Methods for carbon dioxide sequestration

Abstract

To provide a carbon dioxide fixation method that eliminates the need for artificial injection of carbon dioxide, and allows for the removal of carbon dioxide that has naturally dissolved into seawater from the atmosphere, etc., through vapor-liquid equilibrium, by fixing it as carbonate. [Solution] A method for fixing carbon dioxide, characterized by fixing and removing carbon dioxide from seawater as carbonate, comprising the steps of: (A) forming a cyanobacteria mat on the surface of a solid; (B) placing the cyanobacteria mat in seawater; and (C) irradiating the cyanobacteria mat in seawater with light to form carbonate.

Description

[Technical Field]

[0001] This invention relates to a method for fixing carbon dioxide. [Background technology]

[0002] In recent years, carbon dioxide (CO2) emissions from the combustion of fossil fuels in industrial activities have been identified as one of the main causes of global warming, and various CO2 fixation technologies are being considered. One such CO2 fixation technology being researched involves using photosynthetic organisms to fix and remove carbon dioxide from water.

[0003] As such a method for fixing carbon dioxide in water, for example, Japanese Patent Publication No. 2013-540448 (Patent Document 1) discloses a biological method for capturing carbon dioxide using cyanobacteria, which includes the steps of: culturing cyanobacteria in an aqueous medium containing carbonate ions and bicarbonate ions in the presence of calcium; enabling photosynthesis by cyanobacteria based on bicarbonate ions to produce a precipitate of calcium carbonate (CaCO3); and supplying inorganic carbon by injecting CO2 to adjust the pH during the photosynthetic reaction. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2013-540448 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, conventional methods for fixing carbon dioxide in water, such as the one described in Patent Document 1, use cyanobacteria dispersed in a liquid culture to fix carbon dioxide, and require a step of supplying inorganic carbon into the system by injecting carbon dioxide to adjust the pH. Therefore, it was necessary to fix carbon dioxide in a closed system (batch type) in which the cyanobacteria and / or the carbon dioxide injected into the system do not move to the outside of the system. Paragraph

[0049] of Patent Document 1 also states that it is impossible to obtain calcium carbonate precipitate from seawater using a batch-type cyanobacterial culture (finite volume) without supplying additional inorganic carbon.

[0006] This invention has been made in view of the problems of the prior art described above, and aims to provide a carbon dioxide fixation method that does not require the artificial injection of carbon dioxide, and that can fix and remove carbon dioxide that has naturally dissolved in seawater from the atmosphere, etc., by gas-liquid equilibrium, etc., as carbonate. [Means for solving the problem]

[0007] As a result of diligent research to achieve the above objective, the present inventors have discovered that by providing a method for fixing carbon dioxide that includes the steps of (A) forming a cyanobacteria mat on which filamentous cyanobacteria are fixed to the surface of a solid, (B) placing the cyanobacteria mat in seawater, and (C) irradiating the cyanobacteria mat in seawater with light to form carbonates, it is possible to fix and remove carbon dioxide that has naturally dissolved into seawater from the atmosphere, etc., through gas-liquid equilibrium, etc., as carbonates, without the need to artificially inject carbon dioxide, and thus have completed the present invention.

[0008] In other words, the present invention provides the following embodiments.

[0009] [1] A step (A) in which filamentous cyanobacteria are fixed to the surface of a solid, The process of placing the cyanobacteria mat in seawater (B), (C) A step of irradiating the cyanobacteria mat in seawater with light to form carbonates, A method for fixing carbon dioxide, which removes carbon dioxide from seawater by fixing it as carbonates by including [a specific substance / method].

[0010] [2] The method for fixing carbon dioxide according to [1], wherein the cyanobacteria mat is such that, when the proportion of filamentous cyanobacteria among the bacteria present on the surface of the solid is determined by 16S rRNA gene analysis, the proportion of filamentous cyanobacteria is 90% or more.

[0011] [3] The carbon dioxide fixation method according to [1] or [2], wherein the filamentous cyanobacteria comprises at least one species selected from the group consisting of cyanobacteria belonging to the genus Scytonema and cyanobacteria belonging to the genus Calothrix.

[0012] [4] A method for fixing carbon dioxide according to any one of [1] to [3], wherein the filamentous cyanobacteria comprises at least one species of cyanobacteria selected from the group consisting of cyanobacteria belonging to the genus Scytonema and identified in NIES-4073; and cyanobacteria belonging to the genus Calothrix and identified in NIES-267.

[0013] [5] A method for fixing carbon dioxide according to any one of [1] to [4], wherein the filamentous cyanobacteria belongs to the genus Calothrix and is identified in NIES-267.

[0014] [6] A method for fixing carbon dioxide according to any one of [1] to [4], wherein the filamentous cyanobacteria belongs to the genus Scytonema and is identified by NIES-4073.

[0015] [7] A method for fixing carbon dioxide according to any one of [1] to [6], wherein the light irradiated onto the cyanobacteria mat in seawater includes light with a wavelength of 300 nm to 850 nm.

[0016] [8] A method for fixing carbon dioxide according to any one of [1] to [7], wherein the dry weight ratio of CPS (capsular polysaccharide or mucopolysaccharide) of the filamentous cyanobacteria is 1000 μg / g (cell fr. wt) or more per 1 g of wet weight of the cell. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide a carbon dioxide fixation method that does not require the artificial injection of carbon dioxide, and that can fix and remove carbon dioxide that has naturally dissolved in seawater from the atmosphere, etc., through gas-liquid equilibrium, etc., as carbonate. [Brief explanation of the drawing]

[0018] [Figure 1] These are micrographs showing the state of filamentous cyanobacteria fixed on the cyanobacteria mat formed in Example 1 at specific time points (the start time of the experiment, 13 hours after the start time, approximately 38 hours after the start time, and approximately 62 hours after the start time). [Figure 2] The graphs show the relationship between the distance from the surface of the cyanobacteria mat formed in Example 1 and pH for each photon flux density of the irradiated light, and the graphs show the relationship between the distance from the surface of the agar medium used in Comparative Example 1 and pH. [Figure 3] The graphs show the relationship between the distance from the surface of the cyanobacteria mat formed in Example 1 and the concentration of Ca2+ ions for each photon flux density of the irradiated light, and the graphs show the relationship between the distance from the surface of the agar medium used in Comparative Example 1 and pH. [Figure 4]The graphs show the relationship between the distance from the surface of the cyanobacteria mat formed in Example 1 and the O2 concentration for each photon flux density of the irradiated light, and the graphs show the relationship between the distance from the surface of the agar medium used in Comparative Example 1 and the pH. [Figure 5] These are micrographs showing the state of filamentous cyanobacteria fixed on the cyanobacteria mat formed in Example 2 at specific time points (the start time of the experiment, 14 hours after the start time, approximately 39 hours after the start time, and approximately 63 hours after the start time). [Figure 6] The graphs show the relationship between the distance from the surface of the cyanobacteria mat formed in Example 2 and pH for each photon flux density of the irradiated light, and the graphs show the relationship between the distance from the surface of the agar medium used in Comparative Example 2 and pH. [Figure 7] The graphs show the relationship between the distance from the surface of the cyanobacteria mat formed in Example 2 and the concentration of Ca2+ ions for each photon flux density of the irradiated light, and the graphs show the relationship between the distance from the surface of the agar medium used in Comparative Example 2 and pH. [Figure 8] The graphs show the relationship between the distance from the surface of the cyanobacteria mat formed in Example 2 and the O2 concentration for each photon flux density of the irradiated light, and the graphs show the relationship between the distance from the surface of the agar medium used in Comparative Example 2 and the pH. [Figure 9] The graphs show the relationship between the amount of carbon dioxide removed and the photon flux density for the cyanobacteria mats obtained in Examples 1 and 2 and the agar media used in Comparative Examples 1 and 2. [Figure 10] This is a photograph showing the state of the cyanobacteria mat produced in Example 3 (the morphology of filamentous bacteria placed in artificial seawater) as it is placed in a petri dish containing artificial seawater. [Figure 11] This graph shows the relationship between the distance from the surface of the cyanobacteria mat formed in Example 3 and the concentration of Ca2+ ions, for each photon flux density of the irradiated light. [Figure 12]This is a scanning electron microscope (SEM) image of a portion of the surface of the cyanobacteria mat recovered after the light irradiation process in Example 4. [Figure 13] Figure 12 shows a graph of the EDS spectrum of the crystal located within the rectangular area in the scanning electron microscope image. [Modes for carrying out the invention]

[0019] The present invention will be described in detail below with reference to its preferred embodiments. In this specification, unless otherwise specified, the notation "X~Y" for numerical values ​​X and Y means "X or greater and Y or less". If a unit is attached only to the numerical value Y in such notation, that unit shall also apply to the numerical value X.

[0020] The carbon dioxide fixation method of the present invention is The process involves (A) forming a cyanobacteria mat on the surface of a solid, in which filamentous cyanobacteria are fixed, The process of placing the cyanobacteria mat in seawater (B), (C) A step of irradiating the cyanobacteria mat in seawater with light to form carbonates, This method is characterized by its ability to fix and remove carbon dioxide from seawater as carbonates by including [a specific ingredient / method].

