium. Limestone can be added to the growth medium in a solvent-dissolved or solid form, and the order in which the growth medium and limestone are filled in the reactor is irrelevant. The concentration of limestone affects the productivity of biomass and the productivity of useful substances accumulated in the biomass. Accordingly, the optimal limestone concentration may be 80 to 100 mM when the microalgae is Chlorella sorokiniana, and the optimal limestone concentration may be 100 to 120 mM when the microalgae is Haematococcus pluvialis.

[0051]

[0052] The air supply step is the process of supplying air to the growth medium containing limestone inside the reactor. An air pump can be used to supply air inside the reactor. Here, air, a gas constituting the lower layer of the atmosphere, can be used as pure air without any pretreatment. When air is supplied to the growth medium containing limestone, carbon dioxide in the air is adsorbed onto the limestone crystal surface, allowing microalgae to utilize the adsorbed carbon dioxide. Furthermore, limestone contains a large amount of calcium carbonate (CaCO3), which reacts with carbon dioxide and water in the aquatic environment of the growth medium to produce calcium ions and bicarbonate ions. Calcium is an essential trace element for microalgae and influences their growth and development through various functions. First, the initially increased calcium ions act as salt stress due to the excessive concentration compared to the initial cell concentration, increasing reactive oxygen species (ROS). However, as cells grow and adapt to the culture conditions, calcium ions stimulate the cellular antioxidant enzymes (SOD, POD) to alleviate the high ROS. This can prevent cell growth inhibition. Meanwhile, calcium ions are also involved in omega-3 synthesis. Calcium ions bind to pectins, negatively charged molecules in the microalgae cell wall, somewhat reducing the flow of nutrients and stiffening the cell wall. Furthermore, they bind to Ca-binding sites in the cell membrane, organizing the components of the cell membrane and strengthening the cell membrane. Here, microalgae perform homeoviscous adaptation, changing the components of the cell membrane on their own and controlling cell fluidity. At this time, microalgae accumulate more long-chain unsaturated fatty acids to increase the fluidity of cells hardened by calcium ions. In other words, the present invention induces an increase in cell membrane fluidity due to calcium ions, thereby increasing intracellular lipid and omega-3 content.Limestone captures carbon dioxide from the air, providing the same effect as adding carbon dioxide to the growth medium, even without a separate artificial supply. Consequently, the pH of the growth medium can be maintained at 6.5 to 8.0, the optimal range for cultivating Chlorella and Haematococcus.

[0053]

[0054] The first photocultivation stage is a photocultivation process that takes place during the growth phase, when cell division primarily occurs. Here, microalgae are inoculated into a growth medium inside a reactor containing limestone and supplied with air, and light is irradiated to grow the microalgae. The light intensity can be determined depending on the type of microalgae. For example, Chlorella can be cultured under light intensity conditions of 200 to 300 μE / m2 / s, while Haematococcus, which has lower light tolerance than Chlorella species, can be cultured under light intensity conditions of 30 to 50 μE / m2 / s.

[0055] To maintain an appropriate pH for microalgae cultivation, an alkaline solution may be added to the growth medium. For example, the alkaline solution may be a solution containing at least one selected from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), ammonia (NH4OH), calcium hydroxide (Ca(OH)2), and magnesium hydroxide (Mg(OH)2). The alkaline solution may be added not only during the first photo-cultivation step, but also during the limestone addition step or the air supply step. The pH range controlled by this alkaline solution is preferably 6.5 to 8.0.

[0056]

[0057] The microalgae cultivation method according to the present invention can be performed in an environment where no carbon dioxide is supplied other than air. Conventionally, carbon dioxide in combustion exhaust gases emitted from facilities such as power plants and incinerators was captured and artificially supplied with carbon dioxide using separate equipment. However, the present invention enables the cultivation of microalgae by supplying ambient air without the artificial supply of carbon dioxide. Furthermore, in situations where the supply of carbon dioxide is interrupted for a predetermined period of time due to overhaul or equipment failure while artificially supplying carbon dioxide, the microalgae cultivation method according to the present invention can be used to cultivate microalgae.

