Method for identifying carbon sequestration microorganisms

Abstract

The invention discloses a method for identifying carbon sequestration microorganisms, belongs to the technical field of bioengineering, and particularly relates to the method for identifying the carbon sequestration microorganisms. The invention aims to solve the problem that the existing carbon sequestration microbe identification method is difficult to find out marked components. The method comprises the following steps: pre-culturing different-depth soil in wetlands of great khingan and small khingan; the method comprises the following steps: introducing nitrogen-oxygen mixed gas into a transparent closed incubator, respectively introducing equal-volume 13CO2 and 12CO2 to culture soil at different depths for 40 days when the CO2 concentration is close to 0 ppm, extracting soil DNA, adding a CsCl solution and a GB solution, adjusting the refractive index and the buoyancy density, adding a PEG6000 solution, heating in a water bath, detecting the refractive index, and calculating the buoyancy density. After post-treatment, determining the cbbL gene abundance of each density level by utilizing fluorescent quantitative PCR (Polymerase Chain Reaction); therefore, the marking degree of the carbon sequestration microorganisms is judged. According to the method, < 13 > CO2 isotope is used for labeling a carbon sequestration population, and a stable isotope probe labeling method (< 13 > C-DNA-SIP) is used for accurately and rapidly identifying microorganisms with carbon sequestration activity.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for identifying carbon-fixing microorganisms. Background Art

[0002] As global climate change becomes increasingly serious, carbon fixation technology has become an important means to address greenhouse gas emissions and global warming. Microbial carbon fixation, as an important component of wetland carbon cycle, plays an important role in long-term carbon storage. In order to deeply understand the mechanism and potential of carbon fixation microorganisms in wetland environments, an accurate and efficient method is urgently needed to identify carbon fixation microorganisms and their metabolic processes.

[0003] Traditional methods for identifying carbon-fixing microorganisms mostly rely on culture technology and 16S rRNA gene analysis. Although these methods can provide information about microbial communities to a certain extent, they have certain limitations due to the diversity of carbon-fixing microorganisms and the difficulty of culturing some species. In addition, identification methods that rely on 16S rRNA genes can only provide information on microbial classification, but cannot accurately reveal their carbon fixation capacity and metabolic pathways.

[0004] In recent years, as an emerging tool for studying microbial functions, stable isotope probing (DNA-SIP) has shown great potential in the identification and characterization of functional microorganisms. By using labeled stable isotopes, SIP technology can efficiently track the metabolic process of microorganisms on specific substrates, thereby efficiently identifying the microorganisms involved in the metabolic process. Compared with traditional methods, SIP technology can not only provide classification information of microorganisms, but also reveal their functional characteristics under specific environments.

[0005] Although DNA-SIP technology has been applied in a variety of ecosystems, it still faces some challenges in actual operation. For example, it is not easy to find labeled components due to the complexity of the structure and function of soil microbial communities. For specific microorganisms that use carbon as a substrate, the application of DNA-SIP is even more difficult due to changes in carbon valence. Summary of the invention

[0006] The purpose of the present invention is to solve the technical problem that it is difficult to find a marker component in the existing carbon-fixing microorganism identification method, and to provide a method for identifying carbon-fixing microorganisms.

[0007] The method for identifying carbon-fixing microorganisms is carried out according to the following steps:

[0008] S1. Pre-cultivate the soil at different depths in the Greater Khingan Range and Lesser Khingan Range wetlands for one week;

[0009] S2. Introduce nitrogen and oxygen mixed gas into the transparent sealed incubator to expel CO in the box. 2 When CO 2 When the concentration is close to 0 ppm (using CO 2 The analyzer was connected to the outlet of the gas pipe for measurement), and ventilation was stopped to isolate the incubator from the outside environment. 13 CO 2 and 12 CO 2 to 13 CO 2 or 12 CO 2 The concentration reached 500 μmol mol -1 The soils from different depths of the Greater and Lesser Khingan Mountains were cultured for 40 days, and the isotope microcosm experiment was conducted, and then the soil DNA was extracted respectively;

[0010] S3, 2000ng of soil DNA was fixed to 100μl, and then 4.9ml of CsCl solution and 0.9ml of GB solution were added in sequence, and the refractive index was adjusted to 1.4029±0.0002 and the buoyancy density was adjusted to 1.729g / ml (to ensure that most genes are concentrated in the middle part of the density gradient), and then ultra-high-speed centrifugation was performed, and then layered purification was performed to obtain DNA solutions with different buoyancy density levels;

[0011] S4. Add 550 μl of PEG6000 solution to the DNA solutions of different buoyancy density levels, and then heat in a 37°C water bath for 1 h (to ensure that the DNA is fully precipitated). Perform refractive index detection on the DNA solutions of different buoyancy density levels and calculate the buoyancy density. After post-processing, use fluorescent quantitative PCR to determine the abundance of the cbbL gene at each density level.

