Cyanobacteria cultivation method

Abstract

To provide methods for culturing cyanobacteria.SOLUTION: A method for cultivating cyanobacteria includes: providing the cyanobacteria; subjecting the cyanobacteria to physical mutagenesis, so as to obtain primary mutant cyanobacteria; subjecting the primary mutant cyanobacteria to chemical mutagenesis, so as to obtain secondary mutant cyanobacteria; and subjecting the secondary mutant cyanobacteria to a temperature resistance test and an environment resistance test, so as to obtain target cyanobacteria, where the lethal rate of the physical mutagenesis ranges between 50% and 80%, and the lethal rate of the chemical mutagenesis ranges between 40% and 55%.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for culturing cyanobacteria, and more particularly to a method for culturing cyanobacteria that are resistant to high-temperature industrial exhaust gases. [Background technology]

[0002] Cyanobacteria, also known as blue-green algae, are prokaryotes that can perform photosynthesis. Conventional technology utilizes the photosynthetic properties of cyanobacteria to absorb carbon dioxide emitted from factories and achieve the sustainable development goals of zero waste and zero pollution.

[0003] However, industrial exhaust gases contain high concentrations of carbon dioxide as well as other chemicals such as sulfides and nitrogen compounds, which can affect the growth of cyanobacteria and, in some cases, cause their death.In addition, industrial exhaust gases are usually accompanied by high temperatures, which significantly reduce the survival rate and photosynthetic efficiency of cyanobacteria, making it difficult to effectively utilize the carbon fixation properties of cyanobacteria for the treatment of industrial exhaust gases.

[0004] Therefore, the key issue that this project aims to solve is to overcome the above-mentioned drawbacks by providing a method for cultivating cyanobacteria that is resistant to high temperatures and industrial exhaust gases. Summary of the Invention [Problem to be solved by the invention]

[0005] The technical problem to be solved by the present invention is to provide a method for culturing cyanobacteria. [Means for solving the problem]

[0006] The method for culturing cyanobacteria includes providing cyanobacteria, subjecting the cyanobacteria to a physical mutagenesis treatment to obtain a cyanobacteria with primary mutations, subjecting the cyanobacteria with primary mutations to a chemical mutagenesis treatment to obtain a cyanobacteria with secondary mutations, and subjecting the cyanobacteria with secondary mutations to a temperature tolerance test and an environmental tolerance test to obtain a target cyanobacteria, wherein the lethality of the physical mutagenesis treatment is 50% to 80%, and the lethality of the chemical mutagenesis treatment is 40% to 55%.

[0007] In one embodiment, the lethality of the physical mutagenesis treatment is 60% to 80%.

[0008] In one embodiment, the chemical mutagenesis treatment is 45% to 55%.

[0009] In one embodiment, the physical mutagenesis treatment is carried out at an illumination intensity of 0.017 mW / cm 2 ~0.082mW / cm 2 The cyanobacteria are irradiated for 10 to 70 seconds using an ultraviolet light source (UV light source).

[0010] In one embodiment, the chemical mutagenesis treatment involves treatment with nitrosoguanidine (NTG) at a concentration of 50 to 300 μg / ml for 0.5 to 2 minutes.

[0011] In one embodiment, the temperature tolerance test involves placing the secondary mutant cyanobacteria in an environment of 30°C to 60°C, observing their growth, and selecting preselected cyanobacteria.

[0012] In one embodiment, the environmental tolerance test involves placing the preselected cyanobacteria in a mixed gas with an aeration ratio of 0.5% to 9%, thereby selecting the target cyanobacteria.

[0013] In one embodiment, the gas mixture includes hydrogen gas, acetylene, methane, hydrogen sulfide, and acetaldehyde.

[0014] In one embodiment, the mixed gas contains 30 ppm to 50 ppm of hydrogen gas, 150 ppm to 250 ppm of acetylene, 100 ppm to 200 ppm of methane, 0.1 ppm to 1 ppm of hydrogen sulfide, and 1 ppm to 5 ppm of acetaldehyde.

[0015] In one embodiment, the culturing method further comprises subjecting the secondary mutant cyanobacteria to the chemical mutagenesis treatment.