[0021] [Regarding process (A)] Step (A) is a step in which a cyanobacteria mat is formed in which filamentous cyanobacteria are fixed to the surface of a solid.

[0022] Examples of such filamentous cyanobacteria include cyanobacteria belonging to the genera Calothrix, Leptolyngbya, Lyngbya, Microcoleus, Nostoc, Oscillatoria, Phormidium, Plectonema, Pleurocapsa, Pseudanabaena, Rivularia, Schizothrix, Scytonema, and Spirulina.

[0023] Furthermore, as such filamentous cyanobacteria, it is possible to use, for example, the cyanobacteria listed in Tables 1-3 below, with their "NIES number (strain number of the Microbial System Preservation Facility, National Institute for Environmental Studies)" and "scientific name," as appropriate.

[0024] [Table 1]

[0025] [Table 2]

[0026] [Table 3]

[0027] Among these filamentous cyanobacteria, from the viewpoint of rapid growth (short subculturing cycle), cyanobacteria belonging to the genera Scytonema, Calothrix, Leptolyngbya, Nostoc, Oscillatoria, Phormidium, Pleurocapsa, Pseudanabaena, Rivularia, and Spirulina are more preferred, with cyanobacteria belonging to the genera Scytonema being particularly preferred.

[0028] Furthermore, among such filamentous cyanobacteria, those belonging to the genus Scytonema and identified in NIES-4073 are particularly suitable for use because they can achieve high carbon dioxide removal performance even in environments with low light intensity. Also, from another perspective, since carbon dioxide removal performance is higher under strong light irradiation conditions that result in a larger photon flux density, making them more suitable for long-term outdoor use, those belonging to the genus Calothrix and identified in NIES-267 are particularly suitable for use among the aforementioned filamentous cyanobacteria. From these perspectives, it is preferable that the filamentous cyanobacteria consist of at least one species selected from the group consisting of cyanobacteria belonging to the genus Scytonema and cyanobacteria belonging to the genus Calothrix, and more preferably at least one species selected from the group consisting of cyanobacteria belonging to the genus Scytonema and identified in NIES-4073, and cyanobacteria belonging to the genus Calothrix and identified in NIES-267.

[0029] Furthermore, in such filamentous cyanobacteria, the dry weight ratio of CPS (capsular polysaccharides or mucopolysaccharides) is preferably 100 μg / g (cell fr. wt) or more per 1 g of wet weight of the cell, more preferably 450 μg / g (cell fr. wt) or more, even more preferably 1000 μg / g (cell fr. wt) or more, particularly preferably 2000 to 50000 μg / g (cell fr. wt), and most preferably 3000 to 20000 μg / g (cell fr. wt). By setting the CPS value above the aforementioned lower limit, it becomes possible to interact CPS and carbonates extracellularly compared to the case below the lower limit, resulting in even greater effects in terms of facilitating carbonate retention, stabilizing carbonates, and efficiently promoting the growth of carbonate crystals. Here, "CPS" refers to the polysaccharides that constitute the capsule or slime layer located on the outside of the cell wall of filamentous cyanobacteria, i.e., capsular polysaccharides or slime polysaccharides. The dry weight percentage of CPS can be determined by the "Analysis Method for CPS" described below.

[0030] <Methods for analyzing CPS> First, filamentous cyanobacteria are obtained as the target organism for determining the dry weight of CPS, and these filamentous cyanobacteria are cultured using a suitable culture medium (e.g., BG-11, f / 2, etc.). The culture method involves using a 1.5L culture bottle containing 1L of culture medium, irradiating it with fluorescent light (light intensity: 1500 lux), aerating the bottle at a flow rate of 250L / h, and intermittently flowing CO2 to maintain the pH of the medium. After culturing the filamentous cyanobacteria in this manner for 4 weeks, the cells are collected by filtration using filter paper. The collected cells are then suspended in sodium phosphate buffer (100ml) with a pH of 7.0 and a concentration of 20 mmol / L to obtain a suspension. Next, the obtained suspension is subjected to heat treatment by incubation at 60°C for 10 hours. Furthermore, this heat treatment causes the membrane polysaccharides or mucopolysaccharides that constitute the membrane or slime layer located outside the cell wall of the cells to dissolve in the liquid. Next, the cells in the suspension are removed by centrifugation, and the supernatant, which is the liquid in which the membrane polysaccharides or mucopolysaccharides have dissolved, is used as the sample for CPS analysis. Meanwhile, the weight of the cells removed by centrifugation (wet cells) is measured, and the obtained measurement value is adopted as the "wet weight of the cells (unit: g (cell fresh weight))".

[0031] Next, the sample for CPS analysis (the supernatant obtained by centrifugation) is concentrated through a membrane filter with a nominal molecular weight cutoff of 100 kDa (Amicon®, manufactured by Merck Millipore) to a final volume of approximately 50 ml. The concentrated solution obtained in this way is dialyzed with tap water for two days. Then, an equal volume of ethanol is added to the dialyzed liquid, and the resulting alcohol solution is stored overnight at a temperature of -18°C (depending on the type of filamentous cyanobacteria, it may be stored for an appropriate time between 12 and 24 hours) to form a precipitate (ethanol precipitation). Subsequently, the liquid containing the precipitate is centrifuged for 30 minutes under a centrifugal force of 15,300 g to obtain a pellet (precipitate). Next, the obtained pellet is dissolved in hot water (to 1 / 10 of the initial volume) to obtain a solution (hot water dissolution). Then, instead of the dialyzed liquid, the aforementioned solution is used, and the steps of "ethanol precipitation" and "hot water dissolution" described above are repeated multiple times. The resulting solution is then dialyzed with tap water (for 48 hours) and distilled water (for 20 hours), freeze-dried, and the dry weight of the resulting solid is measured. This dry weight of the solid is adopted as the dry weight of CPS (unit: μg). Using the wet weight of the cells and the dry weight of the CPS measured in this way, the ratio of the dry weight of CPS per gram of wet weight of the cells ([dry weight of CPS (unit: μg)] / [wet weight of cells (unit: g (cell fresh weight))]) can be calculated to determine the ratio of the dry weight of CPS (unit: μg / g (cell fr. wt)).

[0032] Furthermore, the dry weight percentage of CPS (capsular polysaccharides or mucopolysaccharides) of the filamentous cyanobacteria obtained in this manner is preferably within the "preferred numerical range for the dry weight percentage of CPS" shown in Table 4 for each strain species of cyanobacteria of the genus shown in Table 4 below.

[0033] [Table 4]

[0034] Furthermore, filamentous cyanobacteria capable of forming stromatolites are preferably used. By using filamentous cyanobacteria capable of forming stromatolites, it becomes possible to form carbonates using the filamentous cyanobacteria, precipitate them, and then stack layers containing the carbonates to form stromatolites. This makes it possible to fix carbon dioxide in seawater into the stromatolites and remove it stably. As such filamentous cyanobacteria capable of forming stromatolites, those that can fix nitrogen on their own even in the ocean where nutrient sources are limited are preferred. Among these, cyanobacteria belonging to the genera Scytonema, Calothrix, Oscillatoria, Nostoc, and Rivularia are preferred. Moreover, from the viewpoint of obtaining high carbon dioxide removal performance even in environments with low light intensity, cyanobacteria belonging to the genus Scytonema are more preferred, and from the viewpoint of being more suitable for long-term outdoor use, cyanobacteria belonging to the genus Calothrix are more preferred.

[0035] Furthermore, in step (A), the filamentous cyanobacteria are fixed to the surface of a solid to form a cyanobacteria mat.

[0036] The solid used to form such a cyanobacteria mat is not particularly limited, as long as it can fix the filamentous cyanobacteria, and known solid culture media (e.g., agar) or known carriers for microbial immobilization (e.g., "Porous α®" manufactured by Tiger Chiyoda Material Co., Ltd., "sterapore" manufactured by Mitsubishi Chemical Corporation, etc.) can be used as appropriate.

[0037] Furthermore, the shape of the solid is not particularly limited, and any shape that can fix filamentous cyanobacteria to its surface is acceptable. Various shapes can be used as appropriate, depending on the situation of use, such as where the cyanobacteria mat is used, including plate-shaped, disc-shaped (disc-shaped plate, disc-shaped mesh, disc-shaped filter, etc.), cubic, and cylindrical shapes. The solid may also be porous, for example, a porous glass filter (glass filter manufactured by Shibata Scientific Co., Ltd.) or a porous glass foam material (product name "Porous α®" manufactured by Tiger Chiyoda Material Co., Ltd.) may be used as appropriate. Thus, the shape of the solid used to form the cyanobacteria mat is not particularly limited, and basically, any known solid (carrier) that can attach and fix microorganisms and can be submerged in seawater can be used as appropriate. Considering that the solid will be placed in seawater, it is preferable that it be made of a material that has a low impact on the environment. For example, materials made from inorganic materials that do not contain plastics or biomass-derived materials can be suitably used.

