Detection method for determining temperature threshold value for maintaining denitrification activity of wetland rhizosphere microorganisms under low-temperature condition

Abstract

The invention relates to the technical field of biological environment. The invention provides a detection method for determining a temperature threshold value for maintaining denitrification activity of wetland rhizosphere microorganisms under a low-temperature condition. The detection method comprises the following steps: (1) planting wetland plants in a test area; (2) gradually reducing the environment temperature of the test area; (3) collecting rhizosphere soil samples and water body samples under different temperature gradient conditions; (4) detecting the denitrification activity of the rhizosphere soil sample, the composition and diversity change of functional microbial communities, the physicochemical properties of soil and the nitrogen concentration change of a water body; and (5) calculating the temperature threshold for maintaining the activity of the functional microorganisms. The method provided by the invention can provide technical support for improving the denitrification effect of the wetland in the low-temperature environment, so that the wetland can play a good role in winter or cold regions, the selection of plant species and the regulation and control of growth conditions in wetland restoration engineering can be guided, and the denitrification capability of the wetland is improved.

Description

Technical Field

[0001] The present invention relates to the field of biological environment technology, and in particular to a detection method for determining a temperature threshold for maintaining denitrification activity of rhizosphere microorganisms. Background Art

[0002] As an important natural ecosystem, wetlands have significant nitrogen pollution reduction capabilities and play a key role in maintaining the ecological balance of water bodies and soil. However, with the acceleration of agricultural activities, industrial emissions and urbanization, a large amount of nitrogen-containing compounds (such as nitrates and ammonia nitrogen) enter wetlands through runoff and drainage, resulting in excessive nitrogen load in wetlands. Long-term nitrogen pollution will not only destroy the structure and function of wetland ecosystems, but also cause a series of environmental problems such as eutrophication of water bodies, loss of biodiversity, and increased emissions of greenhouse gases (such as nitrous oxide). At present, wetland nitrogen pollution control technologies mainly focus on the following aspects: mechanical aeration, chemical treatment and plant regulation, and the core processes of these technologies are microbial-driven nitrification and denitrification processes.

[0003] However, the following key issues exist in existing research and applications: Temperature sensitivity: Microbial denitrification activity is extremely sensitive to ambient temperature, especially under low temperature conditions, the denitrification efficiency of wetlands is significantly reduced. There is currently a lack of research on the temperature threshold of microbial denitrification activity, which leads to a significant weakening of the denitrification function of wetlands in cold seasons or cold regions; Lack of guidance on key parameters: Existing technologies do not conduct in-depth research on the factors affecting the denitrification process of rhizosphere microorganisms in wetlands, especially the lack of systematic analysis of the temperature response characteristics of microbial groups, which cannot provide a scientific basis for wetland restoration projects; Poor ecological adaptability: Many artificial intervention technologies are incompatible with the natural ecological processes of wetlands, which can easily lead to ecological imbalance in wetlands or even further deterioration. Summary of the invention

[0004] The purpose of the present invention is to provide a detection method for determining the temperature threshold for maintaining denitrification activity of rhizosphere microorganisms in wetlands under low temperature conditions. By gradually lowering the ambient temperature, the physical and chemical properties of the rhizosphere soil in the wetland, changes in the microbial community, and nitrification and denitrification activities are systematically collected and detected, and finally the temperature range suitable for maintaining denitrification activity is calculated. The method has the following advantages: (1) Scientificity and operability: The quantitative effect of temperature on the denitrification process of rhizosphere microorganisms is verified by experiments, providing a clear parameter basis for wetland restoration. (2) Wide applicability: This method can be applied in natural wetlands and artificial wetlands, and is particularly suitable for wetland nitrogen pollution control in cold areas or low temperature seasons. (3) Eco-friendly: Relying on the natural effects of plants and microorganisms, it avoids secondary pollution to the wetland ecosystem, while promoting the ecological balance and functional recovery of the wetland.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a detection method for determining the temperature threshold for wetland rhizosphere microorganisms to maintain denitrification activity under low temperature conditions, comprising the following steps:

[0007] (1) Plant wetland plants in the experimental area;

[0008] (2) Gradually reduce the ambient temperature of the test area;

[0009] (3) Collect rhizosphere soil and water samples under different temperature gradient conditions;

[0010] (4) Detect the denitrification activity of rhizosphere soil samples, changes in the composition and diversity of active microbial communities, soil physical and chemical properties, and changes in water nitrogen concentration;

[0011] (5) Calculate the temperature threshold for functional microorganisms to maintain activity.