[0012] S5. Determine the carbon fixation microorganism labeling degree by cbbL gene abundance and buoyancy density. The determination method is as follows: 12 C mark and 13 The buoyant density marked by C is different when using 13 C, the line graph of buoyancy density and cbbL gene abundance will be highly enriched or shifted to the heavy layer. 12 C mark and 13 The difference or offset in cbbL gene abundance caused by C labeling determines whether the microorganism utilizes the labeled carbon for autotrophic growth. The larger the difference or the larger the offset, the higher the degree of labeling, the stronger the microbial activity, and the more substrate utilized.

[0013] The pre-culture temperature in step S1 is 15° C., and water is replenished every 4 days to maintain the original moisture content of the wetland. Pre-culture of the soil can restore the activity of microorganisms in the soil to the greatest extent.

[0014] The different depths of soil described in step S1 are soils with depths of 0-10 cm, 40-50 cm, and 70-80 cm.

[0015] The volume ratio of nitrogen to oxygen in the nitrogen-oxygen mixed gas in step S2 is 21:79.

[0016] In step S2, an equal volume of 13 CO 2 and 12 CO 2 The DNA fragment length was previously detected by 1% agarose gel electrophoresis, and the DNA concentration and DNA purity were determined using NanoDrop 2000 / 2000C UV-visible spectrophotometer.

[0017] The calculation formula of the buoyancy density in steps S3 and S4 is:

[0018] ρ=-75.9318+99.2031x-31.2551x 2 , where ρ represents the buoyancy density and x represents the refractive index.

[0019] The ultra-high speed centrifugation speed in step S3 is 45000 rpm, the centrifugation time is 44 h, and the centrifugation temperature is 20°C;

[0020] The total number of density levels described in S3 is 15.

[0021] Preparation process of the PEG6000 solution in step S4: 150 g of PEG6000 and 46.8 g of NaCl are dissolved in deionized water and the volume is fixed to 500 ml, and then filtered with a 0.2 μm filter membrane and sterilized;

[0022] The post-processing in step S4 is as follows: the DNA solution of each density level is centrifuged to remove the supernatant, then washed and dried, and finally dissolved in 30 μl TE buffer.

[0023] The post-processing in step S4 is as follows: after centrifuging each density level DNA solution, remove the supernatant, then add 70% ethanol to wash twice and centrifuge, each time for 10 minutes, stand at room temperature to fully evaporate the remaining alcohol, and then dissolve in 30 μl TE buffer.

[0024] The primers for cbbL in the fluorescent quantitative PCR in step S4 are k2F: 5'-ACCAYCAAGCCSAAGCTSGG-3' and v2F: 5'-GCCTTCSAGCTTGCCSACCRC-3';

[0025] The quantitative PCR conditions for cbbL were 95°C, pre-denaturation for 15 min, 95°C, 15 s, 59°C, 20 s, 72°C, 20 s, 95°C, 15 s, 60°C, 1 min, 95°C, 15 s, and 40 cycles.

[0026] 12 C mark and 13 C indicates that their buoyancy density is different. 13 C, the line graph of buoyancy density and abundance will be highly enriched or shifted to the heavy layer. 12 C mark and 13 The difference or shift in abundance caused by C labeling determines whether the microorganisms are using the labeled carbon for autotrophic growth. Larger differences or larger shifts indicate a higher degree of labeling, greater microbial activity, and more substrate utilization.

