Methods for cultivating cyanobacteria

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 particularly to a method for culturing cyanobacteria resistant to high-temperature industrial exhaust gases.

Background Art

[0002] Cyanobacteria, also known as blue-green algae, are prokaryotes capable of photosynthesis. In the prior art, the property that cyanobacteria can perform photosynthesis is utilized to absorb carbon dioxide emitted from factories, thereby achieving the sustainable development goals of zero waste and zero pollution.

[0003] However, industrial exhaust gases contain not only high concentrations of carbon dioxide but also other chemical substances such as sulfides and nitrogen compounds. These chemical substances affect the growth of cyanobacteria and may even cause the death of cyanobacteria in some cases. In addition, since industrial exhaust gases usually accompany high temperatures, the survival rate and photosynthesis efficiency of cyanobacteria are significantly reduced, making it difficult to effectively utilize the carbon fixation property of cyanobacteria in the treatment of industrial exhaust gases.

[0004] Therefore, providing a method for culturing cyanobacteria that is resistant to high temperatures and industrial exhaust gases to overcome the above-mentioned drawbacks is an important issue to be solved in this industry.

Summary of the Invention

Problems 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 Problems

[0006] The culturing method of the cyanobacteria includes providing cyanobacteria, obtaining cyanobacteria with primary mutations by performing physical mutagenesis treatment on the cyanobacteria, obtaining cyanobacteria with secondary mutations by performing chemical mutagenesis treatment on the cyanobacteria with primary mutations, and obtaining target cyanobacteria by performing a temperature tolerance test and an environmental tolerance test on the cyanobacteria with secondary mutations. The lethality rate of the physical mutagenesis treatment is 50% - 80%, and the lethality rate of the chemical mutagenesis treatment is 40% - 55%.

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

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

[0009] In one embodiment, in the physical mutagenesis treatment, the cyanobacteria are irradiated with an ultraviolet light source (UV light source) with an illuminance of 0.017 mW / cm 2 ~0.082 mW / cm 2 for 10 - 70 seconds.

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

[0011] In one embodiment, the temperature tolerance test is to place the cyanobacteria with secondary mutations in an environment of 30°C - 60°C, observe the growth status, and select the cyanobacteria for preliminary selection.

[0012] In one embodiment, the environmental tolerance test is to select the target cyanobacteria by placing the cyanobacteria for preliminary selection in a mixed gas with an aeration ratio of 0.5% - 9%.

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

[0014] In one embodiment, the mixed gas includes 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 includes performing the chemical mutagenesis treatment on the cyanobacteria with the secondary mutation.

[0016] To solve the above technical problems, another technical means adopted by the present invention is to provide a method for culturing cyanobacteria. The method for culturing cyanobacteria includes providing cyanobacteria, performing physical mutagenesis treatment on the cyanobacteria so that the lethality rate reaches 50% to 80%, and using the surviving cyanobacteria after the physical mutagenesis treatment as the cyanobacteria with primary mutation. Then, performing chemical mutagenesis treatment on the cyanobacteria with primary mutation so that the lethality rate reaches 40% to 55%, and using the surviving cyanobacteria with primary mutation after the chemical mutagenesis treatment as the cyanobacteria with secondary mutation. Monitoring the growth status of the cyanobacteria with secondary mutation in an environment of 30°C to 60°C to screen the preselected cyanobacteria, and screening the target cyanobacteria by placing the preselected cyanobacteria in a mixed gas with an aeration ratio of 0.5% to 9%. The mixed gas is a mixture including hydrogen gas, acetylene, methane, hydrogen sulfide, and acetaldehyde.

[0017] In one embodiment, in the physical mutagenesis treatment, the cyanobacteria are irradiated for 10 to 70 seconds using an ultraviolet light source (UV light source) with an illuminance of 0.017 mW / cm 2 ~0.082 mW / cm 2 ​

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

[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 includes performing a chemical mutagenesis treatment on the cyanobacteria of the secondary mutation.