[0038] Furthermore, a cyanobacteria mat in which filamentous cyanobacteria are cultured and fixed in a planar manner on a solid surface is more preferable. By using a cyanobacteria mat in which filamentous cyanobacteria are cultured and fixed in a planar (two-dimensional) manner, it becomes possible to create a concentration gradient of substances in seawater near that plane, thereby forming carbonates more efficiently. Moreover, by forming carbonates using a cyanobacteria mat in this way, it becomes possible to precipitate the carbonates on a solid surface and layer them.

[0039] Furthermore, it is preferable that such a cyanobacteria mat has a filamentous cyanobacteria content of 90% or more (more preferably 95% to 100%, and particularly preferably 100%) when the proportion of filamentous cyanobacteria among the bacteria present on the surface of the solid (or, if a bacterial community of multiple species is formed on the surface of the solid, "the proportion of filamentous cyanobacteria among the bacterial community present on the surface of the solid") is determined by 16S rRNA gene analysis. In this way, by making the proportion of filamentous cyanobacteria among the bacteria present on the surface of the cyanobacteria mat (among all types of bacteria present on the surface of the cyanobacteria mat) 90% or more, it becomes possible to more efficiently fix and remove carbon dioxide using the filamentous cyanobacteria. Such 16S rRNA gene analysis can be performed using known methods. For example, the method for 16S rRNA gene analysis described in the known literature (Tomoyo Okumura et al., “Processes Forming Daily Lamination in a Microbe-Rich Travertine Under Low Flow Condition at the Nagano-yu Hot Spring, Southwestern Japan”, Geomicrobiology Journal, 2013, Volume 30, Issue 10) may be used. Furthermore, for such 16S rRNA gene analysis, BLAST (Basic Local Alignment Search Tool) provided by NCBI (National Center for Biotechnology Information) may be used, and the Bellerophon program may be used as a computer program for detecting chimeric sequences. Alternatively, the microbial community analysis tool "QIIME 2 (https: / / qiime2.org / )" may also be used for such 16S rRNA gene analysis.

[0040] Furthermore, as a specific method for determining the proportion of filamentous cyanobacteria among bacteria present on the surface of such a solid, a method of determining the "proportion of filamentous cyanobacteria among bacteria present on the surface of a solid" by 16S rRNA gene analysis employing the steps (procedures) described below can be suitably employed. Specifically, first, a portion of the bacterial mass on the surface of the solid to be measured is collected, a surfactant solution (for example, "Lysis Solution F" manufactured by Nippon Gene Co., Ltd.) is added to the collected bacterial mass, and then the bacterial cells constituting the bacterial mass are pulverized using a cell disruptor (for example, "Shake Master Neo" manufactured by bms Co., Ltd.) to produce a sample. Next, the sample is left to stand at 65°C for 10 minutes, then centrifuged to separate the supernatant solution, and DNA is purified from the separated solution (a commercially available DNA extraction (purification) kit (for example, "Lab-Aid 824s DNA Extraction kit" manufactured by ZEESAN Co., Ltd.) may be used for such DNA purification). Next, the purified DNA is used to perform amplicon sequencing of the V3-V4 region to obtain sequence data (fastq file, etc.) (a preferred step for this is to amplify the V3-V4 region of the 16S rRNA gene using PCR with purified DNA, and then perform sequence analysis (amplicon sequencing analysis of the V3-V4 region) on the obtained PCR product using a next-generation sequencer (e.g., Illumina's "MiSeq") to obtain a fastq file). Then, the obtained sequence data (fastq file, etc.) is analyzed using a microbial community analysis tool (e.g., QIIME 2 (https: / / qiime2.org / )) to calculate the proportion of filamentous cyanobacteria among all bacteria in the sample, and the calculated value is defined as "the proportion of filamentous cyanobacteria among bacteria present on the surface of a solid." In this way, "the proportion of filamentous cyanobacteria among bacteria present on the surface of a solid" can be determined.

[0041] Furthermore, while there are no particular limitations on the method for immobilizing filamentous cyanobacteria on the surface of a solid to form a cyanobacteria mat, a preferred method is to inoculate filamentous cyanobacteria onto the surface of a solid and then culture the filamentous cyanobacteria on the surface of the solid to immobilize them.

[0042] Furthermore, while there are no particular restrictions on the method of culturing filamentous cyanobacteria to be inoculated onto the surface of a solid, it is preferable to adopt a method of culturing filamentous cyanobacteria by three-dimensional culture using a liquid medium, from the viewpoint of rapid growth rate and the ability to more efficiently grow filamentous cyanobacteria used for carbon dioxide fixation. In this way, by efficiently growing filamentous cyanobacteria used for the production of cyanobacterial mats by three-dimensional culture, it becomes possible to produce cyanobacterial mats used for carbon dioxide fixation more efficiently. In other words, by culturing the filamentous cyanobacteria before inoculation onto the surface of a solid using three-dimensional culture, and then culturing the filamentous cyanobacteria on the surface of the solid (two-dimensional culture) after inoculation, the production efficiency of cyanobacterial mats can be improved. As for the liquid medium used for such three-dimensional culture, any known medium (e.g., MDM liquid medium) can be used as appropriate. Furthermore, in the present invention, since filamentous cyanobacteria are fixed to the surface of the solid to form a cyanobacteria mat, it is possible to easily adjust the proportion of filamentous cyanobacteria among the bacteria present on the surface of the solid to 90% or more (more preferably 95% or more and 100%, and especially preferably 100%). Moreover, by using the cyanobacteria mat according to the present invention, it is possible to fix and remove carbon dioxide that has naturally dissolved in seawater as carbonate without the need to artificially inject carbon dioxide. Thus, according to the present invention, it is possible to fix and remove carbon dioxide more efficiently by omitting the step of injecting carbon dioxide.

[0043] [Regarding process (B)] Step (B) is the step of placing the cyanobacteria mat in seawater. By placing the cyanobacteria mat in seawater in this way, it becomes possible to form carbonates using the carbon dioxide and metal ions (magnesium ions, calcium ions, etc.) dissolved in the seawater. Furthermore, in this invention, since carbonates can be formed and carbon dioxide fixed by a simple process such as placing the mat in seawater and irradiating it with light, any region of the Earth's oceans can be used as the region (seawater) in which the cyanobacteria mat is placed. Alternatively, the cyanobacteria mat may be placed in seawater by artificially setting up a seawater reservoir, introducing seawater into it (for example, using a pump), and then placing the cyanobacteria mat in it.

[0044] Furthermore, when placing the cyanobacteria mat in seawater, it is preferable to place it in a region with a water depth of 0 to 200 m (more preferably 0 to 50 m). By placing the cyanobacteria mat in such a water depth region, it becomes possible to irradiate the cyanobacteria mat with light of sufficient intensity from sunlight, making it possible to fix carbon dioxide more reliably without complicated processes by utilizing seawater and sunlight.

[0045] [Regarding process (C)] Step (C) is a step of irradiating the cyanobacteria mat in seawater with light to form carbonates.

[0046] Such a process (C) may be a process of irradiating the cyanobacteria mat with light using an artificial light source (e.g., a halogen lamp, fluorescent lamp, LED, etc.), or a process of irradiating the cyanobacteria mat with light using sunlight. In such a process (C), it is preferable to irradiate with light by sunlight because the process is simpler. The process of irradiating with sunlight can be easily achieved in the above process (B) by placing the cyanobacteria mat in a region of seawater that is exposed to sunlight.

[0047] Furthermore, in step (C), it is preferable that the light irradiated onto the cyanobacteria mat includes light with a wavelength of 300 nm to 850 nm. When light in this wavelength range is included, it becomes possible to efficiently absorb the light and perform photosynthesis regardless of the type of filamentous cyanobacteria, thereby enabling the efficient formation of carbonates. When sunlight is used as the light source, the wavelength conditions of the irradiated light can be efficiently achieved. The light irradiated onto the cyanobacteria mat may be selected to allow for efficient photosynthesis depending on the type of filamentous cyanobacteria, or a suitable wavelength range of light (for example, light mainly in the 350-800 nm wavelength range) may be appropriately selected from the wavelength range of 300 nm to 850 nm depending on the type of filamentous cyanobacteria used.

[0048] Furthermore, in step (C), the photon flux density (light intensity) of the light irradiated onto the cyanobacteria mat should be set within a range that allows carbonate formation depending on the type of filamentous cyanobacteria, and is not particularly limited. For example, if the filamentous cyanobacteria is NIES-4073 of the genus Scytonema, as demonstrated in the examples described later, the photon flux density (1 μE / m²) of light that leaks into the room should be set to a level that allows carbonate formation depending on the type of filamentous cyanobacteria. 2 / s (=1 μmol / m³) 2 Even at approximately 1 μE / m², carbon dioxide can be efficiently removed, indicating that the photon flux density is 1 μE / m². 2 It is preferable to irradiate with light so that the light intensity is 1 / s or more. Furthermore, if the filamentous cyanobacteria is NIES-2119 of the genus Phormidium, as demonstrated in the examples described later, 1000 μE / m 2 / s(=1000μmol / m 2 By setting the photon flux density to 1000 μE / m², it becomes possible to efficiently remove carbon dioxide. In this case, the photon flux density is 1000 μE / m². 2It can be said that it is preferable to irradiate light so that it becomes / s or more. Furthermore, when the filamentous cyanobacteria is NIES-267 of the genus Calothrix, as demonstrated in the examples described later, when the photosynthetic photon flux density is 2000 μE / m 2 / s (= 2000 μmol / m 2 / s), the carbon dioxide removal performance is higher. Therefore, it can be said that it is preferable to irradiate strong light such that the photosynthetic photon flux density becomes a value near 2000 μE / m 2 / s. Thus, it is desirable to appropriately design the magnitude of the photosynthetic photon flux density according to the type of filamentous cyanobacteria.

