Improved device and method for measuring plant biological nitrogen fixation rate by acetylene reduction method

Abstract

The invention discloses an improved device and method for measuring the plant biological nitrogen fixation rate through an acetylene reduction method, and relates to the technical field of detection device.The device comprises a gas circulation box, an injection mechanism is arranged on the front side of the gas circulation box, and a detection mechanism is arranged on the left side of the injection mechanism; the left side and the right side of the gas circulation box are fixedly provided with carrying handles, the bottoms of the carrying handles are communicated with the inner cavity, the tops of the carrying handles are fixedly connected with male connectors penetrating through the inner cavity of the carrying handles, the right side of the top of the gas circulation box is fixedly provided with a storage battery, and the injection mechanism comprises an injector. A large-volume gas circulation box is adopted to directly cover a to-be-detected plant, sealing is achieved by inserting the large-volume gas circulation box into soil or a water body or a water tank base, interference of remote culture is avoided, continuous gas circulation detection can be directly carried out in a field environment, the influence of environmental factors such as illumination and temperature on the nitrogen fixation rate is reduced, and the data accuracy is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection equipment, in particular to an improved device and method for measuring the biological nitrogen fixation rate of plants by acetylene reduction method. BACKGROUND

[0002] Biological nitrogen fixation, as the main source of nitrogen in natural ecosystems, plays an extremely important role in the nitrogen cycle. Common free-living nitrogen-fixing microorganisms include aerobic free-living nitrogen-fixing bacteria (Azotobacter chroococcum), anaerobic free-living nitrogen-fixing bacteria (Clostridium), and nitrogen-fixing cyanobacteria with heterocysts (heterocysts contain nitrogenase and can perform biological nitrogen fixation, such as Anabaena, Nostoc, and Oscillatoria). Traditional methods for studying biological nitrogen fixation include the difference method, acetylene reduction method, and nitrogen balance method. Due to differences in nitrogen fixation ability among plant species, temporal and spatial variations in rhizobial nitrogen fixation ability, and limitations in the precision of the methods themselves, these methods have limited application, especially in the study of the biological nitrogen fixation of perennial plants and woody plants. The acetylene reduction method is based on the principle that nitrogenase produced by nitrogen-fixing bacteria can reduce acetylene to ethylene to measure the nitrogen fixation activity of nitrogen-fixing bacteria. This method involves placing the test object in a sealed container, extracting some air and injecting an equal amount of acetylene, and then measuring the amount of ethylene produced after a certain period of time using a gas chromatograph. This method is sensitive, simple, and easy to use, and can quickly detect nitrogenase activity. The acetylene reduction method is one of the commonly used detection methods. This method is based on the principle of substrate diversity of nitrogenase and indirectly measures the presence and level of nitrogenase activity by detecting whether acetylene is reduced to ethylene and the amount of ethylene produced. Currently, the acetylene reduction method is widely used to measure soil or plant nodule nitrogenase activity in laboratories.

[0003] Chinese patent document 202010520360.X discloses a device and method for measuring nitrogenase activity using the improved acetylene reduction method, and 202122884935.9 discloses a device for measuring soil or strain nitrogenase activity by the acetylene reduction method. The existing technology has the following problems: The existing method's culture system mainly includes a sealed bottle reaction bottle, a bottle plug, a gas sampling tube, and a valve. Due to the size limitation of the culture container, the sample is usually cultured in a different place, and the sample amount is small. The sample collection method is mostly indoor culture test, and a certain amount of gas is collected after the culture is completed using a syringe, which is time-consuming. When performing in-situ detection, the device has a small volume and large error. The air in the culture bottle is still, and the acetylene may not be completely mixed. The culture time is too long and is easily affected by light and air temperature. The detection equipment mainly uses a gas chromatograph to measure the amount of ethylene produced during the culture time. The gas chromatograph needs to be equipped with a special chromatographic column, and the cost is high when a large number of samples are detected. SUMMARY

[0004] The present application provides an improved device and method for measuring the biological nitrogen fixation rate of plants by acetylene reduction method to solve the problems in the background art.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is: The improved device for measuring the biological nitrogen fixation rate of plants by acetylene reduction method comprises a gas circulation box, an injection mechanism is arranged on the front side of the gas circulation box, a detection mechanism is arranged on the left side of the injection mechanism, a hand-held handle is fixedly installed on the left and right sides of the gas circulation box, the bottom of the hand-held handle is in communication with the inner cavity, a male connector is fixedly connected to the top of the hand-held handle and penetrates the inner cavity thereof, a storage battery is fixedly installed on the top right side of the gas circulation box, the injection mechanism comprises a syringe, a two-way valve I is sleeved on the injection end of the syringe, an injection hose is sleeved on the other end of the two-way valve I, a two-way valve II is sleeved on the other end of the injection hose, and the bottom end of the two-way valve II is fixedly connected in communication with the inner cavity of the male connector.