[0012] Preferably, the plant species in step (1) is an emergent plant, and the density during planting is 9 to 25 plants / m 2 .

[0013] Preferably, the reduction in step (2) is 0.8-1.2°C every three days.

[0014] Preferably, the different temperature gradients in step (3) are: 8°C, 4°C, 0°C, -4°C, -10°C and -15°C.

[0015] Preferably, the method for detecting the nitrification and denitrification activity of the rhizosphere soil in step (4) is:

[0016] Soil, KNO3 solution, glucose solution and chloramphenicol solution were mixed and placed in a sealed container. After the sealed container was evacuated, a helium-acetylene mixed gas was injected. After shaking, the gas in the sealed container was extracted to detect the concentration of N2O.

[0017] The usage ratio of the soil, KNO3 solution, glucose solution and chloramphenicol solution is 13-17 g: 23-27 mL: 23-27 mL: 23-27 mL;

[0018] The concentration of the KNO3 solution is 18-22 mg / L;

[0019] The concentration of the glucose solution is 35-45 mg / L;

[0020] The concentration of the chloramphenicol solution is 190-197 mg / L

[0021] The volume ratio of helium to acetylene in the helium-acetylene mixed gas is 85-95:5-15;

[0022] The atmospheric pressure in the sealed container after the helium-acetylene mixed gas is injected is the same as the atmospheric pressure before vacuuming;

[0023] The shaking conditions are 120-130 r / min and 23-27° C.

[0024] The timing of extracting the gas in the sealed container is when the oscillation starts and 110 to 130 minutes after the oscillation starts;

[0025] The concentration of N2O was measured using an Agilent 7890B gas chromatograph.

[0026] Preferably, the method for detecting changes in the composition and diversity of active microbial communities in step (4) is:

[0027] RNA was extracted using the RNAprep Pure Kit total RNA extraction kit, and the purity of the extracted RNA was tested;

[0028] The purity detection method is as follows: PCR amplification of RNA samples, followed by reverse transcription of the amplified RNA into cDNA, high-throughput sequencing of the cDNA samples on an Illumina Hiseq platform, bioinformatics analysis using QIIME2 software, and comparison, classification and annotation of sequencing data.

[0029] Preferably, the soil physical and chemical properties in step (4) include pH value, NO3 - -N, NO2 - -N、NH4 + -N content, organic matter content, total carbon and total nitrogen content;

[0030] The pH value detection method is as follows: dry soil and water are mixed in a mass ratio of 1:2-3, shaken and then allowed to stand, and then measured with a pH meter;

[0031] The NO3 - -N, NO2 - -N、NH4 + -N content was determined by mixing soil and KCl solution, filtering after shaking, and measuring with Smartchem200;

[0032] The concentration of the KCl solution is 0.8-1.2 M, the volume ratio of the soil to the KCl solution is 1:4-6, and the shaking conditions are 180-220 rpm, 50-70 min;

[0033] The organic matter content is detected by low temperature external heating potassium dichromate oxidation-colorimetry;

[0034] The total carbon and total nitrogen contents are detected by an element analyzer.

[0035] Preferably, the detection of the change in nitrogen concentration in the water body in step (4) includes NO3 - -N, NO2 - -N、NH4 + -N concentration detection and TN content detection;

[0036] The NO3 - -N, NO2 - -N、NH4 + -N concentration was detected using a fully automatic chemical analyzer;

[0037] The TN content is detected by using an element analyzer.