[0058]

[0059] Meanwhile, after the growth phase through the first photoculture, a second photoculture can be performed. The second photoculture is a photoculture performed during the induction phase to accumulate useful substances in microalgae. Here, the growth phase medium is replaced with the induction phase medium, and by irradiating light, useful substances can be accumulated in microalgae. The useful substances may include lipids, omega-3, astaxanthin, etc. In the second photoculture step, stronger light than the light irradiated during the first photoculture can be irradiated. For example, in the case of Chlorella, photoculture can be performed under light intensity conditions of 300 to 500 μE / ㎡ / s, and in the case of Haematococcus, photoculture can be performed under light intensity conditions of 100 to 150 μE / ㎡ / s.

[0060] The second photocultivation is performed in the induction phase, and the transition from the growth phase to the induction phase can be determined by the morphological changes of the microalgae or the depletion of the nitrogen source in the medium. In addition, since the induction phase can be performed when the cell density of the microalgae in the growth phase medium is 1.0 g / L or higher, the second photocultivation step can be performed at the light intensity when the cell density of the microalgae reaches 1.0 g / L or higher in the first photocultivation, preferably when it is 1.25 g / L.

[0061] Induction media are media containing nutrients necessary for the induction phase of microalgae. For example, TAP-N can be used as a growth medium for Chlorella, and NIES-N can be used as a growth medium for Haematococcus.

[0062]

[0063] The optimal pH for both Chlorella and Haematococcus is 6.5–8.0, and maintaining a constant pH by supplying carbon dioxide during the culture period is crucial. However, when mass-cultivating in outdoor culture facilities, such as power plants, the pH of the medium can rapidly increase to as high as 10.5 during overhauls and other times when carbon dioxide is not supplied. This causes significant stress on the cells, reducing biomass, lipid, and astaxanthin productivity, as well as carbon dioxide removal efficiency. Because carbon dioxide is difficult to transport, outdoor culture facilities are limited to power plant sites. This makes scaling up the biological conversion process of carbon dioxide using microalgae challenging. Therefore, the need for an effective culture method to address these issues is growing. The present invention utilizes air and limestone to mass-produce biomass with high lipid and astaxanthin content comparable to conventional culture methods. Meanwhile, due to limitations in reducing carbon dioxide already present in the atmosphere, Direct Air Capture (DAC) technology has recently been attracting attention. The present invention cultivates microalgae by utilizing carbon dioxide in the air, so cultivation facilities can be built even when there is no carbon dioxide source nearby, such as a power plant, and can be utilized as a biological removal technology for carbon dioxide, which poses a significant threat to humanity.

[0064]

[0065] Below, the present invention is described in more detail through experimental examples.

[0066] 1. Manufacturing process of components for photosynthetic biological reactions

[0067] Eight 500 mL (width: 5 cm, height: 60 cm) mass cylinders were prepared. To block the inlet of the prepared mass cylinders and enable gas injection and sampling, a multi-purpose inlet / outlet made of silicone was used. To install the gas line and sampling line, a 2-3 mm hole was made in the multi-purpose inlet / outlet made of silicone, and two Teflon tubes were cut to fit the height of the mass cylinder and inserted into the holes. A stone sparger was connected to the bottom of the Teflon tube through which gas was supplied, and an air pump was installed to supply air into the mass cylinder.