Advantages of the Invention

[0021] As an advantageous effect of the present invention, the method for culturing cyanobacteria according to the present invention is characterized by "performing a physical mutagenesis treatment and a chemical mutagenesis treatment on the cyanobacteria", and "the lethality of the physical mutagenesis treatment is 50% to 80%, and the lethality of the chemical mutagenesis treatment is 40% to 55%", which increases the possibility of obtaining cyanobacteria resistant to high-temperature industrial exhaust gas.

Brief Description of the Drawings

[0022]

Figure 1

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Figure 7

Embodiments for Carrying Out the Invention

[0023] In order to better understand the features and technical content of the present invention, please refer to the following detailed description of the present invention and the accompanying drawings. However, the provided accompanying drawings are only for reference and explanation, and are not for limiting the scope of the claims of the present invention.

[0024] Hereinafter, the "method for culturing cyanobacteria" according to the embodiment of the present invention will be described according to a specific embodiment. Those skilled in the art can understand the advantages and effects of the present invention based on the content disclosed in this specification. The present invention can be implemented or applied according to other different specific embodiments, and for each detail in this specification, various modifications and changes can be made based on different viewpoints and uses without departing from the concept of the present invention. Also, as explained in advance, the accompanying drawings of the present invention are simple schematic explanations and are not drawn based on the 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 protection scope of the present invention.

[0025] [First Embodiment] As shown in FIG. 1, the method for culturing cyanobacteria according to the first embodiment of the present invention includes a step S101 of providing cyanobacteria. In one embodiment of the present invention, the cyanobacteria according to the present invention are Synechococcus elongatus PCC7942. In the present invention, by first performing a physical mutagenesis treatment step S102 on cyanobacteria and then performing a chemical mutagenesis treatment step S103, cyanobacteria that are highly heat-resistant and resistant to industrial exhaust gases can be efficiently obtained.

[0026] In the physical mutagenesis treatment step S102, after culturing cyanobacteria in a BG-11 solid medium in an incubator at 30°C and a CO2 concentration of 3% for about 36 hours, a cell counter (Scepter TM Handheld automated cell counter, Millipore) is used to quantify the strain at 10 5 cells / ml. The strain is subjected to physical mutagenesis treatment with a UV light source. After the reaction, the bacterial solution is spread on a 10 cm × 10 cm petri dish and grown in a cell counter at 30°C. After colonies grow, the colonies are transferred to a new BG-11 solid medium using a sterilized toothpick for storage and subsequent analysis.

[0027] As the UV light source of the present invention, the comprehensive dry sterilization and purification device ALL KILL-01 (UVGI, UV-C253.7 nm. Light energy 20000 μw-sec / cm 2 ) manufactured by POWER JADE LINK ENERGY TECHNOLOGY INC. may be used. In order to obtain subsequent cyanobacteria that are highly heat-resistant and resistant to industrial exhaust gases, it is necessary to control the lethality rate in the physical mutagenesis treatment step S102 to 50% - 80%, preferably to 60% - 80%, and more preferably to 75%. If the lethality rate is less than 50% or exceeds 80%, the possibility of obtaining cyanobacteria that are highly heat-resistant and resistant to industrial exhaust gases will be reduced.

[0028] Specifically, the lethality rate in the present invention refers to the number of colonies of the strain smeared on the solid medium after the mutation test / the number of colonies of the strain smeared on the solid medium before the mutation test × 100%. Before the mutation test, the number of strains was calculated with a cell counter, and a bacterial suspension quantified to 10 5 cell / ml was prepared by adding sterilized water. Subsequently, the strains subjected to physical mutagenesis were serially diluted and spread on BG-11 solid medium to calculate the actual number of colonies.

[0029] The present invention controls the lethality rate within a specific range so as to efficiently obtain cyanobacteria with high temperature resistance and environmental tolerance by a specific UV light source and treatment time. In order to control the lethality rate in the physical mutagenesis treatment step S102 to 50% - 80%, in the present invention, the influence of the UV light source at different heights and treatment times on the lethality rate of the strain is examined. As shown in Figure 2, when the vertical height between the UV light source and the strain is 20 cm - 50 cm and the exposure time is 10 seconds - 70 seconds, the lethality rate of the 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 strain is 30 cm - 40 cm and the exposure time is 30 seconds - 40 seconds, the lethality rate of the strain can be controlled to 70% - 75%.