[0006] Further improvement of the technical scheme of the present application is that a mounting square frame is fixedly installed on the inner wall bottom of the gas circulation box, a fan is fixedly installed in the inner cavity of the mounting square frame, a power supply wire is electrically connected to the power supply input end of the fan, and the other end of the power supply wire penetrates to the top of the gas circulation box and is electrically connected to the power supply output end of the storage battery.

[0007] Further improvement of the technical scheme of the present application is that the detection mechanism comprises a portable ethylene detector, an air outlet hose is sleeved on the top output end of the portable ethylene detector, and an air inlet hose is sleeved on the front input end of the portable ethylene detector.

[0008] Further improvement of the technical scheme of the present application is that an air extraction hole penetrating the inner cavity thereof is formed on the top left side of the gas circulation box, an air exhaust hole penetrating the inner cavity thereof is formed on the top right side of the gas circulation box, a Teflon tube is fixedly installed in the inner circle of the air extraction hole and the air exhaust hole, and a connecting hose is sleeved on the top end of the two Teflon tubes.

[0009] Further improvement of the technical scheme of the present application is that one end of the air inlet hose away from the portable ethylene detector is fixedly sleeved with the left connecting hose, and one end of the air outlet hose away from the portable ethylene detector is fixedly sleeved with the right connecting hose.

[0010] The improved method for measuring the biological nitrogen fixation rate of plants by acetylene reduction method comprises the following steps: S1: Covering the plant to be measured with the gas circulation box and sealing it to ensure that the air volume in the box is fixed; S2: Extracting a predetermined volume of air in the inner cavity of the gas circulation box through the injection mechanism and injecting an equal volume of acetylene gas; S3: Start the fan inside the gas circulation box to fully mix the gas in the box; S4: Turn on the portable ethylene detector in the detection mechanism to make the gas in the box pass through the air inlet hose, and return to the box through the air outlet hose after detection, forming a gas circulation; S5: Collect and record ethylene concentration data multiple times within a preset time interval, while monitoring the temperature in the box; S6: Calculate the ethylene production flux according to the slope of the change of ethylene concentration with time, and then calculate the biological nitrogen fixation rate.

[0011] The further improvement of the technical scheme of the application is that the sealing method in S1 includes inserting the gas circulation box into soil or water, or fixing it through a water tank base, and the volume of air extracted in S2 is 10% of the total volume of air in the cavity of the gas circulation box.

[0012] The further improvement of the technical scheme of the application is that the flow rate of the gas circulation in S4 is controlled by the built-in circulation pump of the portable ethylene detector, and the flow rate is 0.5-2 L / min, and the preset time interval in S5 is 1-10 minutes, and the total detection time is within 1 hour.

[0013] Due to the adoption of the above technical scheme, the application has the following technical progress compared with the prior art: The application provides an improved device and method for measuring the biological nitrogen fixation rate of plants by acetylene reduction method, which directly covers the plants to be measured with a large-volume gas circulation box, realizes sealing by inserting into soil, water or a water tank base, avoids the interference of off-site cultivation, realizes continuous gas circulation detection in the field environment, reduces the influence of environmental factors such as light and temperature on the nitrogen fixation rate, and improves the data accuracy.

[0014] The application provides an improved device and method for measuring the biological nitrogen fixation rate of plants by acetylene reduction method, which installs a fan at the bottom of the box, which is driven by a storage battery to forcibly mix the gas, and a built-in circulation pump in the portable ethylene detector forms a closed-loop airflow at a flow rate of 0.5-2 L / min, the gas enters the air inlet hose through the air inlet hole, then enters the detector, and then returns to the box through the air outlet hose and the air outlet hole to complete the circulation, solving the problem of uneven mixing of acetylene in the traditional method, ensuring uniform distribution and real-time circulation of the gas, and improving the reliability of ethylene concentration detection.