[0027] Compared with the prior art, the present invention utilizes 13 CO 2 Isotope labeling of carbon-fixing populations using stable isotope probe labeling method ( 13 C-DNA-SIP) can accurately and quickly identify microorganisms with carbon fixation activity. It can also be combined with cbbL functional sequencing to evaluate the relative contribution of each carbon fixation group to the carbon fixation process, in order to improve the efficiency of carbon source utilization, reduce carbon loss and CO 2 provide a basis for emissions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the relationship between cbbL gene abundance and buoyancy density at different soil depths 1-15 density levels in Xiaoxing'anling.

[0029] Figure 2 This is the relationship between cbbL gene abundance and buoyancy density at different soil depths 1-15 density levels in Daxinganling. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0031] Specific implementation method 1: The method for identifying carbon-fixing microorganisms in this implementation method is carried out according to the following steps:

[0032] S1. Pre-cultivate the soil at different depths in the Greater Khingan Range and Lesser Khingan Range wetlands for one week;

[0033] S2. Introduce nitrogen and oxygen mixed gas into the transparent sealed incubator to expel CO in the box. 2 When CO 2 When the concentration is close to 0 ppm (using CO 2The analyzer was connected to the outlet of the gas pipe for measurement), and ventilation was stopped to isolate the incubator from the outside environment. 13 CO 2 and 12 CO 2 to 13 CO 2 or 12 CO 2 The concentration reached 500 μmol mol -1 The soils from different depths of the Greater and Lesser Khingan Mountains were cultured for 40 days, and the isotope microcosm experiment was conducted, and then the soil DNA was extracted respectively;

[0034] S3, 2000ng of soil DNA was fixed to 100μl, and then 4.9ml of CsCl solution and 0.9ml of GB solution were added in sequence, and the refractive index was adjusted to 1.4029±0.0002 and the buoyancy density was adjusted to 1.729g / ml (to ensure that most genes are concentrated in the middle part of the density gradient), and then ultra-high-speed centrifugation was performed, and then layered purification was performed to obtain DNA solutions with different buoyancy density levels;

[0035] S4. Add 550 μl of PEG6000 solution to the DNA solutions of different buoyancy density levels, and then heat in a 37°C water bath for 1 h (to ensure that the DNA is fully precipitated). Perform refractive index detection on the DNA solutions of different buoyancy density levels and calculate the buoyancy density. After post-processing, use fluorescent quantitative PCR to determine the abundance of the cbbL gene at each density level.

[0036] S5. Determine the carbon fixation microorganism labeling degree by cbbL gene abundance and buoyancy density. The determination method is as follows: 12 C mark and 13 The buoyant density marked by C is different when using 13 C, the line graph of buoyancy density and cbbL gene abundance will be highly enriched or shifted to the heavy layer. 12 C mark and 13 The difference or offset in cbbL gene abundance caused by C labeling determines whether the microorganism utilizes the labeled carbon for autotrophic growth. The larger the difference or the larger the offset, the higher the degree of labeling, the stronger the microbial activity, and the more substrate utilized.

[0037] Specific embodiment 2: This embodiment is different from the specific embodiment 1 in that the pre-culture temperature in step S1 is 15°C, and water is replenished every 4 days to maintain the original water content of the wetland. The rest is the same as the specific embodiment 1.

[0038] Specific implementation method 3: This implementation method is different from specific implementation methods 1 or 2 in that the soils of different depths in step S1 are soils of 0-10 cm, 40-50 cm, and 70-80 cm in depth. The rest is the same as specific implementation methods 1 or 2.

[0039] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the volume ratio of nitrogen to oxygen in the nitrogen-oxygen mixed gas in step S2 is 21:79. The rest is the same as specific embodiments 1 to 3.

[0040] Specific embodiment 5: This embodiment is different from the specific embodiments 1 to 4 in that an equal volume of 13 CO 2 and 12 CO 2 The length of DNA fragments was previously detected by 1% agarose gel electrophoresis, and the DNA concentration and DNA purity were determined by NanoDrop 2000 / 2000C UV-visible spectrophotometer. The rest is the same as that of the first to fourth embodiments.

[0041] Specific implementation method 6: This implementation method is different from specific implementation methods 1 to 5 in that the calculation formula of the buoyancy density in steps S3 and S4 is:

[0042] ρ=-75.9318+99.2031x-31.2551x 2 , wherein ρ represents the buoyancy density, and x represents the refractive index. The rest is the same as that of any one of the first to fifth embodiments.