[0068]

[0069] 2. Microalgae photoculture process

[0070] The media used were TAP-C (Chlorella sorokiniana) and NIES-C (Haematococcus pluvialis), and 450 ml each was injected into a mass cylinder and aeration was performed. Afterwards, 10 mM KOH was injected and the pH was adjusted to 7.5 to 8. This is to maintain the pH between 7.5 and 8 even when carbon dioxide is continuously supplied due to the buffering effect of KOH. Since only air is supplied without artificial carbon dioxide injection, the pH can rise rapidly, so 100 mM limestone was added to solve this problem. Chlorella sorokiniana and Haematococcus pluvialis were used as microalgae, and the initial inoculum amount was 0.1 g L based on biomass. - 1 , and in the growth stage in the mass cylinder, 30 to 50 μ E m -2 s - 1 In the induction phase, 100 to 200 μ E m -2 s - 1 The light was irradiated. The final culture volume in the mass cylinder was 500 mL.

[0071]

[0072] 3. Confirmation of the potential of limestone as a source of bicarbonate ions.

[0073] CaCO3, the main component of limestone, undergoes a series of reactions when it comes into contact with water and CO2 to create calcium ions and bicarbonate ions (CaCO3 (s) + H2O (l) + CO 2 (g) → Ca 2 + (aq) + 2HCO3 - (aq) ).

[0074] Therefore, we conducted a study to integrate a microalgae cultivation system into DAC using limestone. We assumed that as microalgae utilize bicarbonate ions for CCM, an ion sink will be created, allowing more CO2 in the air to dissolve in the medium, and we conducted an experiment to confirm this. Beyond simply dissolving limestone, the experiment aimed to confirm whether sufficient carbon sources for microalgae growth could be supplied from limestone and CO2 in the air at a 1:1 ratio (this will be described later). For the comparison of bicarbonate ions, air, 5% CO2, and N2 were used as gases, and a total of six conditions were set up by including and not adding limestone to the medium.

[0075] First, the theoretical amount of bicarbonate ion that could be dissolved under each condition was calculated. Using the equilibrium constant and pH for four reactions occurring under the condition of bubbling air in the presence of limestone, the amount was calculated to be 65.8 mg L. -1 It was obtained that bicarbonate ions can exist. In the case of bubbling 5% CO2 instead of air under the same conditions, the calculation was done in the same way and 384 mg L -1The bicarbonate ion concentration was obtained. We also checked the case of bubbling nitrogen with a CO2 concentration of 0, unlike air with about 0.04% CO2 and 5% CO2. In this case, the calculation was performed only for the process of limestone dissolving in water, and the solubility of limestone under experimental conditions was used to obtain 61 mg L -1 We obtained the theoretical value called .

[0076] Meanwhile, the condition for bubbling air in the absence of limestone was calculated simply based on the process in which CO2 in the air reacts with water. First, the molar concentration of CO2 gas in an aqueous state was calculated using Henry's law, and then the equilibrium constant for the reaction with water was used for the calculation. As a result, 0.189 mg L -1 Bicarbonate could exist. A similar calculation process was performed for the situation where 5% CO2 was supplied, and 22.57 mg L -1 The concentration of was obtained. In the absence of limestone and the supply of nitrogen, the theoretical value is 0 without the need for calculation.

[0077] In order to compare the theoretical values ​​obtained in this way with the actual measured values, six conditions were set and the amount of bicarbonate ions was measured by sampling at the same time every day. A titration experiment using sulfuric acid was performed, and the results can be confirmed through Figure 2. Figure 2 is a graph evaluating the possibility of limestone as a bicarbonate ion source. The amount of bicarbonate in the medium was significantly higher under the condition with limestone compared to the condition without limestone. In the air condition with limestone, it was 51.91 mg L. -1 , 213.81 mg L under 5% CO2 conditions -1The values ​​were shown. These are 371 and 11 times higher than the conditions without limestone, respectively. When the results were compared with the theoretical maximum values ​​calculated previously, the trends were consistent. Through this, it was confirmed that no matter how much 5% CO2 is continuously bubbling into water, there is a limit to the amount that can be dissolved as carbonate ions in water, and that the carbonate ion concentration is higher depending on the solubility of limestone. It was confirmed that using limestone, it is possible to secure biocarbonate ions equivalent to 5% CO2, which is the condition for cultivating microalgae in the laboratory, even when using air.