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

[0031]

Table 1

[0032] More specifically, as shown in Fig. 3, which is a schematic diagram showing the proportion of strains having a temperature tolerance of 45°C or higher by changing the UV irradiation conditions, in Fig. 3, the highest occurrence probability of strains with a temperature tolerance of 45°C or higher is respectively that the vertical height between the UV light source and the strain is 30 cm and irradiated for 30 seconds, and that the vertical height between the UV light source and the strain is 40 cm and irradiated for 45 seconds. The probability of the temperature-tolerant strains selected under such conditions is about 41.5%. That is, in order to improve the possibility 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% - 80%, preferably controlled to 60% - 80%, and preferably controlled to 75%.

[0033] Subsequently, a chemical mutagenesis treatment step S103 is performed on the surviving strains after the physical mutagenesis treatment step S102. The chemical mutagenesis treatment step S103 according to the present invention employs nitrosoguanidine treatment (NTG treatment), that is, mutagenesis of the strain is performed with N-methyl-N´-nitro-N-nitrosoguanidine having a specific concentration.

[0034] Specifically, first, the strain is placed on a BG-11 solid medium and cultured in an incubator at 30°C for about 36 hours. Then, the strain is quantified to 10 5 cells / ml with a cell counter, and sterilized water or BG-11 is added to prepare a 4 ml bacterial suspension, which is stored in a water bath at 30°C. In this embodiment, for the NTG reaction reagent, 1.5 mg of NTG is supplied to a sterilized centrifuge tube, 1 ml of phosphate buffer (pH 6, 0.2 M) is added to dissolve NTG, and it is stored in a water bath at 30°C.

[0035] When performing the chemical mutagenesis treatment step S103, pour the bacterial suspension into an Eppendorf tube containing the NTG reaction reagent, mix well, and immediately place it in a 30°C water bath to calculate the reaction time. In this embodiment, the final concentration of NTG was 300 μg / ml. After reacting the NTG reaction reagent for a predetermined time, take the Eppendorf tube out of the water bath, centrifuge it at 3500 rpm for 10 minutes, pour the waste liquid into concentrated NaOH, mix the bacterial mass evenly, add 5 ml of physiological saline, then centrifuge it at 3500 rpm for 10 minutes, discard the waste liquid, and then add 5 ml of sterile water to prepare a suspension of mutant bacteria mutagenized with NTG. Subsequently, spread the suspension of mutant bacteria mutagenized with NTG on a 10 cm × 10 cm petri dish, grow the mutant bacteria in an incubator at 25°C, and after the colonies of mutant bacteria have grown, transfer the colonies to a new BG-11 solid medium using a sterilized toothpick for storage and subsequent analysis.

[0036] The present invention controls the lethality rate within a specific range so as to efficiently obtain cyanobacteria with high temperature resistance and environmental tolerance by a specific NTG concentration and treatment time. In order to control the lethality rate in the chemical mutagenesis treatment step S103 to 40% - 55%, in the present invention, the influence of different NTG concentrations and different treatment times on the lethality rate of the strain is examined. As shown in FIG. 4, when treating the strain with NTG at 50 - 300 μg / ml for 0 - 10 minutes and when treating the strain with NTG at 50 - 200 μg / ml for 0.5 - 2 minutes, the lethality rate of the strain can be controlled to 40% - 55%. In one preferred embodiment according to the present invention, when treating the strain with NTG at 50 - 100 μg / ml for 0.5 - 1.5 minutes, the lethality rate of the strain can be controlled to about 50%.

[0037] More specifically, as shown in Fig. 5, which is a schematic diagram showing the ratio of strains selected for high temperature resistance of 45°C or higher by changing the NTG conditions, the highest occurrence probabilities of strains with a temperature resistance of 45°C or higher in Fig. 5 are respectively when the strains are treated with NTG at 50 and 100 μg / ml for 1 minute. The probability of the temperature-resistant strains selected under such conditions is about 30% - 33%. That is, in order to improve the possibility of obtaining strains with a temperature resistance of 45°C or higher, the lethality rate in the chemical mutagenesis treatment step S103 needs to be controlled to 40% - 55%, preferably controlled to 45% - 55%, and more preferably controlled to 50%.