[0049] In this way, in step (C), by irradiating light on the cyanobacterial mat in seawater, carbonates can be formed. Although the mechanism of carbonate formation using filamentous cyanobacteria is not necessarily clear, the inventors of the present invention推测 that it is as follows. That is, first, by irradiating light on the cyanobacterial mat, the filamentous cyanobacteria on the surface of the cyanobacterial mat perform photosynthesis. By such photosynthesis, carbon dioxide is utilized and decreased in the vicinity of the filamentous cyanobacteria. Then, in the vicinity of the filamentous cyanobacteria, an increase in pH occurs due to the decrease in carbon dioxide, and carbonate ions increase. And when carbonate ions increase in the vicinity of the filamentous cyanobacteria, the carbonate ions combine with metal ions (for example, Ca ions) in seawater, thereby forming carbonates. In this way, the inventors of the present invention推测 that carbonates (such as CaCO3) are formed by irradiating light on the cyanobacterial mat.

[0050] Furthermore, in the present invention, if the filamentous cyanobacteria are capable of forming stromatolites, and step (C) is a step of irradiating with sunlight, carbonates are formed by light irradiation during the day and precipitate on the surface of the solid, and at night the filamentous cyanobacteria's mucus (CPS, etc.) helps to retain and stabilize the carbonates (and in some cases it is also possible to induce the growth of some carbonate crystals), and the carbonates are fixed as a sediment layer. Subsequently, during the day the filamentous cyanobacteria become active again on the sediment layer fixed at night, and a new sediment layer is formed at night, and this cycle is repeated, that is, the cycle of carbonate formation during the day and sediment layer formation at night is repeated, thereby forming stromatolites in which layers containing carbonates are stacked, and carbon dioxide can be fixed as carbonate contained in the stromatolite. 2 In cases where carbonates can be formed even with light irradiation at a photon flux density of around 1 / s (for example, NIES-4073 belonging to the genus Scytonema), carbonate formation becomes more stable regardless of weather conditions (sunny, cloudy, rainy, etc.) or time of day, as long as it is daytime. Therefore, it becomes possible to form stromatolites through the aforementioned cycle and fix carbon dioxide as carbonate more efficiently.

[0051] Furthermore, in process (C), for example, if the photon flux density is 2000 μE / m 2 Size near / s (preferably 2000 ± 500 μE / m) 2When irradiating with strong light such as (using sunlight, etc., and placing the cyanobacteria mat in an environment where such strong light is irradiated) the carbon dioxide removal performance can be made more advanced in such an environment, and it is particularly preferable that the cyanobacteria mat irradiated with light is a cyanobacteria mat in which cyanobacteria belonging to the genus Calothrix and identified in NIES-267 are immobilized as filamentous cyanobacteria.

[0052] As described above, the present invention is a method for fixing and removing carbon dioxide in seawater as carbonate through steps (A) to (C) described above. Thus, the present invention makes it possible to form carbonate using carbon dioxide in seawater (carbon dioxide dissolved from the atmosphere, etc.) and cations in seawater through a simple process such as placing a cyanobacteria mat in seawater and irradiating it with light, thereby enabling the fixing and removal of carbon dioxide. With this present invention, there is no need to use batch-type reactors such as culture tanks or bioreactors when fixing carbon dioxide, nor is there a need to carry out complicated processes such as injecting carbon dioxide or increasing its concentration, and it is possible to fix and remove carbon dioxide reproducibly using the sea and sunlight. Furthermore, with this present invention, since carbon dioxide can be fixed and removed using a simple process with a cyanobacteria mat, it is possible to easily scale up the process by simple means such as arranging a large number of cyanobacteria mats (for example, arranging multiple cyanobacteria mats by suspending them in seawater as in shellfish farming). Furthermore, if filamentous cyanobacteria form stromatolites, it is possible to isolate the formed stromatolites on the seabed and sequester carbon dioxide from the atmosphere for a long period of time. Thus, the carbon dioxide fixation method of the present invention is a method that can easily and reproducibly fix and remove carbon dioxide on a large scale, and can therefore be said to be a method that can greatly contribute to carbon neutrality. [Examples]

[0053] The present invention will be described more specifically below based on examples and comparative examples, but the present invention is not limited to the following examples.

[0054] (Example 1) <Formation process of cyanobacteria mats> We prepared NIES-2119 (scientific name: Phormidium ambiguum), a filamentous cyanobacterium belonging to the genus Phormidium. This filamentous cyanobacterium was then cultured in MDM liquid medium at a temperature of 20°C, with a photon flux density of 50 μE / m². 2 The cells were cultured for 6 days by irradiating them with light (wavelength 420-660 nm light (using a white LED (manufactured by MRT Co., Ltd., Sheila-Vegista) as the light source)) at a rate of / s. Next, the filamentous cyanobacteria that had been cultured (growth) in MDM liquid medium were inoculated onto the surface of MDM agar medium (diameter: 30 mm, thickness: approximately 1 cm, agar concentration 0.4%) in a petri dish (inner diameter 30 mm, height 15 mm) (inoculation volume: 100 μL). Subsequently, under a temperature of 20°C, the cells were incubated at 140 μE / m². 2 Light at 1 / s (wavelength 420-660 nm (using a white LED (manufactured by MRT Co., Ltd., Sheila-Vegista) as the light source)) was irradiated onto the surface of the MDM agar medium, and filamentous cyanobacteria were cultured on the surface of the MDM agar medium for 7 days. In a petri dish, the filamentous cyanobacteria formed a cyanobacterial mat fixed to the surface of the agar medium.

[0055] <Preparation process for artificial seawater> A solution was formed by dissolving powder for preparing artificial seawater (product name "Marine Art SF-1" manufactured by Osaka Yakken Co., Ltd., amount used: 1 bag (for 25L)) in Elix water (25L). Then, the solution was allowed to stand until the carbon dioxide in the solution reached equilibrium with the carbon dioxide in the atmosphere, thereby obtaining artificial seawater (calcium chloride dihydrate concentration of 1.5g per liter of water).

[0056] <Process for placing cyanobacteria mats in artificial seawater> First, the laboratory was made dark by preventing as much external light as possible from entering (however, the laboratory was not a completely dark room due to light leaking into the laboratory and light originating from the measuring equipment inside. In this specification, when the laboratory is made dark, the light from which the photon flux density is measured inside the laboratory, without any additional light source being used to illuminate the room, will be referred to as "light leaking into the laboratory" for convenience, and the test procedures will be described below). Next, in such a dark laboratory setting, an acrylic box with a capacity of 414 mL (external dimensions: 14 cm long, 9 cm wide, 6.5 cm high; internal dimensions: 11.5 cm long, 8 cm wide, 4.5 cm high) was placed on the top of a small lab jack (laboratory bench: lifting platform) that had been pre-positioned inside a water tank (18.5 cm long, 31 cm wide, 23 cm high), thereby installing the acrylic box inside the water tank. (Note that the lab jack used was set to a height of 12 cm from the bottom of the water tank (2 cm from the water surface) so that after the artificial seawater was injected, the bottom of the acrylic box would be positioned higher than the water level of the artificial seawater stored in the tank.) Subsequently, in a darkened laboratory, a total of 5 liters of the artificial seawater prepared as described above was poured into the tank containing the acrylic box (the amount of artificial seawater poured was such that any artificial seawater overflowing from the acrylic box would flow into the tank and be stored (allowed)). The temperature of the artificial seawater was then set to 23°C. In the acrylic box and tank into which the artificial seawater had been poured in, a small submersible pump (Putio Co., Ltd., product name "VM2A") placed in the tank was used to circulate the artificial seawater between the tank and the acrylic box, so that the artificial seawater overflowing from the acrylic box and flowing into the tank (artificial seawater stored in the tank) would be returned to the acrylic box at a flow rate of 1 liter / min. Next, the petri dish containing the cyanobacteria mat was placed in the artificial seawater in the acrylic box so that its edge was positioned 5 mm above the water surface (at a water depth of 5 mm).In this case, the cyanobacteria mat was positioned approximately in the center of the acrylic box when viewed from above (perpendicular to the water surface). The petri dish was then placed in the artificial seawater to distribute the cyanobacteria mat, and it was left undisturbed at a constant temperature of 23°C for 13 hours (incubation).