[0043] Specific embodiment 7: This embodiment differs from any one of specific embodiments 1 to 6 in that the ultra-high speed centrifugation speed in step S3 is 45000 rpm, the centrifugation time is 44 h, and the centrifugation temperature is 20°C;

[0044] The total number of density levels in S3 is 15. The rest is the same as that in Specific Embodiments 1 to 6.

[0045] Specific embodiment eight: This embodiment differs from specific embodiments one to seven in that the PEG6000 solution is prepared in step S4 as follows: 150 g of PEG6000 and 46.8 g of NaCl are dissolved in deionized water and the volume is fixed to 500 ml, then filtered with a 0.2 μm filter membrane and sterilized;

[0046] The specific post-processing of step S4 is: centrifuging each density level DNA solution to remove the supernatant, then washing and drying, and finally dissolving in 30 μl TE buffer. The rest is the same as the specific embodiments 1 to 7.

[0047] Specific embodiment 9: This embodiment is different from specific embodiments 1 to 8 in that the post-processing in step S4 is as follows: after centrifuging each density level DNA solution, the supernatant is removed, and then 70% ethanol is added to wash twice and centrifuged, each centrifugation for 10 minutes, and the remaining alcohol is fully volatilized at room temperature, and then dissolved in 30 μl TE buffer. The rest is the same as specific embodiments 1 to 8.

[0048] Specific embodiment ten: The difference between this embodiment and any one of specific embodiments one to nine is that the primers for cbbL in the fluorescent quantitative PCR in step S4 are k2F: 5'-ACCAYCAAGCCSAAGCTSGG-3' and v2F: 5'-GCCTTCSAGCTTGCCSACCRC-3';

[0049] The quantitative PCR conditions of cbbL are 95°C, pre-denaturation for 15 min, 95°C, 15 s, 59°C, 20 s, 72°C, 20 s, 95°C, 15 s, 60°C, 1 min, 95°C, 15 s, 40 cycles. The rest is the same as in the first to ninth embodiments.

[0050] The following examples are used to verify the effects of the present invention:

[0051] Embodiment 1:

[0052] The method for identifying carbon-fixing microorganisms is carried out according to the following steps:

[0053] S1. Pre-cultivate the soil at different depths in the Greater Khingan Range and Lesser Khingan Range wetlands for one week: replenish water every 4 days at 15°C to maintain the original moisture content of the wetlands.

[0054] S2. In each transparent sealed incubator, introduce a CO-free 2 Nitrogen and oxygen mixture (O 2 21%, N 2 79%) for 10 minutes to exhaust the CO in the box 2 , when the concentration is close to 0 ppm (using CO 2 The analyzer is connected to the outlet of the gas pipe for measurement), and ventilation is stopped to isolate the incubator from the outside environment. Then, an equal volume of 13 CO 2 and 12 CO 2 Gas (0.05%; 500 μmol mol -1), and then cultured for 40 days, respectively, and then soil DNA samples were extracted, wherein the soil DNA samples were extracted using MP soil extraction kit (MP Biomedicals, Santa Ana, CA, USA), and after extraction, the DNA fragment length was detected by 1% agarose gel electrophoresis and the DNA concentration and purity were determined by NanoDrop 2000 / 2000C UV-visible spectrophotometer.

[0055] Table 1: DNA concentration and volume of DNA to be added to a 5.1 ml centrifuge tube

[0056]

[0057]

[0058]

[0059] Note: DP stands for Lesser Khingan Range, CP stands for Greater Khingan Range, 0-10, 40-50 and 70-80 stand for soil depths of 0-10 cm, 40-50 cm and 70-80 cm respectively.

[0060] S3. Adjust the volume of 2000ng soil DNA to 100μl, then add 4.9ml CsCl and 0.9ml GB solution in turn, and adjust the refractive index to 1.4029±0.0002. Put it into an ultra-high-speed centrifuge tube and seal it, then perform ultra-high-speed centrifugation, and then perform layered purification to obtain DNA solutions of different density levels; the ultra-high-speed centrifugation speed is set to 45000rpm, the centrifugation time is 44h, and the centrifugation temperature is 20℃.