[0038] Preferably, the method for calculating the temperature threshold in step (4) comprises the following steps:

[0039] A. Prepare soil microbial community dataset and environmental factor dataset;

[0040] B. In the microbial community data, standardization is performed to ensure that the minimum number of sampling sequences for each sample is the same;

[0041] C. Use the titan function in the TITAN2 package to analyze the temperature thresholds of positive and negative response groups in the microbial community;

[0042] The method for obtaining the soil microbial community dataset in step A is as follows: comparing the microbial sequencing data in step (5) with the SILVA database to generate an OTU table of denitrification microbial community composition;

[0043] The method for obtaining the environmental factor data set in step A is as follows: the temperature gradient in step (3) is used as the environmental factor.

[0044] The present invention also provides a device for the detection method, comprising an inner frame and a heat-insulating outer layer;

[0045] The inner frame has a length of 30 to 50 cm, a width of 30 to 50 cm, and a height of 50 to 70 cm;

[0046] The heat-insulating outer layer is wrapped around the periphery and bottom of the inner frame.

[0047] In the present invention, the material of the inner frame is transparent plastic, and the material of the heat-insulating outer layer is EPP.

[0048] The present invention also provides a method for using the device, comprising the following steps:

[0049] When the device is used for waterless environment simulation, the soil is filled to 50-70% of the height of the inner frame, and the test is performed after the plants are planted on the soil;

[0050] When the device is used for simulating a water environment, soil is filled to 40-60% of the height of the inner frame, and water is then injected into the inner frame until the water level reaches 80-100% of the height of the inner frame. Plants are planted on the soil and then tested.

[0051] In the present invention, when the device is used for simulating a water environment, the surface of the water layer is cooled into an ice layer after the temperature drops, and the water layer below the ice layer keeps flowing.

[0052] The present invention provides a detection method for determining the temperature threshold for maintaining denitrification activity of rhizosphere microorganisms in wetlands under low temperature conditions, comprising the following steps: (1) planting wetland plants in a test area; (2) gradually lowering the ambient temperature of the test area; (3) collecting rhizosphere soil samples and water samples under different temperature gradient conditions; (4) detecting the denitrification activity, functional microbial community composition and diversity changes, soil physical and chemical properties, and water nitrogen concentration changes of rhizosphere soil samples; (5) calculating the temperature threshold for maintaining the activity of functional microorganisms. The decrease in denitrification efficiency of wetlands under cold climate conditions is a common problem. The technical solution of the present invention can provide technical support for improving the denitrification effect of wetlands in low temperature environments, so that it can also play a good role in winter or cold areas. By clarifying the temperature range of rhizosphere microbial denitrification activity, the selection of plant species and the regulation of growth conditions in wetland restoration projects can be guided, thereby improving the denitrification capacity of wetlands. The present method optimizes the functions of indigenous microorganisms by promoting plant-microorganism interactions, and can also reduce exogenous intervention and save governance costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 A small test device used in the embodiments of the present invention;

[0054] Figure 2 The response curves indicate the mutation points of the total scores of negative response species (Z-) and positive response species (Z+) of the microbial community along the temperature gradient. DETAILED DESCRIPTION

[0055] The present invention provides a detection method for determining the temperature threshold for wetland rhizosphere microorganisms to maintain denitrification activity under low temperature conditions, comprising the following steps:

[0056] (1) Plant wetland plants in the experimental area;

[0057] (2) Gradually reduce the ambient temperature of the test area;

[0058] (3) Collect rhizosphere soil and water samples under different temperature gradient conditions;

[0059] (4) Detect the denitrification activity of rhizosphere soil samples, changes in the composition and diversity of active microbial communities, soil physical and chemical properties, and changes in water nitrogen concentration;

[0060] (5) Calculate the temperature threshold for functional microorganisms to maintain activity.

[0061] In the present invention, the plant species in step (1) is preferably emergent plants, and the density during planting is preferably 9 to 25 plants / m 2 .