[0078]

[0079] 4. Overall biomass process of Chlorella sorokiniana

[0080] Two mass cylinders were used for each condition, and the biomass concentration was 1 to 1.2 g L -1 At the same time, the medium was changed to TAP-N and NIES-N, respectively. First, in order to find the optimal limestone concentration for culturing Chlorella sorokinia, limestone was added from 0 mM to 100 mM in a mass cylinder and cultured for 10 days, and the results are shown in Fig. 3. Fig. 3 shows the biomass concentration (g L) according to limestone concentration produced through mass cylinder culture of Chlorella sorokinia wild bacteria. -1 ) is a graph representing the

[0081] When only limestone was added in an environment with only air supply, the biomass was 0.38 g L -1 It was confirmed that the growth was almost non-existent. However, when limestone was injected at 50 mM and 100 mM and cultured, the biomass was 2.02 g L each. -1 , 2.38 g L -1 It was confirmed that the improvement was 5.32 times and 6.26 times compared to the control (see Fig. 3).

[0082] Since the samples obtained through cultivation may contain dissolved limestone or solid limestone fragments, an effective method to measure only the biomass of Chlorella sorokinia was needed. For this purpose, the following method was used. First, two samples of 10 mL each were sampled, and then one sample was loaded onto a dried weight filter of known weight and dried for a day. The difference in the weights of the two samples was used to calculate the weight of the sample including biomass and other impurities (since the dry weight is 10 mL, the difference was multiplied by 100 to obtain g L). - 1(Converted to .) Another sample had to be washed using distilled water with the pH lowered from 4 to 4.5 using hydrochloric acid. This was to remove any calcium carbonate that might be present by converting it into calcium chloride, water, and carbon dioxide with the weakly acidic distilled water. After washing twice or more, the biomass was loaded onto a dried weight filter of known weight in the same way as before, and dried for a day. The weight difference was used to measure the weight of the biomass itself. In addition, the weight of impurities was also measured by subtracting the weight of the biomass from the previously measured weight including the impurities. As for the limestone remaining after cultivation, the impurities except for the limestone were removed using distilled water with a pH of 8 to 8.5, in contrast to the previous washing process, and only the weight of the limestone itself was measured after the removal of impurities. In order to confirm that the effective biomass production when limestone was used for cultivation was not simply due to the effect of pH control but rather to some effect caused by limestone, the cultivation experiment was conducted in an environment with air supply, with the pH constant maintained between 7 and 7.5 without limestone injection. Figure 4 is a graph showing the experimental results confirming whether the effect of limestone on biomass production is due to its pH control function. The cultivation results confirmed that even when the pH was maintained constant, biomass was 5.08 times higher when limestone was used (see Figure 4). Therefore, limestone not only serves as a pH regulator but also creates the environment necessary for microalgae growth. Ultimately, it was confirmed that limestone effectively adsorbs carbon dioxide from the air and utilizes it for microalgae cultivation.

[0083] To confirm that limestone and air are the carbon sources that affect the growth of microalgae biomass. 13An isotope experiment was conducted using CaCO3 labeled with C. Generally, carbon dioxide in air has a C12 atomic number, and limestone has a C13 atomic number. The results of the isotope experiment are shown in [Table 1] below, and it can be confirmed precisely that the carbon source affecting the growth of microalgal biomass was derived from limestone and air in a 1:1 ratio.

[0084] [Table 1]

[0085]

[0086] First, the initial H 13 CO3 - Half of the sheep are Ca 13 In CO3, the other half came from air, so HCO3 in the medium just before cell inoculation - Under conditions of cultivation with only value and air 13 C / 12 Calculations were performed using the C value. HCO3 in the medium - The value was 5.86 mg, so Ca 13 The value from CO3 is half that, 2.93 mg. The remaining 2.93 mg would have come from air. 13 The C ratio is δ 13 C PDB From (‰) value 13 C / 12 Using the point that C is 0.01053, it was calculated to be 0.03 mg. Therefore, the initial H 13 CO3 - The value is 2.96 mg HCO3 - Since 1 kg of biomass can be obtained from 2.6 kg of H 13 CO3 - The biomass that can grow is 1.13 mg. Since 2.25 mg of biomass can grow with the 5.86 mg that was initially present in the medium, 1.12 mg, which is 1.13 mg minus H 12 CO3 - You can see that it came from .