[0016] Another technical solution adopted by the present invention to solve the above technical problems provides a method for culturing cyanobacteria. The method includes providing cyanobacteria, subjecting the cyanobacteria to physical mutagenesis to achieve a mortality rate of 50% to 80%, and designating the cyanobacteria that survive the physical mutagenesis as primary mutant cyanobacteria, and subjecting the primary mutant cyanobacteria to chemical mutagenesis to achieve a mortality rate of 40% to 55%, and designating the primary mutant cyanobacteria that survive the chemical mutagenesis as secondary mutant cyanobacteria. Monitoring the growth of the secondary mutant cyanobacteria in an environment between 30°C and 60°C to select preselected cyanobacteria, and placing the preselected cyanobacteria in a gas mixture with an aeration ratio of 0.5% to 9%, thereby selecting the target cyanobacteria. The gas mixture is a mixture containing hydrogen gas, acetylene, methane, hydrogen sulfide, and acetaldehyde.

[0017] In one embodiment, the physical mutagenesis treatment is carried out at an illumination intensity of 0.017 mW / cm 2 ~0.082mW / cm 2 The cyanobacteria are irradiated for 10 to 70 seconds using an ultraviolet light source (UV light source).

[0018] In one embodiment, the chemical mutagenesis treatment involves treatment with nitrosoguanidine (NTG) at a concentration of 50 μg / ml to 300 μg / ml for 0.5 to 2 minutes.

[0019] In one embodiment, the mixed gas contains 30 ppm to 50 ppm of hydrogen gas, 150 ppm to 250 ppm of acetylene, 100 ppm to 200 ppm of methane, 0.1 ppm to 1 ppm of hydrogen sulfide, and 1 ppm to 5 ppm of acetaldehyde.

[0020] In one embodiment, the culturing method further comprises subjecting the secondary mutant cyanobacteria to a chemical mutagenesis treatment. [Effects of the Invention]

[0021] As an advantageous effect of the present invention, the method for culturing cyanobacteria according to the present invention increases the possibility of obtaining cyanobacteria that are resistant to high-temperature industrial exhaust gases by virtue of the technical features that "physical mutagenesis treatment and chemical mutagenesis treatment are performed on the cyanobacteria" and "the lethality rate of the physical mutagenesis treatment is 50% to 80%, and the lethality rate of the chemical mutagenesis treatment is 40% to 55%." [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a flowchart of a method for culturing cyanobacteria according to a first embodiment of the present invention. [Figure 2] FIG. 1 is a curve diagram showing the effect of UV irradiation height and time on the lethality of bacterial strains. [Figure 3] FIG. 1 is a schematic diagram showing the percentage of bacterial strains that can withstand temperatures above 45° C. by changing UV irradiation conditions. [Figure 4] FIG. 1 is a curve diagram showing the effect of NTG concentration and treatment time on the lethality of strains. [Figure 5]FIG. 1 is a schematic diagram showing the proportion of strains selected for high-temperature tolerance of 45° C. or higher by changing NTG conditions. [Figure 6] 1 is a flowchart of a method for culturing cyanobacteria according to a second embodiment of the present invention. [Figure 7] FIG. 1 is a schematic diagram showing the amount of CO2 consumed by cyanobacteria selected by the method of the present invention when treating industrial exhaust gas. DETAILED DESCRIPTION OF THE INVENTION

[0023] To better understand the features and technical contents of the present invention, please refer to the following detailed description of the present invention and the accompanying drawings, which are provided for reference and explanation only and are not intended to limit the scope of the present invention.

[0024] The "method for culturing cyanobacteria" according to an embodiment of the present invention will be described below using certain specific embodiments, and those skilled in the art will be able to understand the advantages and effects of the present invention based on the content disclosed herein. The present invention can be implemented or applied using other different specific embodiments, and various modifications and changes can be made to the details herein based on different perspectives and applications without departing from the concept of the present invention. Also, as previously explained, the accompanying drawings of the present invention are for simple schematic illustrations and are not drawn to actual size. The technical content of the present invention will be described in more detail based on the following embodiments, but the disclosed content does not limit the scope of protection of the present invention.

[0025] [First embodiment] As shown in Figure 1, a method for culturing cyanobacteria according to a first embodiment of the present invention includes providing cyanobacteria in step S101. In one embodiment of the present invention, the cyanobacteria according to the present invention is Synechococcus elongatus PCC7942. In the present invention, the cyanobacteria are first subjected to physical mutagenesis in step S102, and then to chemical mutagenesis in step S103, thereby efficiently obtaining cyanobacteria that are resistant to high temperatures and industrial exhaust gases.