[0057] <Light irradiation process for cyanobacteria mats> Thirteen hours after the start of incubation (containment) immediately following the placement of the cyanobacteria mat in artificial seawater in the aforementioned placement process (hereinafter sometimes referred to as the "experiment start time"), the cyanobacteria mat (the cyanobacteria mat immediately after incubation) was irradiated with light under a temperature of 23°C as follows. For this light irradiation, the incubated cyanobacteria mat was first left undisturbed in a dark laboratory to allow light to leak into the laboratory (photon flux density of light irradiated onto the cyanobacteria mat: approximately 1 μE / m²). 2 After irradiating the cyanobacteria mat with light using a ( / s) method, the cyanobacteria mat was further irradiated with light primarily in the 350-800 nm wavelength range using a halogen lamp (SCHOTT KL1500) with a daylight filter as the light source. Therefore, the photon flux density of the light emitted from the halogen lamp during irradiation was 0-2000 μE / m². 2 This means the change was made within the range of / s.

[0058] In this type of light irradiation, first, without irradiating from a halogen lamp, light leaking into the laboratory (photon quantum flux density approximately 1 μE / m²) is used. 2 Under conditions utilizing ( / s), the cyanobacteria mat immediately after incubation had a photon flux density of approximately 1 μE / m². 2 The lamp was irradiated with light at / s for 5 minutes (the photon flux density of the light emitted from the halogen lamp was 0 μE / m²). 2( / s). Next, light irradiation from the halogen lamp is started, and the cyanobacteria mat is first exposed to light from the halogen lamp at a photon flux density of 100 μE / m². 2 Light was irradiated for 5 minutes under the condition of / s, and then the photon flux density was 1000 μE / m 2 Light was irradiated for 5 minutes under the condition of / s, and then the photon flux density was 2000 μE / m². 2 The light was irradiated at a speed of / s for 5 minutes. After irradiating the cyanobacteria mat with light for 20 minutes while varying the photon flux density of the irradiated light in this manner, the photon flux density reached 1000 μE / m². 2 The photon flux density is fixed to a constant value of / s, and a photon flux density of 1000 μE / m² is obtained from a halogen lamp. 2 The cyanobacteria mat was irradiated with light at a rate of / s for 49 hours. In this manner, the cyanobacteria mat was irradiated with light while varying the photon flux density.

[0059] <Observation using a polarizing microscope> Immediately after placing the cyanobacteria mat in artificial seawater (at the start of incubation (experiment start time)), 13 hours after the start of the experiment (immediately after incubation), and approximately 38 hours after the start of the experiment (when the photon flux density was set to 1000 μE / m² during the light irradiation process) 2 Approximately 24 hours after fixing the speed to / s, and approximately 62 hours after the start of the experiment (when the photon flux density in the light irradiation process is set to 1000 μE / m²), and approximately 62 hours after the start of the experiment. 2 At each of the following time points (approximately 48 hours after fixing to / s), the state of the filamentous cyanobacteria fixed on the cyanobacteria mat was observed using a polarizing microscope (Keyence, product name: VHX-X1) with plane polarization (PPL) and cross-polarized light (XPL). Microscopic images (plane polarization (PPL) and cross-polarized light (XPL)) of the filamentous cyanobacteria at the start of the experiment, 13 hours after the start of the experiment, approximately 38 hours after the start of the experiment, and approximately 62 hours after the start of the experiment are shown in Figure 1.

[0060] <Measurement of substance concentration and pH using microelectrodes> In the aforementioned light irradiation process, the photon flux density of the irradiated light is set to approximately 1 μE / m 2 / s (when using light leaking into the laboratory without irradiation from a halogen lamp), 100 μE / m 2 / s (when using light from a halogen lamp), 1000 μE / m 2 / s (when using light from a halogen lamp), 2000 μE / m 2 The light is irradiated in the order of / s (when using light from a halogen lamp), and at a timing of 5 minutes after the start of light irradiation at each photon flux density, Ca near filamentous cyanobacteria was observed. 2+ The concentrations of ions and oxygen, as well as the pH changes of seawater near filamentous cyanobacteria, were measured using microelectrodes. As a result of these measurements, Figure 2 shows a graph illustrating the relationship between the distance from the surface of the cyanobacteria mat and pH, and the relationship between the distance from the surface of the cyanobacteria mat and Ca 2+ Figure 3 shows a graph illustrating the relationship between ion concentrations, and Figure 4 shows a graph illustrating the relationship between the distance from the surface of the cyanobacteria mat and the O2 concentration.

[0061] <16S rRNA gene analysis of cyanobacteria mats> A portion of the bacterial mass present on the surface of the solid cyanobacteria mat (MDM agar medium) obtained through the aforementioned cyanobacteria mat formation process was collected, and 16S rRNA gene analysis was performed as follows to determine the proportion of filamentous cyanobacterium NIES-2119 among the bacteria present on the surface of the solid (MDM agar medium). Specifically, first, a portion of the bacterial mass was collected from the solid surface of the cyanobacteria mat. Next, a surfactant solution (product name "Lysis Solution F" manufactured by Nippon Gene Co., Ltd.) was added to the collected bacterial mass, and the cells were pulverized for 2 minutes at 1,500 rpm using a cell disruptor called "Shake Master Neo" manufactured by bms Co., Ltd. to prepare a sample. After that, the sample was allowed to stand at 5°C for 10 minutes, and then centrifuged for 2 minutes at a centrifugal force of 12,000 x g, and the supernatant solution was collected. Next, DNA was purified from the separated solution using a DNA extraction kit (product name "Lab-Aid 824s DNA Extraction kit" manufactured by ZEESAN). Then, PCR amplification targeting the V3-V4 region of the 16S rRNA gene was performed using the purified DNA by performing PCR and purification twice (using the 2-step tailed PCR method). Subsequently, the obtained PCR product (amplicon) was analyzed using a next-generation sequencer ("MiSeq" manufactured by Illumina) (amplicon sequencing analysis of the V3-V4 region) to obtain a fastq file. Next, the obtained fastq file was analyzed using a microbial community analysis tool (QIIME 2 (https: / / qiime2.org / )) to calculate the proportion of filamentous cyanobacterium NIES-2119 among all bacteria in the sample, and the proportion of filamentous cyanobacterium NIES-2119 among bacteria present on the surface of the solid (MDM agar medium) was determined. The results of this analysis confirmed that the proportion of filamentous cyanobacteria NIES-2119 among the bacteria present on the surface of the solid (MDM agar medium) was 100%.

[0062] (Comparative Example 1) Except for not using filamentous cyanobacteria and using the same artificial seawater as in Example 1, and using a petri dish containing the same MDM agar medium (diameter: 30 mm, thickness: approximately 1 cm, agar concentration 0.4%) as used in Example 1 instead of the petri dish with the cyanobacteria mat (using MDM agar medium instead of cyanobacteria mat), the MDM agar medium was placed in the artificial seawater in the same arrangement procedure as in Example 1. Subsequently, a halogen lamp (SCHOTT: KL1500) with a daylight filter was used as the light source, and under a temperature of 23°C, light mainly with a wavelength of 350-800 nm was emitted at a rate of 2000 μE / m² to the MDM agar medium in the artificial seawater. 2 The MDM agar medium was irradiated with a photon flux density of / s for 5 minutes. Then, 5 minutes after the start of light irradiation to the MDM agar medium, the measurement was taken near the surface of the MDM agar medium using the same method as "Measurement of substance concentration and pH using a microelectrode" used in Example 1, to measure the Ca near the surface of the MDM agar medium. 2+ Changes in ion and oxygen concentrations, as well as changes in the pH of seawater near the surface of the MDM agar medium, were measured. The results obtained, along with those from Example 1, are shown in Figures 2-4, respectively.

[0063] (Example 2) The type of filamentous cyanobacteria was changed from NIES-2119 (scientific name: Phormidium ambiguum) to NIES-4073 (scientific name: Scytonema sp.), which belongs to the genus Scytonema. In the cyanobacteria mat formation process, the filamentous cyanobacteria were inoculated onto the surface of the MDM agar medium, and the photosynthetic photon flux density was 140 μE / m². 2Except for changing the period of irradiation with light at / s (the period for culturing filamentous cyanobacteria after inoculation onto the surface of MDM agar medium) from 7 days to 16 days, and changing the incubation time at constant temperature during the process of placing the cyanobacteria mat in artificial seawater from 13 hours to 14 hours, the same process as in Example 1 was carried out: "formation of cyanobacteria mat," "preparation of artificial seawater," "placement of cyanobacteria mat in artificial seawater," and "light irradiation of cyanobacteria mat."