[0087] Meanwhile, the final biomass of the cells obtained was 86 mg and HCO3 - 83.75 mg, excluding 2.25 mg grown under air conditions, is the value grown under air-derived carbon source after cell inoculation. 13 C / 12 Using the C value, 82.88 mg out of 83.75 mg 12 0.87 mg from C 13 You can see that it grew from C.

[0088] To summarize, among biomass 13 The amount from C is 2mg, which is 1.13mg and 0.87mg added together. 12 The amount from C is 84 mg, which is 1.12 mg and 82.88 mg added together. Through this 13 C / 12 If we calculate the C value, it is 0.0238, which is obtained from an actual isotope analysis experiment. 13 C / 12 It can be seen that the C value of 0.01924 is theoretically established. Furthermore, previous studies have shown that some CO2 is re-released during photorespiration by the oxygenase activity of Rubisco. This phenomenon increases as the CO2 concentration decreases, and the result showed that approximately 20.5% of CO2 is re-released at 1% CO2. Accordingly, 13 C / 12 Approximately 23.7% of the difference in C values ​​can be attributed to photorespiration.

[0089] Through this, it was found that in an airless environment, cells grew not using limestone as a carbon source, but using carbon dioxide in the air for growth. In order to confirm how much limestone was actually utilized in the culture of Chlorella sorokiniana, the weight of limestone before and after cultivation was measured, and the results are shown in Figure 5. Figure 5 is a graph showing the change in limestone weight over time in the culture of Chlorella sorokiniana using air and limestone. The weight of limestone used was 0.352 g L. -1 Through this, 0.155 g L -1 (0.352 X CO2 molecular weight / 100) carbon dioxide can be generated and with this amount 0.085 g L -1 It can only produce 0.155 / 1.83 (conversion factor) biomass. The final biomass of the cell is 2.38 g L -1 As limestone only contributed 3.49% to biomass production, it was found that carbon dioxide in the air was responsible for the remaining 96.51% (see Fig. 5).

[0090] Figure 6 is a graph showing the lipid content and lipid productivity of Chlorella sorokiniana under various conditions. Referring to Figure 6, the lipid content and lipid productivity were similar to those observed when supplied with 5% carbon dioxide, and were 5.6 and 14 times higher, respectively, than those observed in cells grown in air- and limestone-free environments.

[0091] Figure 7 is a photograph showing the limestone-based Chlorella sorokiniana cultivation process using a 20L photobioreactor in an outdoor cultivation facility. As shown in Figure 7, to verify whether limestone-based cultivation can be effectively operated outdoors, cultivation was conducted using a 20L reactor in the outdoor cultivation system of the Korea District Heating Corporation in Pangyo. The cultivation results confirmed that biomass increased 20.17 times when limestone was added in a situation where only air was supplied compared to when limestone was not added. In fact, theoretical calculations were performed to verify whether biomass growth was theoretically possible using 0.04% carbon dioxide in the air. It was assumed that cultivation was conducted in an environment where air was supplied at 0.1 vvm in a 5L incubator. In this case, the total amount of air supplied to the reactor was 720 L day. -1 And since the proportion of carbon dioxide in the air is 0.04%, the amount of carbon dioxide supplied is 0.288 L day. -1 It can be seen that. Since V = 22.4 L when 1 mol of gas is supplied at room temperature and pressure, 0.0129 mol of carbon dioxide is supplied. Since the molecular weight of carbon dioxide is 44, the total amount of carbon dioxide supplied for 16 days is 9.05 g. Therefore, since 4.81 g of biomass can grow with 9.05 g of carbon dioxide, it can be seen that the biomass obtained through cultivation is a theoretically possible value (biomass × (44 (carbon dioxide molecular weight) / 12 (carbon molecular weight)) × 0.496 (carbon content in Chlorella sorokiniana)). In reality, it was confirmed that when only air was supplied and limestone was not injected, the cells eventually died.