[0026] In the physical mutagenesis treatment step S102, the cyanobacteria were cultured in a BG-11 solid medium in an incubator at 30°C with a CO2 concentration of 3% for about 36 hours, and then analyzed using a cell counter (Scepter TM Handheld automated cell counter (Millipore) was used for 10 5 The strains were quantified in terms of cells / ml. The strains were subjected to physical mutagenesis using a UV light source. After the reaction, the bacterial solution was spread onto a 10cm x 10cm Petri dish and grown at 30°C using a cell counter. After colonies grew, they were transferred to new BG-11 solid medium using a sterile toothpick for storage and subsequent analysis.

[0027] The UV light source used in this invention is the ALL KILL-01 comprehensive dry disinfection, sterilization and purification device (UVGI, UV-C 253.7 nm, light energy 20,000 μW-sec / cm) manufactured by POWER JADE LINK ENERGY TECHNOLOGY INC. 2 ) may be used. In order to obtain cyanobacteria that are resistant to high temperatures and industrial exhaust gases, the mortality rate in the physical mutagenesis treatment step S102 must be controlled to 50% to 80%, preferably 60% to 80%, and more preferably 75%. If the mortality rate is less than 50% or more than 80%, the possibility of obtaining cyanobacteria that are resistant to high temperatures and industrial exhaust gases is low.

[0028] Specifically, the lethality rate in the present invention refers to the number of bacterial strains smeared on the solid medium after the mutation test divided by the number of bacterial strains smeared on the solid medium before the mutation test × 100%. Before the mutation test, the number of bacterial strains was counted using a cell counter, and sterilized water was added to the solid medium to count 10 5 Bacterial suspensions quantified in cells / ml were prepared, and the physically mutagenized strains were then serially diluted and plated onto BG-11 solid medium to calculate the actual colony counts.

[0029] The present invention controls the lethality within a specific range by using a specific UV light source and treatment time to efficiently obtain high-temperature- and environmentally resistant cyanobacteria. To control the lethality in the physical mutagenesis treatment step S102 to 50%-80%, the present invention examines the effects of different UV light source heights and treatment times on the lethality of bacterial strains. As shown in Figure 2, when the vertical height between the UV light source and the bacterial strain is 20 cm-50 cm and the exposure time is 10 seconds-70 seconds, the lethality of the bacterial strain can be controlled to 50%-80%. In one preferred embodiment of the present invention, when the vertical height between the UV light source and the bacterial strain is 30 cm-40 cm and the exposure time is 30 seconds-40 seconds, the lethality of the bacterial strain can be controlled to 70%-75%.

[0030] When using a UV-C 253.7 nm UV light source, the relationship between the vertical height of the UV light source and the bacterial strain and the received illuminance is as shown in Table 1 below.

[0031] [Table 1]

[0032] To explain further, Figure 3 is a schematic diagram showing the percentage of strains with a temperature tolerance of 45°C or higher by changing UV irradiation conditions. In Figure 3, the highest probability of generating strains with a temperature tolerance of 45°C or higher is obtained when the UV light source is irradiated for 30 seconds at a vertical height of 30 cm from the strain, and when the UV light source is irradiated for 45 seconds at a vertical height of 40 cm from the strain. The probability of generating temperature-tolerant strains selected under these conditions is approximately 41.5%. In other words, to increase the probability of obtaining strains with a temperature tolerance of 45°C or higher, the lethality rate of the physical mutagenesis treatment step S102 needs to be controlled to 50% to 80%, preferably 60% to 80%, and more preferably 75%.

[0033] Subsequently, the strains that survived the physical mutagenesis step S102 are subjected to the chemical mutagenesis step S103. The chemical mutagenesis step S103 of the present invention employs nitrosoguanidine treatment (NTG treatment), i.e., mutagenesis of the strains is performed using N-methyl-N'-nitro-N-nitrosoguanidine at a specific concentration.

[0034] Specifically, the strain was first placed on a BG-11 solid medium and cultured in an incubator at 30°C for approximately 36 hours. After that, the strain was counted at 10 5 The bacterial suspension is quantified to cells / ml, and sterilized water or BG-11 is added to prepare 4 ml of bacterial suspension, which is then stored in a water bath at 30°C. In this embodiment, the NTG reaction reagent is prepared by placing 1.5 mg of NTG in a sterilized centrifuge tube, adding 1 ml of phosphate buffer (pH 6, 0.2 M) to dissolve the NTG, and storing the tube in a water bath at 30°C.