[0062] In the present invention, the reduction in step (2) is preferably 0.8-1.2° C. every three days.

[0063] In the present invention, the different temperature gradients in step (3) are preferably: 8°C, 4°C, 0°C, -4°C, -10°C and -15°C.

[0064] In the present invention, the detection method of rhizosphere soil nitrification and denitrification activity described in step (4) is preferably: weigh a fresh soil sample equivalent to 15g dry soil weight and put it into a 250mL culture bottle, add 25mL of a 20mg / L KNO3 solution, 40mg / L glucose and 193.8mg / L chloramphenicol mixed solution to the bottle, then quickly seal the culture bottle, and evacuate the upper air chamber in the bottle, then inject a high-purity helium and acetylene mixed gas (the volume fraction of acetylene is about 10%) balanced with the external atmospheric pressure into the bottle, and perform light-free shaking culture at a speed of 125r / min and 25°C, extract 5mL of gas in the culture bottle after 0 and 2h of culture, and promptly determine the N2O concentration in the gas. The N2O concentration is determined using an Agilent 7890B gas chromatograph (Agilent, USA).

[0065] In the present invention, the method for detecting changes in the composition and diversity of the microbial community in step (4) is preferably: RNA is extracted by RNAprep Pure Kit total RNA extraction kit (Tiangen Biotech Co., Beijing). To detect the purity of the extracted RNA, the RNA sample was PCR amplified using the 16S rRNA gene universal primer (27F / 1492R). After verifying the purity, the RNA was reverse transcribed into cDNA using the PrimeScript RT kit (Perfect Real Time, TaKaRa, Japan) and stored at -80°C for standby use. The cDNA sample was sequenced by NovaSeq-PE250 (Illumina, USA). The sequences were clustered into operational taxonomic units (OTUs) with a sequence similarity of 97% using QIIME 2.0 software.

[0066] In the present invention, the method for detecting the physical and chemical properties of the soil in step (4) is preferably:

[0067] (1) pH determination: weigh 10 g of air-dried soil sample passed through a 2 mm sieve, add 25 ml of distilled water, shake in an oscillating incubator at 180 rpm for 5 min, let stand for 30 min, and measure with a pH meter.

[0068] (2) NO3 - -N, NO2 - -N、NH4 + -N was determined by extracting fresh soil (volume ratio of soil to water was 1:5) using 1M KCl (Chinese medicine, premium grade) solution, shaking at 200 rpm for 1 h, filtering, and the concentration of the obtained extract was determined using Smartchem200;

[0069] (3) For the determination of organic matter, use the low-temperature external heating potassium dichromate oxidation-colorimetric method, weigh 0.5g of air-dried soil sample that has passed through a 100-mesh sieve, and place it in a 50ml colorimetric tube. At the same time, make a blank without a soil sample. Add 5ml of potassium dichromate solution and 5ml of concentrated sulfuric acid to the sample, blank, and standard sample, and shake well. Cover with a small funnel and place in a 100℃ constant temperature box. After 90 minutes, take out and place in a cold water bath to cool down. After the temperature drops to room temperature, add deionized water to 50ml, shake well, and let it stand for 3 hours. Take the supernatant and add it to a 96-well plate. Measure 3 parallels for each sample and measure the absorbance at a wavelength of 590nm. Calculate the corresponding carbon content by formulating a standard curve;

[0070] (4) Total carbon (TC) and total nitrogen (TN) were determined by weighing 0.1 g of air-dried soil sample passed through a 100-mesh sieve and using an elemental analyzer (VarioEL III, Elementar, Germany).

[0071] In the present invention, the method for detecting the change in nitrogen concentration in step (4) is preferably: filtering the water sample using a 0.45 μm microporous filter membrane, and immediately measuring NO3 using a SmartChem 200 fully automatic chemical analyzer (WESTCO, USA). - -N, NO2 - -N、NH4 + -N concentration; Vario ELⅢ element analyzer (Elementar, Germany) was used to determine the TN content.