[0092] Fig. 8 is a photograph showing a limestone-based Chlorella sorokiniana cultivation process using a 100 L photobioreactor in an outdoor cultivation facility. An experiment was conducted to scale up the cultivation process of the present invention, which utilizes limestone for the cultivation of Chlorella sorokiniana with excellent growth potential, to 100 L. Fig. 9 is a graph showing the change in biomass concentration in a limestone-based Chlorella sorokiniana cultivation process using a 100 L photobioreactor in an outdoor cultivation facility. The cultivation results in Fig. 9 confirmed that the biomass production was 8.2 times higher when limestone was present than when it was not present in a situation where air was not supplied.

[0093]

[0094] 5. Overall biomass process of Haematococcus pluvialis

[0095] In order to confirm whether astaxanthin can be effectively produced using air even in a situation where artificial carbon dioxide is not supplied, an experiment was conducted on Haematococcus, similar to the experiment on Chlorella. Fig. 10 is a graph showing the change in biomass concentration of Haematococcus pluvialis according to the presence or absence of limestone under various conditions, Fig. 11 is a graph showing the astaxanthin concentration of Haematococcus pluvialis according to the presence or absence of limestone under various conditions, and Fig. 12 is a graph showing the astaxanthin productivity of Haematococcus pluvialis according to the presence or absence of limestone under various conditions.

[0096] Referring to Figure 10, in an environment supplied only with air, the Haematococcus biomass was 0.24 g L -1 It was confirmed that the growth was almost non-existent. However, when limestone was additionally added at 100 mM and cultured, the biomass was 2.65 g L -1 It was confirmed that it was 11.04 times higher than the control group and 4.73 times higher than the experimental group in which the pH was kept constant without limestone.

[0097] Referring to Figure 11, the concentration of astaxanthin when 5% CO2 was supplied and when air and limestone were used was 101 mg L each. -1 , 94 mg L -1 There was no significant difference. However, in an air-supplied environment, the astaxanthin concentration showed a significant difference depending on the presence or absence of limestone. When limestone was present, the astaxanthin concentration was more than 18 times higher. As shown in Figure 12, astaxanthin productivity was found to be more than 25 times higher in the presence of limestone than in its absence. This demonstrated that limestone can be effectively applied to Haematococcus culture.

[0098] Figure 13 is a graph showing the weight change of limestone over time in the cultivation of Haematococcus pluvialis using air and limestone. In order to confirm how much limestone was actually utilized in the cultivation of Haematococcus pluvialis, the weight of limestone used was measured before and after cultivation, and the weight of limestone used was 0.492 g L. -1 , through this 0.216 g L -1 (0.492 X CO2 molecular weight / 100) carbon dioxide can be generated and with this amount 0.116 g L -1 It can only produce 0.116 / 1.83 (conversion factor) biomass. The final biomass of the cell is 2.65 g L -1 As such, it can be seen that limestone only contributed 4.45% of biomass to production, so carbon dioxide in the air, which is a carbon source, contributed to the remaining 95.55% (see Figure 13).

[0099] Figure 14 is a graph showing the fatty acid and omega-3 content of Haematococcus pluvialis under various conditions. The fatty acid content increased by 24.26% when cultured using limestone compared to when supplied with 5% CO2. In particular, the omega-3 content was confirmed to have increased by 24%.