[0035] During the chemical mutagenesis step S103, the bacterial suspension was poured into a centrifuge tube containing the NTG reaction reagent, thoroughly mixed, and immediately placed in a 30°C water bath to calculate the reaction time. In this embodiment, the final NTG concentration was 300 μg / ml. After the NTG reaction reagent was allowed to react for the specified time, the centrifuge tube was removed from the water bath and centrifuged at 3500 rpm for 10 minutes. The waste solution was poured into concentrated NaOH, the bacterial mass was homogenized, 5 ml of saline was added, and the mixture was centrifuged at 3500 rpm for 10 minutes. After discarding the waste solution, 5 ml of sterile water was added to prepare a suspension of mutant bacteria mutated with NTG. The suspension of mutant bacteria mutated with NTG was then spread onto a 10 cm x 10 cm Petri dish and grown in an incubator at 25°C. After the mutant colonies had grown, they were transferred to new BG-11 solid medium using a sterilized toothpick for storage and subsequent analysis.

[0036] The present invention controls the lethality within a specific range by using a specific NTG concentration and treatment time to efficiently obtain high-temperature- and environmentally tolerant cyanobacteria. To control the lethality in the chemical mutagenesis treatment step S103 to 40%-55%, the present invention examines the effects of different NTG concentrations and treatment times on the lethality of bacterial strains. As shown in Figure 4, when a bacterial strain is treated with 50-300 μg / ml NTG for 0-10 minutes and with 50-200 μg / ml NTG for 0.5-2 minutes, the lethality of the bacterial strain can be controlled to 40%-55%. In one preferred embodiment of the present invention, when a bacterial strain is treated with 50-100 μg / ml NTG for 0.5-1.5 minutes, the lethality of the bacterial strain can be controlled to approximately 50%.

[0037] More specifically, as shown in FIG. 5, which is a schematic diagram showing the proportion of strains selected for thermotolerance of 45°C or higher by changing NTG conditions, the highest probability of generating strains with a thermotolerance of 45°C or higher is obtained by treating the strains with 50 and 100 μg / ml NTG for 1 minute. The probability of generating thermotolerant strains selected under these conditions is approximately 30% to 33%. In other words, to improve the probability of obtaining strains with a thermotolerance of 45°C or higher, the lethality rate of the chemical mutagenesis treatment step S103 needs to be controlled to 40% to 55%, preferably 45% to 55%, and more preferably 50%.

[0038] Subsequently, the strains surviving the chemical mutagenesis treatment step S103 are subjected to a temperature tolerance test S104. In one embodiment of the present invention, the strains subjected to the temperature tolerance test S104 are 12.5% ​​of the native cyanobacterial strains provided in step S101.

[0039] [Second embodiment] In a second embodiment of the present invention, a method for culturing cyanobacteria includes a step S201 of providing cyanobacteria. In step S202, the cyanobacteria are subjected to physical mutagenesis to achieve a mortality rate of 50% to 80%, and 20% to 50% of the cyanobacteria that survive the physical mutagenesis are designated as primary mutant cyanobacteria. In a preferred embodiment, in step S202, the cyanobacteria are subjected to physical mutagenesis to achieve a mortality rate of 60% to 80%, i.e., 20% to 40% of the cyanobacteria that survive the physical mutagenesis are designated as primary mutant cyanobacteria. In a more preferred embodiment, in step S202, the cyanobacteria are subjected to physical mutagenesis to achieve a mortality rate of 75%, i.e., 25% of the cyanobacteria that survive the physical mutagenesis are designated as primary mutant cyanobacteria.

[0040] In step S203, the primary mutant cyanobacteria are subjected to chemical mutagenesis to achieve a mortality rate of 40% to 55%, and 45% to 60% of the primary mutant cyanobacteria that survive the chemical mutagenesis are designated as secondary mutant cyanobacteria. In one preferred embodiment, in step S203, the primary mutant cyanobacteria are subjected to chemical mutagenesis to achieve a mortality rate of 45% to 55%, and 45% to 55% of the primary mutant cyanobacteria that survive the chemical mutagenesis are designated as secondary mutant cyanobacteria. In a more preferred embodiment, in step S203, the primary mutant cyanobacteria are subjected to chemical mutagenesis to achieve a mortality rate of 50%, and 50% of the primary mutant cyanobacteria that survive the chemical mutagenesis are designated as secondary mutant cyanobacteria.