[0061] In the present invention, the total volume of the ultra-high speed centrifuge tube is 5.1 ml. In order to ensure that the volume of each layer is 340 μl, the total number of density layers is set to 15. Too few layers will increase the identification cost, and too many layers will make it difficult to separate the density layers, resulting in large errors.

[0062] S4. After centrifugation, take out the ultracentrifuge tube. Divide the sample into 15 layers, and measure the refractive index of each layer to calculate the buoyancy density of each layer (ρ = -75.9318 + 99.2031x - 31.2551x 2 ρ represents buoyant density, and x represents refractive index). 550 μl of PEG6000 solution was added to the DNA solution of each layer, heated in a 37°C water bath for 1 h, and then centrifuged for 30 min to remove the supernatant. Then, 500 μl of 70% ethanol was added to wash twice, and each centrifugation was 10 min. The remaining alcohol was allowed to evaporate at room temperature, and then dissolved in 30 μl of TE buffer. Fluorescence quantitative PCR was then used to determine the abundance of cbbL genes at the buoyant density level;

[0063] The primers for cbbL in the fluorescent quantitative PCR in step S4 are k2F: 5'-ACCAYCAAGCCSAAGCTSGG-3' and v2F: 5'-GCCTTCSAGCTTGCCSACCRC-3';

[0064] The 20μl quantitative system of cbbL is 10μl of TB enzyme, 0.5μl of upstream and downstream primers, 5μl of DNA template, and the rest is made up with water. The quantitative PCR conditions of cbbL are 95℃, pre-denaturation for 15min, 95℃, 15s, 59℃, 20s, 72℃, 20s, 95℃, 15s, 60℃, 1min, 95℃, 15s, 40 cycles.

[0065] The preparation process of the PEG6000 solution is as follows: 150 g of PEG6000 and 46.8 g of NaCl are dissolved in deionized water and the volume is fixed to 500 ml, and then filtered with a 0.2 μm filter membrane and sterilized.

[0066] S5. Determine the degree of carbon fixation microbial labeling by cbbL gene abundance and buoyancy density.

[0067] Table 2: Buoyancy density of different buoyancy density levels

[0068] Lesser Xing'an Mountains (0-10) Layers Buoyancy density g / ml First floor 1.79 Second floor 1.78 Third floor 1.77 Fourth floor 1.77 Fifth floor 1.76 Sixth floor 1.75 Seventh floor 1.74 Eighth Floor 1.73 Ninth Floor 1.72 Tenth floor 1.71 Eleventh Floor 1.70 12th Floor 1.69 Thirteenth Floor 1.68 14th floor 1.67 Fifteenth floor 1.56

[0069]

[0070]

[0071] Lesser Xing'an Mountains (70-80) Layers Buoyancy density g / ml First floor 1.78 Second floor 1.77 Third floor 1.77 Fourth floor 1.76 Fifth floor 1.75 Sixth floor 1.74 Seventh floor 1.74 Eighth Floor 1.73 Ninth Floor 1.72 Tenth floor 1.71 Eleventh Floor 1.70 12th Floor 1.70 Thirteenth Floor 1.69 14th floor 1.68 Fifteenth floor 1.63

[0072]

[0073]

[0074]

[0075]

[0076] Daxinganling (70-80) layers Buoyancy density g / ml First floor 1.78 Second floor 1.77 Third floor 1.76 Fourth floor 1.76 Fifth floor 1.75 Sixth floor 1.74 Seventh floor 1.74 Eighth Floor 1.73 Ninth Floor 1.72 Tenth floor 1.71 Eleventh Floor 1.70 12th Floor 1.69 Thirteenth Floor 1.69 14th floor 1.67 Fifteenth floor 1.52

[0077] Figure 1-2 The cbbL gene abundance and buoyancy density relationship diagrams of the density levels 1-15 at different soil depths in the Greater and Lesser Khingan Mountains in Example 1 are shown respectively. When the initial refractive index is set to 1.4029±0.0002, it can be ensured that most genes are concentrated in the middle part of the density gradient. Microorganisms use carbon as a substrate, and determine the specific microbial groups that metabolize related carbon sources, which directly proves that complete carbon fixation bacteria can utilize 13 C grows autotrophically.

[0078] In summary, the present invention utilizes 12 CO 2 and 13 CO 2 Isotope labeling of carbon-fixing populations using stable isotope probe labeling method ( 13 C-DNA-SIP) can accurately and rapidly identify microorganisms with carbon fixation activity.