[0100] Fig. 15 is a photograph showing a limestone-based Haematococcus pluvialis cultivation process using a 20 L photobioreactor in an outdoor cultivation facility. To confirm whether limestone-based cultivation can be effectively operated outdoors, cultivation was performed using a 20 L reactor in the outdoor cultivation system of the Korea District Heating Corporation in Pangyo. Fig. 16 is a graph showing the biomass concentration, astaxanthin content, astaxanthin productivity, and astaxanthin concentration in a limestone-based Haematococcus pluvialis cultivation process using a 20 L photobioreactor in an outdoor cultivation facility. The cultivation results showed that the biomass increased 23.2 times when limestone was added in a situation where only air was supplied compared to when it was not added. The astaxanthin concentration and productivity were also confirmed to be 10 times and 32 times higher, respectively, when limestone was present in a situation where air was supplied compared to when it was not present. In order to confirm whether biomass growth is theoretically possible using 0.04% carbon dioxide in the air, the following calculations were performed and it was found that 4.34 g of biomass can be accumulated with the carbon dioxide in the supplied air, which means that cultivation is sufficient. In the absence of limestone when air is supplied, it was confirmed that the pH increased rapidly, leading to cell death. In addition, it was found that contamination by contaminants such as bacteria and fungi occurred seriously. Therefore, a constant supply of carbon dioxide is essential in Haematococcus culture, and an effective cultivation technique utilizing limestone in a situation where the carbon dioxide supply is interrupted is expected to be an important source technology for the commercialization of astaxanthin.

[0101] Fig. 17 is a photograph showing a limestone-based Haematococcus pluvialis cultivation process using a 100 L photobioreactor in an outdoor cultivation facility, and Fig. 18 is a graph showing biomass concentration, astaxanthin content, and astaxanthin productivity in a limestone-based Haematococcus pluvialis cultivation process using a 100 L photobioreactor in an outdoor cultivation facility. As shown in Fig. 17, cultivation was performed by scaling up to 100 L. As a result of the cultivation, it was confirmed that when limestone was present in a situation where air was not supplied, the Haematococcus biomass production was increased by 15 times, the astaxanthin content was increased by 8.75 times, and the astaxanthin productivity was increased by 30 times compared to when limestone was not present (see Fig. 18).

[0102]

[0103] Although the present invention has been described in detail through specific examples, this is intended to specifically explain the present invention, and the present invention is not limited thereto, and it is clear that modifications and improvements can be made by those skilled in the art within the technical spirit of the present invention.

[0104] 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.

[0105]

[0106] The present invention relates to a technology for mass-producing biomass containing high lipid content and astaxanthin by adding limestone to a microalgae culture medium and supplying air to culture microalgae, and thus, the possibility of industrial application is recognized.

Claims

1. In a microalgae cultivation method for cultivating microalgae inside a reactor, (a) a step of adding growth medium and limestone to the inside of the reactor; (b) a step of supplying air to the inside of the reactor to which the growth medium and the limestone are added using an air pump; and (c) A method for culturing microalgae, comprising the step of inoculating microalgae into the interior of the reactor to which the air is supplied and performing a first light culture to grow the microalgae.

2. In claim 1, The above microalgae are, A method for culturing microalgae, such as Chlorella sorokiniana or Haematococcus pluvialis.

3. In claim 2, If the above microalgae is Chlorella sorokiniana, A microalgae cultivation method wherein the concentration of the limestone above is 80 to 100 mM.

4. In claim 2, If the above microalgae is Haematococcus pluvialis, A microalgae cultivation method wherein the concentration of the limestone above is 100 to 120 mM.

5. In claim 1, The above microalgae cultivation method is, A method for cultivating microalgae performed in an environment where no separate carbon dioxide is supplied other than the above air.

6. In claim 1, (d) A method for culturing microalgae, further comprising the step of replacing the growth medium with an induction medium and performing a second light culture to induce accumulation of useful substances in the microalgae.

7. In claim 6, Step (d) above, A microalgae cultivation method performed when the cell density of the microalgae reaches 1.0 g / L or more through the above first light culture.

Images