[0041] In step S204, the secondary mutant cyanobacteria are placed in an environment of 30°C to 60°C, and the growth status is observed to select pre-selected cyanobacteria. Specifically, by culturing the secondary mutant cyanobacteria at 5°C intervals, pre-selected cyanobacteria adapted to growth environments of 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C can be obtained.

[0042] In step S205, the target cyanobacteria is selected by placing the preselected cyanobacteria in a mixed gas with an aeration ratio of 0.5% to 9%. Specifically, the preselected cyanobacteria that are tolerant to the target temperature are selected in step S204, and the preselected cyanobacteria are cultured in mixed gas with aeration ratios of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 5.0%, 7.0%, and 9.0%. The growth status is observed to obtain the target cyanobacteria that are tolerant to specific aeration conditions.

[0043] In the present invention, specific aeration conditions include "30-50 ppm hydrogen gas, 150-250 ppm acetylene, 100-200 ppm methane, 0.1-1 ppm hydrogen sulfide, and 1-5 ppm acetaldehyde." Specifically, specific aeration conditions may be "40 ppm hydrogen gas, 200 ppm acetylene, 150 ppm methane, 0.5 ppm hydrogen sulfide, and 3 ppm acetaldehyde." Furthermore, the growth temperature of the target cyanobacteria can be further increased by further subjecting the secondary mutant cyanobacteria to chemical mutagenesis. Thus, the cyanobacterial cultivation method of the present invention effectively improves the possibility of obtaining cyanobacteria that are resistant to high temperatures and industrial exhaust gases.

[0044] To demonstrate that "mixed mutation," in which UV mutation is first performed followed by NTG mutation, improves the likelihood of obtaining a thermotolerant strain, (A) a strain that has undergone UV mutation alone, (B) a strain that has undergone NTG mutation alone, and (C) a strain that has undergone mixed mutation, were each transferred to BG-11 liquid medium and cultured at different temperatures in a 1% CO2 environment. Growth of the strains was monitored over time to obtain a strain adapted to growth at high temperatures. The experimental results are shown in Table 2 below (temperature units: °C).

[0045] [Table 2]

[0046] In Table 2, the growth status is evaluated as excellent (+++) if it is similar to or better than the growth rate of the native strain (a strain not subjected to mutagenesis treatment), as good (++) if it is lower than 0-25%, and as fair (+) if it is lower than 25-50%. Here, similar growth status means that the time to achieve the same number of strains is comparable. In the present invention, the growth rate is measured by the OD 730It is expressed as the time required for the growth rate to increase from 0 to 1. The specific growth rate is shown in Table 3 below.

[0047] [Table 3]

[0048] According to the criteria in Table 3, the strains that underwent only UV mutation were rated as excellent in growth at 35°C and good in growth at 40°C. The strains that underwent only NTG mutation were able to adapt to temperatures between 40°C and 45°C, but when the temperature rose to 60°C, their growth was rated poor. However, the strains that underwent mixed mutation were able to adapt to a growth environment between 40°C and 45°C, and their growth at 55°C was rated good. Thus, the results in Table 2 show that the strains that underwent mixed mutation had excellent growth at high temperatures.

[0049] Furthermore, strains that showed "excellent" or "good" growth at relatively high temperatures in the temperature tolerance test S104 were selected and subjected to the environmental tolerance test S105. In the environmental tolerance test S105, high-temperature-tolerant strains were grown at the appropriate temperature and tested with CO2 mixed gas at aeration rates of 0.5% to 9%. The growth of the strains was monitored over time, and strains adapted to growth at various CO2 mixed gas concentrations were identified. Here, at an aeration rate of 1%, 1 milliliter of CO2 mixed gas was supplied per minute per liter of culture volume.

[0050] In this invention, a CO2 mixed gas was prepared based on the composition of actual industrial exhaust gas. Specifically, the CO2 mixed gas contains, in addition to carbon dioxide (CO2), 40 ppm of hydrogen gas (H2), 200 ppm of acetylene (C2H2), 150 ppm of methane (CH4), 0.5 ppm of hydrogen sulfide (HS), and 3 ppm of acetaldehyde (CH3CHO). The strains numbered A66, B25, B5, C33, C15, and C27 in Table 1 were subjected to environmental resistance tests. The experimental results are shown in Table 4 below, and the growth status evaluation is shown in Table 3.