[0038] Subsequently, a temperature resistance test S104 is performed on the surviving strains after the chemical mutagenesis treatment step S103. In one embodiment of the present invention, the strains on which the temperature resistance test S104 is performed are 12.5% of the wild strains of cyanobacteria provided in step S101.

[0039] [Second Embodiment] In the second embodiment of the present invention, the culture method of cyanobacteria includes a step S201 of providing cyanobacteria. In step S202, physical mutagenesis treatment is performed on the cyanobacteria so that the lethality rate reaches 50% - 80%, and 20% - 50% of the surviving cyanobacteria after the physical mutagenesis treatment are used as the cyanobacteria with primary mutations. In one preferred embodiment, in step S202, physical mutagenesis treatment is performed on the cyanobacteria so that the lethality rate reaches 60% - 80%, that is, 20% - 40% of the surviving cyanobacteria after the physical mutagenesis treatment are used as the cyanobacteria with primary mutations. In one more preferred embodiment, in step S202, physical mutagenesis treatment is performed on the cyanobacteria so that the lethality rate reaches 75%, that is, 25% of the surviving cyanobacteria after the physical mutagenesis treatment are used as the cyanobacteria with primary mutations.

[0040] In step S203, the cyanobacteria with primary mutations are subjected to chemical mutagenesis so that the lethality rate reaches 40% - 55%. After the chemical mutagenesis treatment, 45% - 60% of the surviving cyanobacteria with primary mutations are used as cyanobacteria with secondary mutations. In one preferred embodiment, in step S203, the cyanobacteria with primary mutations are subjected to chemical mutagenesis so that the lethality rate reaches 45% - 55%. After the chemical mutagenesis treatment, 45% - 55% of the surviving cyanobacteria with primary mutations are used as cyanobacteria with secondary mutations. In one more preferred embodiment, in step S203, the cyanobacteria with primary mutations are subjected to chemical mutagenesis so that the lethality rate reaches 50%. After the chemical mutagenesis treatment, 50% of the surviving cyanobacteria with primary mutations are used as cyanobacteria with secondary mutations.

[0041] In step S204, the cyanobacteria with secondary mutations are placed in an environment of 30°C - 60°C, and their growth status is observed to select the preselected cyanobacteria. Specifically, by culturing the cyanobacteria with secondary mutations at 5°C intervals, preselected cyanobacteria adapted to the 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 preselected cyanobacteria are placed in a mixed gas with an aeration ratio of 0.5% - 9% to select the target cyanobacteria. Specifically, preselected cyanobacteria resistant to the target temperature are selected from step S204, and these preselected cyanobacteria are placed and cultured in a 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%, and the growth status is observed to obtain the target cyanobacteria resistant to specific aeration conditions.

[0043] In the present invention, specific ventilation conditions include "30 to 50 ppm of hydrogen gas, 150 to 250 ppm of acetylene, 100 to 200 ppm of methane, 0.1 to 1 ppm of hydrogen sulfide, and 1 to 5 ppm of acetaldehyde". Specifically, as the specific ventilation conditions, it may be "40 ppm of hydrogen gas, 200 ppm of acetylene, 150 ppm of methane, 0.5 ppm of hydrogen sulfide, and 3 ppm of acetaldehyde". Further, by further performing a chemical mutagenesis treatment on the cyanobacteria of the secondary mutation, the growth temperature of the target cyanobacteria can be further improved. Thus, the culture method of cyanobacteria according to the present invention can effectively improve the possibility of obtaining cyanobacteria that are resistant to high temperatures and industrial exhaust gases.

[0044] After performing the mutation by UV first and then the mutation by NTG, the "mixed mutation", in order to prove that the possibility of obtaining a strain with high-temperature resistance is improved, (A) the strain that has only undergone the mutation by UV, (B) the strain that has only undergone the mutation by NTG, and (C) the strain that has undergone the mixed mutation are each transferred to BG-11 liquid medium, and high-temperature culture is performed at different temperatures in an environment of 1% CO2, and the growth of the strain is monitored over time so as to obtain a strain adapted to growth at high temperatures. The experimental results are as shown in Table 2 below (temperature unit: °C).