[0072] In the present invention, the calculation method of the temperature threshold in step (5) is preferably:

[0073] A. Prepare soil microbial community dataset and environmental factor dataset;

[0074] B. In the microbial community data, standardization is performed to ensure that the minimum number of sampling sequences for each sample is the same;

[0075] C. Use the titan function in the TITAN2 package to analyze the temperature thresholds of positive and negative response groups in the microbial community;

[0076] The method for obtaining the soil microbial community dataset in step A is as follows: comparing the microbial sequencing data in step (5) with the SILVA database to generate an OTU table of denitrification microbial community composition;

[0077] The method for obtaining the environmental factor data set in step A is as follows: the temperature gradient in step (3) is used as the environmental factor.

[0078] The present invention also provides a device for the detection method, comprising an inner frame and a heat-insulating outer layer;

[0079] The inner frame has a length of 30 to 50 cm, a width of 30 to 50 cm, and a height of 50 to 70 cm;

[0080] The heat-insulating outer layer is wrapped around the periphery and bottom of the inner frame.

[0081] The present invention also provides a method for using the device, comprising the following steps:

[0082] When the device is used for waterless environment simulation, the soil is filled to 50-70% of the height of the inner frame, and the test is performed after the plants are planted on the soil;

[0083] When the device is used for simulating a water environment, soil is filled to 40-60% of the height of the inner frame, and water is then injected into the inner frame until the water level reaches 80-100% of the height of the inner frame. Plants are planted on the soil and then tested.

[0084] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0085] Example 1

[0086] In order to explore the response of reed rhizosphere denitrification process to cooling, the following experiments were conducted:

[0087] Step 1: Plant healthy reed (Phragmites australis) plants with the same growth conditions at a rate of 10 plants / m 2 The density was planted in the wetland soil in the pilot device, and the water depth was set to 15 cm to ensure that the plant roots could be immersed in the water and obtain sufficient water and oxygen when the water level changed.

[0088] Step 2: Use an artificial climate chamber for precise temperature control to simulate the natural cooling process. The temperature gradient is set to: 8℃, 4℃, 0℃, -4℃, -10℃, -15℃, and each stage lasts 72 hours. The temperature drop is set to 1℃ every 3 days to simulate the natural gradual cooling. In addition, the humidity of the wetland environment is maintained between 50% and 70%, and low light conditions are set to simulate the natural environment of low temperature and weak light in the winter wetland.

[0089] Step 3: At the end of each temperature stage, collect soil samples from the upper soil layer of the plant rhizosphere area (10 cm below the soil surface), ensuring that sampling is repeated three times for each temperature stage. Use a 5 cm diameter PVC tube for sampling, and take 100 g of soil sample each time. The collected soil samples should be refrigerated or quickly frozen immediately to prevent changes in microbial and enzyme activity. Use a water quality sampler to collect samples from the water bodies in the test area, and analyze the nitrogen concentration and other water quality indicators in the water, including pH, dissolved oxygen, ammonia nitrogen, nitrate nitrogen, etc. The water samples are analyzed immediately after filtration to avoid deviations in results caused by long-term storage.

[0090] Step 4: Detect the denitrification activity and functional microbial community composition of rhizosphere soil samples. Take 15g of dry soil sample, add 25mL of 20mg / L KNO3 solution, 40mg / L glucose and 193.8mg / L chloramphenicol mixed solution, seal the culture bottle, evacuate and inject helium and acetylene mixed gas (acetylene volume fraction 10%). Place the culture bottle in a light-free shaking incubator at 25°C and a speed of 125r / min for culture. Extract 5mL of gas after 0 hour and 2 hours of culture, and use Agilent 7890B gas chromatograph (USA) to measure the concentration change of N2O. The amount of N2O-N generated per unit time (h) per unit dry soil (kg) represents the soil denitrification activity. The measured denitrification activity at each temperature stage is: 0.67±0.07mg g -1 h -1 , 0.44±0.06mg g -1 h -1 , 0.31±0.04mg g -1 h -1 , 0.24±0.08mg g -1 h -1 , 0.13±0.03mg g -1 h -1 , 0.08±0.02mg g -1 h -1 .