[0064] Furthermore, the timing of observations using a polarizing microscope will be determined as follows: at the start of the experiment, 14 hours after the start of the experiment (immediately after incubation), and approximately 39 hours after the start of the experiment (when the photon flux density is 1000 μE / m² during the light irradiation process). 2 Approximately 24 hours after fixing the speed to / s, and approximately 63 hours after the start of the experiment (when the photon flux density in the light irradiation process is set to 1000 μE / m²), and approximately 63 hours after the start of the experiment. 2 Except for changing the timing to approximately 48 hours after fixing the time to / s, the state of the filamentous cyanobacteria was observed using a polarizing microscope in the same manner as the "observation using a polarizing microscope" adopted in Example 1. Microscopic images (plane polarized (PPL) and cross-polarized (XPL)) of the filamentous cyanobacteria at the start of the experiment, 14 hours after the start of the experiment, approximately 39 hours after the start of the experiment, and approximately 63 hours after the start of the experiment are shown in Figure 5.

[0065] Furthermore, using the same method as "measurement of substance concentration and pH using a microelectrode" employed in Example 1, Ca present near filamentous cyanobacteria was measured. 2+ The concentrations of ions and oxygen, as well as the pH changes of seawater near filamentous cyanobacteria, were measured using microelectrodes. As a result of these measurements, Figure 6 shows a graph illustrating the relationship between the distance from the surface of the cyanobacteria mat and pH, and the relationship between the distance from the surface of the cyanobacteria mat and Ca 2+ Figure 7 shows a graph illustrating the relationship between ion concentrations, and Figure 8 shows a graph illustrating the relationship between the distance from the surface of the cyanobacteria mat and the O2 concentration.

[0066] Furthermore, except that the type of cyanobacteria mat was changed to the one obtained in Example 2, and the proportion of filamentous cyanobacteria NIES-4073 was determined instead of the proportion of filamentous cyanobacteria NIES-2119, 16S rRNA gene analysis was performed using the same method as in Example 1's "16S rRNA gene analysis of cyanobacteria mat," and the proportion of filamentous cyanobacteria NIES-4073 among the bacteria present on the surface of the solid (MDM agar medium) was determined. As a result of this analysis, it was confirmed that the proportion of filamentous cyanobacteria NIES-4073 among the bacteria present on the surface of the solid (MDM agar medium) was 99.9%.

[0067] (Comparative Example 2) Except for not using filamentous cyanobacteria and using the same artificial seawater as in Example 2, and instead of using a petri dish to form a cyanobacteria mat, a petri dish containing the same MDM agar medium (diameter: 30 mm, thickness: approximately 1 cm, agar concentration 0.4%) as used in Example 2 was used (using MDM agar medium instead of a cyanobacteria mat), the arrangement process was the same as in Example 2, and the MDM agar medium was placed in the artificial seawater. Subsequently, a halogen lamp (SCHOTT: KL1500) with a daylight filter was used as the light source, and under a temperature of 23°C, light mainly with a wavelength of 350-800 nm was emitted at a rate of 2000 μE / m² to the MDM agar medium in the artificial seawater. 2 The MDM agar medium was irradiated with a photon flux density of / s for 5 minutes. Then, 5 minutes after the start of light irradiation to the MDM agar medium, the measurement was taken near the surface of the MDM agar medium using the same method as "Measurement of substance concentration and pH using a microelectrode" used in Example 2, to measure the Ca near the surface of the MDM agar medium. 2+ Changes in ion and oxygen concentrations, as well as changes in the pH of seawater near the surface of the MDM agar medium, were measured. The results obtained are shown in Figures 6-8, along with the results from Example 2.

[0068] [Discussion of the measurement results for Examples 1-2 and Comparative Examples 1-2] <Comparison between Example 1 and Comparative Example 1> In the micrographs shown in Figure 1 (results of observations using a polarizing microscope performed in Example 1: micrographs of filamentous cyanobacteria of NIES-2119 in artificial seawater at specific time points), the presence of white precipitates, thought to be carbonates, was confirmed, particularly in the photographs taken approximately 38 hours after the XPL observation.

[0069] Furthermore, the results shown in Figures 2 and 4 confirm that, near the surface of the cyanobacteria mat (NIES-2119) used in Example 1, as the photon flux density increased, the pH value and oxygen concentration increased more significantly near the cyanobacteria mat, indicating that the increase in pH value and oxygen concentration is dependent on the photon flux density. In addition, the results shown in Figure 3 confirm that, near the surface of the cyanobacteria mat (NIES-2119) used in Example 1, Ca was released upon irradiation with light. 2+ A decrease in ion concentration was confirmed.

[0070] In contrast, the results of Comparative Example 1 shown in Figures 2-4 indicate that the result was 2000 μE / m², as in Comparative Example 1. 2 Even when the surface of MDM agar is irradiated with light at a photon flux density of / s, the pH value, oxygen concentration, Ca 2+ It was confirmed that no significant changes in ion concentration occurred.

[0071] As shown in Figures 1-4, the results indicate that by placing the cyanobacteria mat formed in Example 1 in seawater and irradiating it with light, calcium carbonate, a type of carbonate, can be formed, and carbon dioxide can be fixed as a carbonate.

[0072] <Comparison between Example 2 and Comparative Example 2> Furthermore, in the micrographs shown in Figure 5 (results of observations using a polarizing microscope performed in Example 2: micrographs of filamentous cyanobacteria of NIES-4073 in artificial seawater at specific time points), the presence of white precipitates, thought to be carbonates, was confirmed, particularly in the photographs taken approximately 39 hours after the XPL observation.

[0073] Furthermore, the results shown in Figures 6 and 8 confirm that, near the surface of the cyanobacteria mat (NIES-4073) used in Example 2, as the photon flux density increased, the pH value and oxygen concentration increased more significantly near the cyanobacteria mat, indicating that the increase in pH value and oxygen concentration is dependent on the photon flux density. In addition, the results shown in Figure 7 confirm that, near the surface of the cyanobacteria mat (NIES-4073) used in Example 2, Ca was released upon irradiation with light. 2+ A decrease in ion concentration was confirmed. Note that the results shown in Figure 7 assume a photon flux density of 2000 μE / m². 2 Ca in light irradiation at / s 2+ The decrease in ion concentration is related to the Ca ion concentration when irradiated with light of a different photon flux density. 2+ The degree of decrease was smaller than the decrease in ion concentration. Based on these results, for NIES-4073, a photon flux density of 2000 μE / m² is recommended. 2 By irradiating with light at a value lower than / s, Ca can be treated more efficiently. 2+ It was found that it is possible to reduce the ion concentration.

[0074] In contrast, the results of Comparative Example 2, shown in Figures 6-8, indicate that the result was 2000 μE / m², as in Comparative Example 2. 2 Even when the surface of MDM agar is irradiated with light at a photon flux density of / s, the pH value, oxygen concentration, Ca 2+ It was confirmed that no significant changes in ion concentration occurred.

[0075] As shown in Figures 5-8, the results indicate that by placing the cyanobacteria mat formed in Example 2 in seawater and irradiating it with light, calcium carbonate, a type of carbonate, can be formed, and carbon dioxide can be fixed as a carbonate.

[0076] [Estimation of carbon dioxide fixation] The distance from the surface of the cyanobacteria mat and Ca were determined by the "measurement of substance concentration and pH using microelectrodes" performed in Examples 1 and 2. 2+ Graphs showing the relationship between ion concentrations (Figures 3 and 7: Ca 2+ Using data on changes in ion concentration, and based on Fick's first law, approximately 1 μE / m 2 / s (Photon quantum flux density when light is emitted using light leaking into the laboratory without irradiation from a halogen lamp), 100 μE / m 2 / s (photon quantum flux density of light from a halogen lamp), 1000 μE / m 2 / s (photon flux density of light from a halogen lamp) and 2000 μE / m 2 The amount of carbon dioxide fixed (removed) during light irradiation for each photon flux density ( / s) from a halogen lamp was calculated.

[0077] Furthermore, for Comparative Examples 1 and 2, the 2000 μE / m² values ​​were used. 2 Because only light irradiation with a photon flux density of / s was performed, the result was 2000 μE / m 2 Ca near the surface of MDM agar medium upon irradiation with light photon flux density / s 2+ Based on the data on the change in ion concentration (Figures 3 and 7), the amount of carbon dioxide fixed was calculated using Fick's first law.

[0078] In performing such calculations, use the following formula (I) to determine the Ca flux (unit: mol / m³). 2Assuming that the Ca flux ( / s) is equivalent to the amount of CO2 fixed, the amount of CO2 fixed was determined. In this process, the Ca concentration at distances from the surface to 400 μm (diffusion boundary layer) was checked in 100 μm increments, and the value where this concentration difference was maximized was defined as dC (the difference between 100 μm and 0 μm, 200 μm and 100 μm, 300 μm and 200 μm, and 400 μm and 300 μm was calculated, and the maximum of these differences was defined as dC). In this way, when determining the amount of CO2 fixed, considering that the Ca flux would be a negative value, the amount of CO2 fixed was obtained as a positive value by multiplying the Ca flux by a negative value. Furthermore, the following conditions (A) and (B): [Condition (A)] Ca at a distance of 0 μm from the surface 2+ The concentration is Ca at a distance of 400-1000 μm from the surface. 2+ Average Ca concentration 2+ The condition that the concentration is greater than the stated concentration; [Condition (B)] Ca at a distance of 0 μm from the surface 2+ The concentration is Ca at a distance of 400-1000 μm from the surface. 2+ Average Ca concentration 2+ When the concentration is less than the difference, the difference is Ca at a distance of 400-1000 μm from the surface. 2+ The condition that the concentration is within the standard deviation; If either of the following conditions is met, Ca will be released from the water mass. 2+ Assuming that the concentration had not decreased, the amount of CO2 fixed was considered to be 0. Figure 9 shows a graph illustrating the relationship between the amount of carbon dioxide fixed and the photon flux density, calculated in this manner based on Fick's first law. For reference, Figure 9 also includes the amount of carbon dioxide removed by the forest per second (5.3 × 10⁻¹⁰). -7 mol / m 2 The dotted line representing the value of ( / s) and the graph of the amount removed are shown together. The amount of carbon dioxide removed by forests like this was calculated (converted) from data on the annual amount of carbon dioxide absorbed by forests (approximately 8 t / ha / y).