[0045]

Table 2

[0046] In Table 2, when the growth state is approximate or better than the growth rate of the wild strain (the strain that has not undergone mutagenesis treatment), it is excellent (+++), when it is lower than 0 to 25%, it is good (++), and when it is lower than 25% to 50%, it is fair (+). Here, the growth state being approximate means that the time to achieve the same number of the same strain is comparable. In the present invention, the growth rate is OD 730It is shown by the time required to improve from 0 to 1. The specific growth rate is as shown in Table 3 below.

[0047]

Table 3

[0048] According to the criteria in Table 3, the strain that only underwent UV-induced mutation was evaluated to have excellent growth at 35°C and good growth at 40°C. The strain that only underwent NTG-induced mutation could adapt to temperatures from 40°C to 45°C, but when the temperature rose to 60°C, the growth state was evaluated as inferior. However, the strain that underwent mixed mutation adapted to the growth environment of 40°C to 45°C and was evaluated to have good growth at 55°C. Thus, according to the results in Table 2, it was shown that the strain that underwent mixed mutation had excellent growth at high temperatures.

[0049] Furthermore, in the temperature tolerance test S104, those with "excellent" or "good" growth states at relatively high temperatures were selected and the environmental tolerance test S105 was conducted. In the environmental tolerance test S105, the strain with high temperature tolerance was grown at the corresponding temperature, and the test was carried out with a CO2 mixed gas with an aeration ratio of 0.5% - 9%. The growth of the strain was monitored over time, and strains adapted to growth at various CO2 mixed gas concentrations were obtained. Here, when the aeration ratio was 1%, 1 milliliter of CO2 mixed gas was supplied per minute for a 1-liter culture volume.

[0050] In the present 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 (H2S), and 3 ppm of acetaldehyde (CH3CHO). Strains with numbers A66, B25, B5, C33, C15, and C27 in Table 1 were subjected to the environmental tolerance test. The experimental results are as shown in Table 4 below, and the evaluation of the growth state is as shown in Table 3.

[0051]

Table 4

[0052] As shown in Table 4 above, the most excellent strain is the strain numbered C33. The strain numbered C33 was evaluated as "good" in terms of growth state even in an environment of a high temperature of 55°C and a CO2 mixed gas with an aeration ratio of 1.5%. Therefore, the strain numbered C33 was shown to have high temperature resistance and resistance to industrial exhaust gas. The strain numbered A66 has resistance to a 2% CO2 mixed gas, but the applicable growth temperature is relatively low. Also, among the strains that can adapt to 45°C, only the strain numbered C15 can withstand a CO2 mixed gas of up to 9%. In order to further improve the high temperature resistance of the strain numbered C15, NTG mutagenesis was further performed on it.

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

[0054]

Table 5

[0055] As shown in Table 5, the strains subjected to the second NTG treatment can adapt to temperatures of 45°C or higher, and particularly to 50°C to 55°C. Further explaining, those with an "excellent" growth state at 55°C were selected for an environmental resistance test. That is, the strains numbered C15-17, C15-31, and C15-87 in Table 5 were selected for an environmental resistance test. The experimental results are as shown in Table 6 below. The criteria for evaluating the growth state are the same as those shown in Table 3.

[0056]

Table 6

[0057] As shown in Table 6, the strain that has undergone the second NTG treatment can adapt to 55°C and can improve its environmental tolerance characteristics. For example, the strain numbered C15-17 can adapt to temperatures of 55°C or higher and has a growth state similar to that of the wild strain in an environment where the ventilation ratio of the CO2 mixed gas is 0.5 to 5%. Also, it has an excellent growth state even in an environment of a high-concentration CO2 mixed gas (7% to 9%).

[0058] Furthermore, the industrial exhaust gas was treated with the strain selected by the method of the present invention (taking C15-17 as an example). As shown in FIG. 7, the strain can grow rapidly with the passage of time even in an environment of a CO2 mixed gas at 45°C. In the first 12 hours, the amount of the strain was small and only about 10% of the CO2 mixed gas was consumed, but along with the rapid growth of the strain, after 24 hours, about 50% of the CO2 mixed gas was consumed. Furthermore, after 36 hours, more than about 90% of the CO2 mixed gas was consumed. That is, since the strain selected by the method of the present invention is effective for the treatment of the CO2 mixed gas, it can be applied to the treatment of industrial exhaust gas.