Claims

1. A method for identifying carbon-fixing microorganisms, characterized in that The method for identifying carbon-fixing microorganisms is carried out according to the following steps: S1. Pre-cultivate the soil at different depths in the Greater Khingan Range and Lesser Khingan Range wetlands for one week; S2. Introduce nitrogen and oxygen mixed gas into the transparent sealed incubator. When the CO2 concentration in the transparent sealed incubator approaches 0ppm, stop ventilation and introduce equal volumes of 13 CO2 and 12 CO2 to 13 CO2 or 12 CO2 concentration reaches 500 μmol mol -1 Soil from different depths of the Greater Khingan Range and Lesser Khingan Range wetlands was cultured for 40 days, and soil DNA was extracted from each of them. S3, 2000ng of soil DNA was fixed to 100μl, and then 4.9ml of CsCl solution and 0.9ml of GB solution were added in sequence, and the refractive index was adjusted to 1.4029±0.0002 and the buoyancy density was adjusted to 1.729g / ml, and then ultra-high-speed centrifugation was performed, and then layered purification was performed to obtain DNA solutions with different buoyancy density levels; S4. Add 550 μl of PEG6000 solution to the DNA solutions of different buoyancy density levels, and then heat them in a 37°C water bath for 1 h. Perform refractive index detection on the DNA solutions of different buoyancy density levels and calculate the buoyancy density. After post-processing, use fluorescent quantitative PCR to determine the abundance of cbbL genes in each density level. S5. Determine the carbon fixation microorganism labeling degree by cbbL gene abundance and buoyancy density. The determination method is as follows: 12 C mark and 13 The difference or offset in cbbL gene abundance caused by C labeling determines whether the microorganism utilizes the labeled carbon for autotrophic growth. The larger the difference or the larger the offset, the higher the degree of labeling, the stronger the microbial activity, and the more substrate utilized.

2. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that The pre-cultivation temperature in step S1 is 15° C., and water is replenished every 4 days to maintain the original moisture content of the wetland.

3. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that The different depths of soil described in step S1 are soils with depths of 0-10 cm, 40-50 cm, and 70-80 cm.

4. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that The volume ratio of nitrogen to oxygen in the nitrogen-oxygen mixed gas in step S2 is 21:

79.

5. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that In step S2, an equal volume of 13 CO2 and 12 The DNA fragment length was detected by 1% agarose gel electrophoresis before CO2 addition, and the DNA concentration and purity were determined by NanoDrop 2000 / 2000C UV-visible spectrophotometer.

6. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that The calculation formula of the buoyancy density in steps S3 and S4 is: ρ=-75.9318+99.2031x-31.2551x 2 , where ρ represents the buoyancy density and x represents the refractive index.

7. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that In step S3, the ultra-high speed centrifugation speed is 45000 rpm, the centrifugation time is 44 h, and the centrifugation temperature is 20°C; The total number of density levels described in S3 is 15.

8. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that Preparation process of the PEG6000 solution in step S4: 150 g of PEG6000 and 46.8 g of NaCl are dissolved in deionized water and the volume is fixed to 500 ml, and then filtered with a 0.2 μm filter membrane and sterilized; The post-processing in step S4 is as follows: the DNA solution of each density level is centrifuged to remove the supernatant, then washed and dried, and finally dissolved in 30 μl TE buffer.

9. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that The post-processing in step S4 is as follows: after centrifuging each density level DNA solution, remove the supernatant, then add 70% ethanol to wash twice and centrifuge, each centrifugation for 10 minutes, stand at room temperature to fully evaporate the remaining alcohol, and then dissolve in 30 μl TE buffer.

10. The method for identifying carbon-fixing microorganisms according to claim 1, characterized in that The primers for cbbL in the fluorescent quantitative PCR in step S4 are k2F: 5'-ACCAYCAAGCCSAAGCTSGG-3' and v2F: 5'-GCCTTCSAGCTTGCCSACCRC-3'; The quantitative PCR conditions for cbbL were 95°C, pre-denaturation for 15 min, 95°C, 15 s, 59°C, 20 s, 72°C, 20 s, 95°C, 15 s, 60°C, 1 min, 95°C, 15 s, and 40 cycles.

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