[0051] [Table 4]

[0052] As shown in Table 4 above, the best strain was strain C33. Even at a high temperature of 55°C and a 1.5% CO2 mixed gas environment, the growth condition of strain C33 was rated "good," demonstrating its resistance to high temperatures and industrial exhaust gases. Strain A66 was tolerant to a 2% CO2 mixed gas environment, but its adaptable growth temperature was relatively low. Furthermore, among the strains adaptable to 45°C, only strain C15 could tolerate a maximum CO2 mixed gas of 9%. To further improve the high-temperature tolerance of strain C15, NTG mutagenesis was performed.

[0053] In one embodiment of the present invention, the strain was treated with 100 μg / ml NTG for 1 minute to control the mortality rate to approximately 50%. In one embodiment of the present invention, strain C15 was subjected to a second NTG treatment (second NTG mutagenesis), and the mutated strain was subjected to a temperature tolerance test. The experimental results are shown in Table 5 below. The criteria for evaluating the growth status were the same as those shown in Table 3.

[0054] [Table 5]

[0055] As shown in Table 5, the strains that underwent the second NTG treatment were able to adapt to temperatures above 45°C, and were particularly adapted to temperatures between 50°C and 55°C. More specifically, strains with "excellent" growth conditions at 55°C were selected and subjected to environmental resistance tests. Specifically, strains numbered C15-17, C15-31, and C15-87 in Table 5 were selected and subjected to environmental resistance tests. The experimental results are shown in Table 6 below. The evaluation criteria for growth conditions were the same as those in Table 3.

[0056] [Table 6]

[0057] As shown in Table 6, the strains that underwent the second NTG treatment were able to adapt to temperatures above 55°C and showed improved environmental tolerance. For example, strain C15-17 was able to adapt to temperatures above 55°C and showed growth characteristics similar to those of conventional strains in an environment with a CO2 mixed gas aeration ratio of 0.5 to 5%. It also showed excellent growth characteristics in an environment with a high CO2 mixed gas concentration (7% to 9%).

[0058] More specifically, industrial exhaust gas was treated with a bacterial strain (C15-17 as an example) selected by the method of the present invention. As shown in Figure 7, the strain grew rapidly over time even in a CO2-mixed gas environment at 45°C. During the first 12 hours, the amount of the strain was small, consuming only about 10% of the CO2-mixed gas. However, as the strain grew rapidly, after 24 hours it consumed about 50% of the CO2-mixed gas. Furthermore, after 36 hours it consumed more than 90% of the CO2-mixed gas. Thus, the bacterial strain selected by the method of the present invention is effective in treating CO2-mixed gas and can therefore be applied to the treatment of industrial exhaust gas.

[0059] [Advantageous Effects of the Embodiments] As an advantageous effect of the present invention, the method for culturing cyanobacteria according to the present invention increases the possibility of obtaining cyanobacteria that are resistant to high-temperature industrial exhaust gases by virtue of the technical features that "physical mutagenesis treatment and chemical mutagenesis treatment are performed on the cyanobacteria" and "the lethality rate of the physical mutagenesis treatment is 50% to 80%, and the lethality rate of the chemical mutagenesis treatment is 40% to 55%."

[0060] To further explain, the present invention provides an efficient mutation strategy. By further subjecting the secondary mutant cyanobacteria of the present invention to NTG mutagenesis, the growth temperature of the cyanobacteria can be further improved, and the concentration of industrial exhaust gas that the cyanobacteria can tolerate can be increased. This makes it possible to efficiently obtain cyanobacteria that are tolerant to high-temperature industrial exhaust gas.