[0059] [Advantageous Effects According to 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 resistant to high-temperature industrial exhaust gas by technical features such as "performing physical mutagenesis treatment and chemical mutagenesis treatment 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] More specifically, in the present invention, an efficient mutation strategy is provided. By further performing NTG mutagenesis treatment on the secondary mutant cyanobacteria in the present invention, the growth temperature adaptable to the cyanobacteria can be further improved, and the concentration of industrial exhaust gas tolerable by the cyanobacteria can be improved. Thereby, cyanobacteria resistant to high-temperature industrial exhaust gas can be efficiently obtained.

[0061] The content disclosed above is only a preferred executable embodiment of the present invention, and the scope of the claims of the present invention is not limited thereto. Therefore, all equivalent technical changes made using the content of the specification and drawings of the present invention are included in the scope of the claims of the present invention.

Description of Reference Signs

[0062] S101~S105, S201~S205... Manufacturing processes of the cultivation method of cyanobacteria

Claims

1. Providing cyanobacteria; subjecting the cyanobacteria to a physical mutagenesis treatment to obtain a primary mutant cyanobacterium; subjecting the first mutant cyanobacteria to a chemical mutagenesis treatment to obtain a second mutant cyanobacteria; and subjecting the secondary mutant cyanobacteria to a temperature resistance test and an environmental resistance test to obtain a target cyanobacterium. A method for culturing cyanobacteria, wherein the mortality rate of the physical mutagenesis treatment is 50% to 80%, and the mortality rate of the chemical mutagenesis treatment is 40% to 55%.

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

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

4. In the physical mutagenesis treatment, the illuminance was 0.017 mW / cm ２ ~0.082mW / cm ２ The method for culturing cyanobacteria according to claim 1, wherein the cyanobacteria are irradiated for 10 to 70 seconds using an ultraviolet light source (UV light source).

5. 2. The method for culturing cyanobacteria according to claim 1, wherein the chemical mutagenesis treatment is performed with nitrosoguanidine (NTG) at a concentration of 50 to 300 μg / ml for 0.5 to 2 minutes.

6. The method for culturing cyanobacteria according to claim 1, further comprising the steps of: placing the secondary mutation cyanobacteria in an environment of 30°C to 60°C; observing the growth state; and selecting the preselected cyanobacteria.

7. The method for culturing cyanobacteria according to claim 6, wherein the environmental resistance test is to select the target cyanobacteria by placing the preselected cyanobacteria in a mixed gas having an aeration ratio of 0.5% to 9%.

8. The method for culturing cyanobacteria according to claim 7 , wherein the mixed gas contains hydrogen gas, acetylene, methane, hydrogen sulfide, and acetaldehyde.

9. The method for culturing cyanobacteria according to claim 8, 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.

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

11. Providing a cyanobacterium; subjecting the cyanobacteria to a physical mutagenesis treatment so as to achieve a mortality rate of 50% to 80%, and determining the cyanobacteria that survive the physical mutagenesis treatment as primary mutant cyanobacteria; subjecting the first mutant cyanobacteria to a chemical mutagenesis treatment so as to achieve a mortality rate of 40% to 55%, and the first mutant cyanobacteria surviving the chemical mutagenesis treatment are regarded as second mutant cyanobacteria; monitoring the growth of the secondary mutant cyanobacteria at 30°C to 60°C to select a preselected cyanobacterium; screening the target cyanobacteria by placing the preselected cyanobacteria in a gas mixture having 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.

12. In the physical mutagenesis treatment, the illuminance was 0.017 mW / cm ２ ~0.082mW / cm ２ The method for culturing cyanobacteria according to claim 11, wherein the cyanobacteria are irradiated for 10 to 70 seconds using an ultraviolet light source (UV light source).

13. The method for culturing cyanobacteria according to claim 11, wherein the chemical mutagenesis treatment is performed with nitrosoguanidine (NTG) at a concentration of 50 μg / ml to 300 μg / ml for 0.5 to 2 minutes.

14. The method for culturing cyanobacteria according to claim 11, 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.

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

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