[0079] <Calculation formula (I) based on Fick's first law> J = -D·dC / dz (I) (In equation (1), J is Ca flux (unit: mol / m³) 2 It shows ( / s), and D is Ca-CO3 2- Diffusion coefficient (m 2 dC represents the difference in Ca concentration (mol / m³) ( / s). 3 ) is shown, and dz indicates the distance (in meters) from the surface of the cyanobacteria mat (Examples 1-2) or MDM agar medium (Comparative Examples 1-2).

[0080] As shown in Figure 9, in Examples 1 and 2, the amount of carbon dioxide removed from the forest was (5.3E-07:5.3 × 10⁻¹⁰). -7 mol / m 2 It can be understood that there are irradiation conditions with photon flux density that show higher removal performance than 1 μE / m², and it can be seen that by placing the cyanobacteria mats formed in Examples 1 and 2 in seawater and irradiating them with light, it is possible to efficiently fix and remove carbon dioxide. Furthermore, comparing the measurement results of Example 1 and Example 2 based on the results shown in Figure 9, it can be seen that the filamentous cyanobacteria (NIES-4073) used in Example 2 exhibits higher carbon dioxide removal performance than the filamentous cyanobacteria (NIES-2119) used in Example 1 under the same photon flux density conditions. In addition, the filamentous cyanobacteria used in Example 2 have a photon flux density of 1 μE / m². 2 It can also be seen that it exhibits high carbon dioxide removal performance even under light irradiation conditions where the value is / s (dark conditions).

[0081] Furthermore, in this invention, by using a cyanobacteria mat on which filamentous cyanobacteria are immobilized, Ca is used in the region near the cyanobacteria mat. 2+The inventors surmise that by causing changes in the concentration of substances such as ions and an increase in pH, it becomes possible to efficiently create an environment near the cyanobacteria mat that promotes the formation of carbonates. Therefore, without the complicated process of artificially adding carbon dioxide to maintain pH, carbonates can be efficiently formed and carbon dioxide fixed by a simple process such as irradiating with light. In contrast, when using a suspension in which cyanobacteria are dispersed in liquid culture as described in Patent Document 1, the overall conditions of the bulk water used for carbon dioxide fixation must be changed to conditions in which carbonates can be formed (in other words, the composition of the entire liquid phase in the area where the suspension is added and carbon dioxide fixation is performed must be changed to cause an increase in pH, etc., as occurs near the cyanobacteria mat used in the embodiment of the present invention). Therefore, it is clear that carbonate formation is less likely to occur compared to the present invention, and the inventors surmise that it is not possible to efficiently form carbonates by a simple process such as irradiating with light, as in the present invention.

[0082] (Example 3) <Formation process of cyanobacteria mats> NIES-267 (scientific name: Calothrix parasitica), a filamentous cyanobacterium belonging to the genus Calothrix, was prepared. Separately, an artificial seawater solution was prepared by dissolving Daigo Artificial Seawater SP for Marine Microalgae (Fujifilm Wako Pure Chemical Industries, product number 395-01343) at a ratio of 1 sachet per 1 liter of water. To this artificial seawater solution, Gillard (F / 2) Seawater Nutrient Solution (Merck, product number G0154-500M) was added in a volume ratio ([F / 2 Seawater Nutrient Solution]: Artificial Seawater Solution) of 1:19 to prepare an F / 2 liquid culture medium. Since the filamentous cyanobacterium NIES-267 used is a seawater cyanobacterium, a seawater F / 2 liquid culture medium was prepared in this example. The filamentous cyanobacterium was then cultured in the F / 2 liquid culture medium for 2 months. This type of culture is performed using a temperature gradient incubator (TG-180WLED-5LE, manufactured by Nippon Ika Kikai Seisakusho) at a temperature of 25°C, with a photon flux density of 60 μE / m². 2 The experiment was carried out by irradiating light for two months under conditions where the photon flux density was 60 μE / m² and the light-dark cycle consisted of 12 hours of light and 12 hours of darkness. Subsequently, the bacterial solution obtained by culturing the filamentous cyanobacteria was dropped onto a glass filter (Shibata Scientific, product number A13010-011, glass filter P250, diameter: 19.5) to allow the filamentous cyanobacteria to adhere to (inoculate) the glass filter. After that, the glass filter was immersed in an F / 2 liquid culture medium and, under a temperature of 25°C, the photon flux density was 60 μE / m². 2 By culturing the cyanobacteria for two weeks in a temperature gradient incubator under conditions where the temperature was 12 hours / s and the light-dark cycle consisted of 12 hours of light and 12 hours of dark, filamentous cyanobacteria were fixed (formed) on a glass filter to obtain a cyanobacteria mat.

[0083] <Preparation process for artificial seawater> Artificial seawater (with a calcium chloride dihydrate concentration of 1.5 g per liter of water) was obtained by employing the same process as the "artificial seawater preparation process" used in Example 1.

[0084] <Process for placing cyanobacteria mats in artificial seawater> In the cyanobacteria mat formation process described above, the cyanobacteria mat (with filamentous cyanobacteria immobilized on a glass filter) produced was placed in a petri dish (30 mm inner diameter, 15 mm height) to prepare a "petri dish with cyanobacteria mat," and this prepared "petri dish with cyanobacteria mat" was placed in the artificial seawater in the acrylic box in place of the "petri dish in which the cyanobacteria mat was formed." Except for this, the same process as the "process of placing the cyanobacteria mat in artificial seawater" used in Example 1 was followed, and the cyanobacteria mat (with filamentous cyanobacteria immobilized on a glass filter) was placed in the artificial seawater and left at a constant temperature of 23°C for 13 hours (incubation). Figure 10 shows a photograph of the state of the cyanobacteria mat (morphology of filamentous cyanobacteria) placed in the artificial seawater.

[0085] <Light irradiation process for cyanobacteria mats> In the aforementioned placement process, 13 hours after the start of constant-temperature incubation immediately following the placement of the cyanobacteria mat in artificial seawater, the cyanobacteria mat (the cyanobacteria mat immediately after incubation) was irradiated with light in a dark laboratory at a temperature of 23°C as follows: A halogen lamp (SCHOTT KL1500) with a daylight filter was used as the light source, and the cyanobacteria mat was irradiated with light mainly consisting of light with a wavelength of 350 to 800 nm. During this light irradiation, the cyanobacteria mat was first irradiated with light from the halogen lamp at a photon flux density of 1000 μE / m². 2 Light was irradiated for 5 minutes under the condition of / s, and then the photon flux density was 2000 μE / m 2 Light was irradiated for 5 minutes under the condition of / s. In this way, the photon flux density of the light irradiated onto the cyanobacteria mat was set to 1000 μE / m². 2 / s, 2000 μE / m 2 The cyanobacteria mat was irradiated with light while changing the order of the / s.

[0086] <Measurement of Substance Concentration Using a Microelectrode> In the above-described light irradiation step, the condition of the photon flux density of the irradiated light was changed in the order of 1000 μE / m 2 / s and 2000 μE / m 2 / s, and light was irradiated. At the timing 5 minutes after the start of light irradiation at each photon flux density, the change in the concentration of Ca 2+ ions present in the vicinity of the filamentous cyanobacteria was measured using a microelectrode. As a result of such measurement, a graph showing the relationship between the distance from the surface of the cyanobacterial mat and the concentration of Ca 2+ ions is shown in FIG. 11.

[0087] <16S rRNA Gene Analysis of Cyanobacterial Mats> The type of the cyanobacterial mat was changed to the cyanobacterial mat obtained in Example 3, and the same method as the "16S rRNA gene analysis of cyanobacterial mats" adopted in Example 1 was adopted except that the proportion of the filamentous cyanobacterium NIES-267 was determined instead of the proportion of the filamentous cyanobacterium NIES-2119. 16S rRNA gene analysis was performed to determine the proportion of the filamentous cyanobacterium NIES-267 present in the bacteria existing on the surface of the glass filter. As a result of such analysis, it was confirmed that the proportion of the filamentous cyanobacterium NIES-267 present in the bacteria existing on the surface of the glass filter was 100%.