[0015] The application provides an improved device and method for measuring the biological nitrogen fixation rate of plants by using the acetylene reduction method, so as to replace a gas chromatograph with a portable ethylene detector, realize gas circulation through a Teflon tube connected by a hose, control the total detection time within 1 hour, automatically collect data every 1-10 minutes, only need to extract 10% of the gas in the box to inject acetylene through the injection mechanism, avoid the consumption of an expensive chromatographic column, and reduce the equipment cost; the operation is simplified and time-consuming, and the method is suitable for rapid detection of a large number of samples in the field. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a schematic view of the structure of the application; Figure 2 is a schematic view of the injection mechanism of the structure of the application; Figure 3 is a schematic view of the inner cavity of the gas circulation box of the structure of the application; Figure 4 is a schematic view of the detection mechanism of the structure of the application; Figure 5 is a schematic view of the method flow of the application.

[0017] In the figure: 1, gas circulation box; 11, hand-held handle; 12, male connector; 13, battery; 14, mounting box; 141, fan; 142, power supply wire; 15, air extraction hole; 16, air exhaust hole; 17, Teflon tube; 171, connecting hose; 2, injection mechanism; 21, syringe; 22, two-way valve one; 23, injection hose; 24, two-way valve two; 3, detection mechanism; 31, portable ethylene detector; 32, air inlet hose; 33, air outlet hose. DETAILED DESCRIPTION

[0018] The application will be further described in detail below in combination with examples: Example

[0019] As Figures 1-5As shown, the present application provides an improved acetylene reduction method for determining the rate of plant biological nitrogen fixation device, including gas circulation box 1, the front side of the gas circulation box 1 is provided with injection mechanism 2, the left side of the injection mechanism 2 is provided with detection mechanism 3, the left and right sides of the gas circulation box 1 are both fixedly installed with hand-held handle 11, the bottom of the hand-held handle 11 is in communication with the inner cavity, the top of the hand-held handle 11 is fixedly connected with the male connector 12 penetrating the inner cavity thereof, the top right side of the gas circulation box 1 is fixedly installed with the storage battery 13, the injection mechanism 2 comprises a syringe 21, the injection end of the syringe 21 is sleeved with a two-way valve one 22, the other end of the two-way valve one 22 is sleeved with an injection hose 23, the other end of the injection hose 23 is sleeved with a two-way valve two 24, the bottom end of the two-way valve two 24 is fixedly connected with the inner cavity of the male connector 12 in communication, the inner wall bottom of the gas circulation box 1 is fixedly installed with the mounting square frame 14, the inner cavity of the mounting square frame 14 is fixedly installed with the fan 141, the power supply input end of the fan 141 is electrically connected with the power supply wire 142, the other end of the power supply wire 142 penetrates to the top of the gas circulation box 1 and is electrically connected with the power supply output end of the storage battery 13, for power supply of the fan 141.

[0020] In this embodiment, when in use, the whole gas circulation box 1 can be held by the hand-held handle 11, then the gas circulation box 1 can be inserted into the soil or water where the plant is located, so that the inner cavity is kept sealed, then the male connector 12 and the two-way valve one 22 are opened, so that 10% of the gas in the box can be extracted by the syringe 21, and an equal amount of acetylene gas is replaced, and the fan 141 in the mounting square frame 14 in the box is opened, so that the internal air is mixed uniformly, and the detection quality is ensured. Embodiment

[0021] As Figures 1-5 shown, on the basis of embodiment 1, the present application provides a technical scheme: preferably, the detection mechanism 3 comprises a portable ethylene detector 31, the top output end of the portable ethylene detector 31 is sleeved with an air outlet hose 33, the front input end of the portable ethylene detector 31 is sleeved with an air inlet hose 32, the top left side of the gas circulation box 1 is provided with an air extraction hole 15 penetrating the inner cavity thereof, the top right side of the gas circulation box 1 is provided with an air exhaust hole 16 penetrating the inner cavity thereof, the inner circle of the air extraction hole 15 and the air exhaust hole 16 are both fixedly installed with a Teflon tube 17, the top end of the two Teflon tubes 17 is sleeved with a connecting hose 171, the end of the air inlet hose 32 away from the portable ethylene detector 31 is fixedly sleeved with the left connecting hose 171, and the end of the air outlet hose 33 away from the portable ethylene detector 31 is fixedly sleeved with the right connecting hose 171.