[0091] To obtain the data of microbial community composition, RNA extraction and reverse transcription were first performed. Soil RNA was extracted using RNAprep PureKit (Tiangen, Shanghai), and PCR amplification was performed using nirS and nirK gene primers F1aCu / R3Cu and cd3aF / R3cd. After verifying the purity, RNA was reverse transcribed into cDNA using PrimeScript RT reagent kit. cDNA sequencing was performed using NovaSeq-PE250 (Illumina, USA) to generate sequence data. QIIME 2.0 software was used to cluster the sequences into operational taxonomic units (OTUs) with a sequence similarity of 97%.

[0092] Step 5: Calculate the temperature threshold for functional microorganisms to maintain activity. The microbial community sequencing data obtained in the above step 4 was compared with the SILVA database to generate an OTU table of denitrifying microbial community composition. Use the temperature gradient in step (3) as the environmental factor to establish a data set. Standardize the microbial community data to ensure that the minimum number of extracted sequences for each sample is the same. Use the titan function in the TITAN2 package to analyze the temperature thresholds of positive and negative response groups in the microbial community, and obtain the response changes of functional microorganisms at different temperatures. The results showed that low temperature had a significant effect on the denitrifying microbial community and enzyme activity in the rhizosphere soil of reed, especially when the temperature was below 4.85℃, the relative abundance of denitrifying microorganisms decreased significantly, and the denitrification activity was significantly inhibited.

[0093] As can be seen from the above embodiments, the present invention provides a detection method for determining the temperature threshold for maintaining denitrification activity of rhizosphere microorganisms in wetlands under low temperature conditions, comprising the following steps: (1) planting wetland plants in the test area; (2) gradually lowering the ambient temperature of the test area; (3) collecting rhizosphere soil samples and water samples under different temperature gradient conditions; (4) detecting the denitrification activity, functional microbial community composition and diversity changes, soil physical and chemical properties, and water nitrogen concentration changes of rhizosphere soil samples; (5) calculating the temperature threshold for maintaining the activity of functional microorganisms. The decrease in denitrification efficiency of wetlands under cold climate conditions is a common problem. The technical solution of the present invention can provide technical support for improving the denitrification effect of wetlands in low temperature environments, so that it can also play a good role in winter or cold areas. By clarifying the temperature range of rhizosphere microbial denitrification activity, the selection of plant species and the regulation of growth conditions in wetland restoration projects can be guided to improve the denitrification capacity of wetlands. This method optimizes the functions of indigenous microorganisms by promoting plant-microorganism interactions, and can also reduce exogenous intervention and save governance costs.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the temperature threshold for wetland rhizosphere microorganisms to maintain denitrification activity under low temperature conditions, characterized in that: The steps include: (1) Plant wetland plants in the experimental area; (2) Gradually reduce the ambient temperature of the test area; (3) Collect rhizosphere soil and water samples under different temperature gradient conditions; (4) Detect the denitrification activity of rhizosphere soil samples, changes in the composition and diversity of active microbial communities, soil physical and chemical properties, and changes in water nitrogen concentration; (5) Calculate the temperature threshold for functional microorganisms to maintain activity.

2. The detection method according to claim 1, characterized in that: The plant species in step (1) is an emergent plant, and the density during planting is 9 to 25 plants / m 2 .

3. The detection method according to claim 2, characterized in that: The reduction in step (2) is 0.8-1.2°C every three days; The different temperature gradients in step (3) are: 8°C, 4°C, 0°C, -4°C, -10°C and -15°C.