[0088] [Discussion on the Measurement Results of Example 3] [Estimation of Carbon Dioxide Fixation Amount] Using the same method as described in the column of "Estimation of carbon dioxide fixation amount" in the column of "Discussion on the measurement results of Examples 1 to 2 and Comparative Examples 1 to 2" above, in Example 3 as well, based on Fick's first law, using the graph (FIG. 11: data on the change in the concentration of Ca 2+ ions) showing the relationship between the distance from the surface of the cyanobacterial mat and the concentration of Ca 2+ ions obtained by "Measurement of substance concentration using a microelectrode", at 1000 μE / m 2 / s and 2000 μE / m 2 The amount of carbon dioxide fixed (removed) during irradiation of light with each photon flux density of / s was determined by calculation. The results are shown in Table 5. In addition, in Table 5, data on the amount of carbon dioxide removed by forests per second are also shown for reference.

[0089]

Table 5

[0090] From the results shown in Table 5, it can be understood that the cyanobacterial mat formed in Example 3 can exhibit a carbon dioxide removal performance higher than that of the forest (5.3E-07: 5.3×10 2 mol / m 2 / s) when the photon flux density is 1000 μE / m -7 mol / m 2 / s and when the photon flux density is 2000 μE / m

[0091] From such results as well, by using a cyanobacterial mat in which filamentous cyanobacteria are fixed, it becomes possible to cause a change in the concentration of substances such as Ca<QQQ000099>ions in the region near the cyanobacterial mat, and the inventors推测 that it is possible to efficiently form carbonates and immobilize carbon dioxide by performing a simple process such as irradiating light without performing a complicated process of artificially adding carbon dioxide to maintain the pH.

[0092] In Example 3, a glass filter was used to form the cyanobacteria mat. This demonstrates that even when using a solid material like a glass filter capable of adsorbing filamentous cyanobacteria, the filamentous cyanobacteria can be attached to and fixed to the surface of the solid. Similar to Examples 1 and 2, this allows for carbon dioxide removal performance higher than that of a forest per second through light irradiation. Furthermore, by immobilizing filamentous cyanobacteria on a relatively hard solid material like a glass filter to form a cyanobacteria mat, handling of the mat becomes easier during transport and immersion in the sea (making the handling of the cyanobacteria mat significantly easier).

[0093] Furthermore, comparing the results shown in Table 5 and Figure 9, Examples 1-3 show a photon flux density of 2000 μE / m². 2 Under conditions of strong light such as 1 / s, the cyanobacteria mat formed in Example 3 achieved the highest carbon dioxide removal rate among Examples 1-3. From this fact, it can be inferred that the cyanobacteria mat formed in Example 3 has greater durability against strong sunlight, making it more suitable for long-term outdoor use. Furthermore, considering the fixation of carbon dioxide in natural seawater using strong sunlight, a material capable of long-term outdoor use is considered more suitable. From this perspective, the cyanobacteria mat formed in Example 3 is the most suitable among Examples 1-3. On the other hand, among Examples 1-3, the filamentous cyanobacteria used in Example 2 had a photon flux density of 1 μE / m². 2It can be seen that the cyanobacteria mat exhibits high carbon dioxide removal performance even under light irradiation conditions of such low light levels (dark conditions). Therefore, when considering the fixation of carbon dioxide in seawater in areas where strong light is difficult to reach, the cyanobacteria mat formed in Example 2 is more suitable among Examples 1 to 3. Considering these points, it can be seen that by appropriately selecting filamentous cyanobacteria according to the usage environment (for example, appropriately selecting suitable cyanobacteria from the genus Scytonema and the genus Calothrix) and forming and using a cyanobacteria mat, it is possible to efficiently utilize the advantages of the filamentous cyanobacteria used and fix carbon dioxide more efficiently.

[0094] (Example 4) <Process for forming cyanobacteria mats, process for preparing artificial seawater, and process for placing cyanobacteria mats into artificial seawater> Using the same steps as in Example 3, namely the "formation of cyanobacteria mat," the "preparation of artificial seawater," and the "placement of cyanobacteria mat in artificial seawater," a cyanobacteria mat (with filamentous cyanobacteria (NIES-267 belonging to the genus Calothrix) immobilized on a glass filter) and artificial seawater were formed. After preparing the artificial seawater, the cyanobacteria mat was placed in the artificial seawater and left at a constant temperature of 23°C for 13 hours (incubation).

[0095] <Light irradiation process for cyanobacteria mats> The aforementioned cyanobacteria mat (the cyanobacteria mat immediately after incubation during the process of placing the cyanobacteria mat in artificial seawater) was subjected to a photon flux density of 1000 μE / m² in a dark laboratory at a temperature of 23°C, using a halogen lamp (SCHOTT KL1500) with a daylight filter as the light source. 2Under conditions of / s, light primarily consisting of wavelengths of 350-800 nm was irradiated for 88 hours. After 88 hours of light irradiation of the cyanobacteria mat in seawater in this manner, the cyanobacteria mat was removed from the seawater and collected, and washed with a 50% ethanol solution (a solution diluted with water).

[0096] <Measurement by SEM-EDS> The cyanobacteria mat, after washing, was coated with carbon using a carbon coater (CADE, manufactured by Meiwa Forsis Co., Ltd.) to prepare a sample for measurement. Subsequently, the sample was measured using a scanning electron microscope (JSM-6390A, manufactured by JEOL Corporation) equipped with an energy-dispersive X-ray spectrometer (EDS analyzer) under the conditions of acceleration voltage: 15kV and current: 53μA, and the constituent elements of the crystals formed on the cyanobacteria mat were confirmed by SEM-EDS analysis. As an example of the results of such analysis, Figure 12 shows a scanning electron microscope image of the surface of the cyanobacteria mat containing the measured crystals, and Figure 13 shows a graph of the EDS spectrum of these crystals (crystals located within the area enclosed by a rectangle in the scanning electron microscope image shown in Figure 12).

[0097] [Discussion regarding the measurement results of Example 4] As is clear from the results shown in Figures 12-13, the EDS analysis (EDS spectrum) of the crystals formed on the cyanobacteria mat mainly showed peaks of Ca, C, and O, confirming that CaCO3 was formed on the cyanobacteria mat by the light irradiation process described above. This confirms that carbon dioxide can be fixed and removed as stable calcium carbonate by placing the cyanobacteria mat in seawater and irradiating it with light.

[0098] From the results of Examples 1 to 4 described above, it was found that by forming a cyanobacteria mat on the surface of a solid, immobilizing filamentous cyanobacteria, and then placing it in seawater and irradiating it with light, it is possible to immobilize and remove (isolate) carbon dioxide in seawater as stable carbonates (such as calcium carbonate). [Industrial applicability]

[0099] As described above, the present invention provides a carbon dioxide fixation method that eliminates the need for artificial injection of carbon dioxide, and allows for the fixation and removal of carbon dioxide that has naturally dissolved in seawater from the atmosphere, etc., through gas-liquid equilibrium, etc., as carbonates. Therefore, the carbon dioxide fixation method of the present invention can be applied to various areas of the ocean to fix and remove carbon dioxide, and thus can be said to be a method that contributes to carbon neutrality.

Claims

1. Step (A) is to form a cyanobacteria mat on the surface of a solid, in which filamentous cyanobacteria are fixed. The process of placing the cyanobacteria mat in seawater (B), The process includes (C) irradiating the cyanobacteria mat in seawater with light to form carbonates, A method for fixing carbon dioxide, characterized by removing carbon dioxide from seawater by fixing it as carbonates by including [a specific substance].

2. The method for fixing carbon dioxide according to claim 1, characterized in that the cyanobacteria mat is such that, when the proportion of filamentous cyanobacteria among the bacteria present on the surface of the solid is determined by 16S rRNA gene analysis, the proportion of filamentous cyanobacteria is 90% or more.

3. The carbon dioxide fixation method according to claim 1, characterized in that the filamentous cyanobacteria consist of at least one species selected from the group consisting of cyanobacteria belonging to the genus Scytonema and cyanobacteria belonging to the genus Calothrix.

4. The carbon dioxide fixation method according to claim 1, characterized in that the filamentous cyanobacteria consist of at least one cyanobacterium selected from the group consisting of cyanobacteria belonging to the genus Scytonema and identified in NIES-4073, and cyanobacteria belonging to the genus Calothrix and identified in NIES-267.

5. The carbon dioxide fixation method according to claim 1, characterized in that the filamentous cyanobacteria belong to the genus Calothrix and are identified in NIES-267.

6. The carbon dioxide fixation method according to claim 1, characterized in that the filamentous cyanobacteria belong to the genus Scytonema and are identified by NIES-4073.

7. The carbon dioxide fixation method according to claim 1, characterized in that the light irradiated onto the cyanobacteria mat in seawater includes light with a wavelength of 300 nm to 850 nm.

8. The carbon dioxide fixation method according to claim 1, characterized in that the dry weight ratio of CPS (capsular polysaccharide or mucopolysaccharide) of the filamentous cyanobacteria is 1000 μg / g (cell fr. wt) or more per 1 g of wet weight of the cells.

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