[0061] The above disclosure is merely a preferred embodiment of the present invention, and the scope of the claims of the present invention is not limited thereto. Therefore, all equivalent technical modifications made using the specification and drawings of the present invention are included in the scope of the claims of the present invention. [Explanation of symbols]

[0062] S101~S105, S201~S205...Manufacturing steps for the cyanobacteria cultivation method

Claims

1. Providing a cyanobacterium that is Synechococcus elongatus PCC7942; subjecting the cyanobacteria to a physical mutagenesis treatment to obtain a cyanobacterium with primary mutations, and irradiating the cyanobacteria with an ultraviolet light source (UV light source) having an illuminance of 0.017 mW / cm 2 to 0.082 mW / cm 2 for 10 to 70 seconds in the physical mutagenesis treatment; subjecting the primary mutated cyanobacteria to a chemical mutagenesis treatment to obtain a secondary mutated cyanobacteria, and treating the primary mutated cyanobacteria with nitrosoguanidine (NTG) at a concentration of 50 to 300 μg / ml for 0.5 to 2 minutes in the chemical mutagenesis treatment; and subjecting the secondary mutant cyanobacteria to a temperature tolerance test and an environmental tolerance test to obtain a target cyanobacterium; A method for culturing cyanobacteria, wherein the lethality rate of the physical mutagenesis treatment is 50% to 80%, and the lethality rate of the chemical mutagenesis treatment is 40% to 55%.

2. 2. The method for culturing cyanobacteria according to claim 1, wherein the mortality rate of the physical mutagenesis treatment is 60% to 80%.

3. 2. The method for culturing cyanobacteria according to claim 1, wherein the chemical mutagenesis treatment is 40% to 55%.

4. The method for culturing cyanobacteria according to claim 1, wherein the temperature tolerance test comprises placing the secondary mutant cyanobacteria in an environment of 30°C to 60°C, observing their growth, and selecting cyanobacteria that require 225 minutes or less for their OD730 to increase from 0 to 1, thereby selecting them as pre-selected cyanobacteria.

5. 5. The method for culturing cyanobacteria according to claim 4, wherein the environmental tolerance test involves placing the preselected cyanobacteria in a mixed gas with an aeration ratio of 0.5% to 9%, and selecting cyanobacteria that require 225 minutes or less for their OD730 to increase from 0 to 1, and using this as the target cyanobacteria.

6. The method for culturing cyanobacteria according to claim 5 , wherein the mixed gas contains hydrogen gas, acetylene, methane, hydrogen sulfide, and acetaldehyde.

7. 7. The method for culturing cyanobacteria according to claim 6, wherein the mixed gas contains 30 ppm to 50 ppm of hydrogen gas, 150 ppm to 250 ppm of acetylene, 100 ppm to 200 ppm of methane, 0.1 ppm to 1 ppm of hydrogen sulfide, and 1 to 5 ppm of acetaldehyde.

8. The method for culturing cyanobacteria according to claim 1 , further comprising subjecting the secondary mutant cyanobacteria to the chemical mutagenesis treatment.

9. Providing a cyanobacterium that is Synechococcus elongatus PCC7942; subjecting the cyanobacteria to a physical mutagenesis treatment so as to achieve a mortality rate of 50% to 80%, and designating the cyanobacteria that survive the physical mutagenesis treatment as primary mutant cyanobacteria; and irradiating the cyanobacteria with an ultraviolet light source (UV light source) having an illuminance of 0.017 mW / cm 2 to 0.082 mW / cm 2 for 10 to 70 seconds during the physical mutagenesis treatment; subjecting the primary mutant cyanobacteria to a chemical mutagenesis treatment so as to achieve a mortality rate of 40% to 55%, and the primary mutant cyanobacteria surviving the chemical mutagenesis treatment are designated as secondary mutant cyanobacteria, and in the chemical mutagenesis treatment, treating with nitrosoguanidine (NTG) at a concentration of 50 to 300 μg / ml for 0.5 to 2 minutes; Observing the growth of the secondary mutant cyanobacteria in an environment of 30°C to 60°C, and selecting cyanobacteria that require 225 minutes or less for OD730 to increase from 0 to 1, as pre-selected cyanobacteria; and screening the target cyanobacteria by placing the preselected cyanobacteria in a gas mixture with an aeration ratio of 0.5% to 9%; A method for culturing cyanobacteria, wherein the mixed gas is a mixture containing hydrogen gas, acetylene, methane, hydrogen sulfide, and acetaldehyde.

10. 10. The method for culturing cyanobacteria according to claim 9, wherein the mixed gas contains 30 ppm to 50 ppm of hydrogen gas, 150 ppm to 250 ppm of acetylene, 100 ppm to 200 ppm of methane, 0.1 ppm to 1 ppm of hydrogen sulfide, and 1 ppm to 5 ppm of acetaldehyde.

11. The method for culturing cyanobacteria according to claim 9, further comprising subjecting the secondary mutant cyanobacteria to the chemical mutagenesis treatment.

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