4. The detection method according to claim 3, characterized in that: The detection method of denitrification activity in step (4) is: Soil, KNO3 solution, glucose solution and chloramphenicol solution were mixed and placed in a sealed container. After the sealed container was evacuated, a helium-acetylene mixed gas was injected. After shaking, the gas in the sealed container was extracted to detect the concentration of N2O. The usage ratio of the soil, KNO3 solution, glucose solution and chloramphenicol solution is 13-17 g: 23-27 mL: 23-27 mL: 23-27 mL; The concentration of the KNO3 solution is 18-22 mg / L; The concentration of the glucose solution is 35-45 mg / L; The concentration of the chloramphenicol solution is 190-197 mg / L The volume ratio of helium to acetylene in the helium-acetylene mixed gas is 85-95:5-15; The atmospheric pressure in the sealed container after the helium-acetylene mixed gas is injected is the same as the atmospheric pressure before vacuuming; The shaking conditions are 120-130 r / min and 23-27° C. The timing of extracting the gas in the sealed container is when the oscillation starts and 110 to 130 minutes after the oscillation starts; The concentration of N2O was measured using an Agilent 7890B gas chromatograph.

5. The detection method according to claim 4, characterized in that: The method for detecting changes in the composition and diversity of active microbial communities in step (4) is: RNA was extracted using the RNAprep Pure Kit total RNA extraction kit, and the purity of the extracted RNA was tested; The purity detection method is as follows: PCR amplification of RNA samples, followed by reverse transcription of the amplified RNA into cDNA, high-throughput sequencing of the cDNA samples on an Illumina HiSeq platform, bioinformatics analysis using QIIME2 software, and comparison, classification and annotation of sequencing data.

6. The detection method according to claim 5, characterized in that: The soil physical and chemical properties in step (4) include pH value, NO3 - -N, NO2 - -N、NH4 + -N content, organic matter content, total carbon and total nitrogen content; The pH value detection method is as follows: dry soil and water are mixed in a mass ratio of 1:2-3, shaken and then allowed to stand, and then measured with a pH meter; The NO 3- -N, NO 2- -N、NH4 + -N content was determined by mixing soil and KCl solution, shaking, filtering, and measuring with Smartchem200; The concentration of the KCl solution is 0.8-1.2 M, the volume ratio of the soil to the KCl solution is 1:4-6, and the shaking conditions are 180-220 rpm, 50-70 min; The organic matter content is detected by low temperature external heating potassium dichromate oxidation-colorimetry; The total carbon and total nitrogen contents are detected by an element analyzer.

7. The detection method according to claim 6, characterized in that: The detection of the change in nitrogen concentration in the water body in step (4) includes NO 3- -N, NO 2- -N、NH4 + -N concentration detection and TN content detection; The NO 3- -N, NO 2- -N、NH4 + -N concentration was detected using a fully automatic chemical analyzer; The TN content is detected by using an element analyzer.

8. The detection method according to any one of claims 1 to 7, characterized in that: The method for calculating the temperature threshold in step (4) comprises the following steps: A. Prepare soil microbial community dataset and environmental factor dataset; B. In the microbial community data, standardization is performed to ensure that the minimum number of sampling sequences for each sample is the same; C. Use the titan function in the TITAN2 package to analyze the temperature thresholds of positive and negative response groups in the microbial community; The method for obtaining the soil microbial community dataset in step A is as follows: comparing the microbial sequencing data in step (5) with the SILVA database to generate an OTU table of denitrification microbial community composition; The method for obtaining the environmental factor data set in step A is as follows: the temperature gradient in step (3) is used as the environmental factor.

9. A device for the detection method according to any one of claims 1 to 8, characterized in that: It includes an inner frame and an outer insulation layer; The inner frame has a length of 30 to 50 cm, a width of 30 to 50 cm, and a height of 50 to 70 cm; The heat-insulating outer layer is wrapped around the periphery and bottom of the inner frame.

10. The method for using the device according to claim 9, characterized in that: The steps include: When the device is used for waterless environment simulation, the soil is filled to 50-70% of the height of the inner frame, and the test is performed after the plants are planted on the soil; When the device is used for simulating a water environment, soil is filled to 40-60% of the height of the inner frame, and water is then injected into the inner frame until the water level reaches 80-100% of the height of the inner frame. Plants are planted on the soil and then tested.

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