[0022] In this embodiment, the portable ethylene detector 31 is connected to the Teflon tube 17 inside the exhaust port 15 and exhaust port 16 via the connecting hose 171, the inlet hose 32, and the outlet hose 33. The portable ethylene detector 31 has a built-in circulation pump that generates negative pressure to extract gas. The gas in the chamber enters the Teflon tube 17 and the inlet hose 32 through the left exhaust port 15 and then enters the portable ethylene detector 31. The detected gas returns to the chamber through the outlet hose 33 and the Teflon tube 17 inside the right exhaust port 16, completing the circulation. Example

[0023] like Figures 1-5 As shown, this invention provides a modified acetylene reduction method for determining the rate of biological nitrogen fixation in plants, comprising the following steps: S1: Cover the plant to be tested with the gas circulation box 1 and seal it to ensure that the air volume inside the box is fixed; S2: A predetermined volume of air is drawn from the inner cavity of the gas circulation box 1 through the injection mechanism 2, and an equal volume of acetylene gas is injected. S3: Start the fan 141 inside the gas circulation box 1 to fully mix the gas inside the box; S4: Turn on the portable ethylene detector 31 in the detection mechanism 3, so that the gas in the box enters the inlet hose 32 through the air extraction port 15, and after detection, it returns to the box through the outlet hose 33 and the exhaust port 16, forming a gas cycle. S5: Collect and record ethylene concentration data multiple times within a preset time interval, while monitoring the temperature inside the chamber; S6: Calculate the ethylene production flux based on the slope of the change in ethylene concentration over time, and then deduce the biological nitrogen fixation rate. The sealing method in S1 includes inserting the gas circulation box 1 into the soil or water body, or fixing it through the water tank base. The volume of air extracted in S2 is 10% of the total volume of air in the inner cavity of the gas circulation box 1. The flow rate of gas circulation in S4 is controlled by the built-in circulation pump of the portable ethylene detector 31, with a flow rate of 0.5-2 L / min. The preset time interval in S5 is 1-10 minutes, and the total detection time is within 1 hour.

[0024] In this embodiment, since light and temperature affect the rate of biological nitrogen fixation, the interval detection time can be selected at different time periods according to the weather conditions. For example, if the detection time is set to once every 10 minutes, six samples can be taken continuously within 1 hour. The ethylene concentration in the chamber increases with time. At the same time, the temperature in the chamber and the air temperature are recorded. In order to reduce the influence of environmental factors, the interval time can also be shortened. The ethylene production flux is calculated by calculating the slope of ethylene concentration versus time.

[0025] The working principle of the improved acetylene reduction method for determining the rate of biological nitrogen fixation in plants will be explained in detail below.

[0026] like Figures 1-5 As shown, the gas circulation box 1 is placed in a base with a water tank, on grass, or inserted into a body of water. It is connected to the portable ethylene detector 31 through the connecting hose 171 on the Teflon tube 17 inside the exhaust port 15 and exhaust port 16, as well as the air inlet hose 32 and the air outlet hose 33. Ensure that the box is sealed and that the air inside the box is kept at a constant volume. Use the syringe 21 to extract 10% of the air volume inside the box and replace it with an equal amount of acetylene gas. Turn on the fan 141 inside the box's mounting frame 14 to mix the internal air. After the gas inside the box is mixed and stable, turn on the portable ethylene detector 31. The portable ethylene detector 31 has a built-in circulation pump that generates negative pressure to extract the gas. The gas inside the box enters the portable ethylene detector 31 through the left exhaust port 15, the Teflon tube 17 and the air inlet hose 32, and then enters the portable ethylene detector 31. The detected gas returns to the box through the air outlet hose 33 and the Teflon tube 17 inside the right exhaust port 16, completing the circulation.

[0027] The present invention has been described in detail above. However, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. An improved apparatus for measuring the rate of biological nitrogen fixation in plants by the acetylene reduction method, comprising a gas circulation chamber (1), characterised in that: The front side of the gas circulation box (1) is provided with an injection mechanism (2), the left side of the injection mechanism (2) is provided with a detection mechanism (3), the left and right sides of the gas circulation box (1) are both fixedly installed with a hand-held handle (11), the bottom of the hand-held handle (11) is in communication with the inner cavity, the top of the hand-held handle (11) is fixedly connected with a male connector (12) penetrating through the inner cavity, the top right side of the gas circulation box (1) is fixedly installed with a storage battery (13), the injection mechanism (2) comprises a syringe (21), the injection end of the syringe (21) is sleeved with a two-way valve I (22), the other end of the two-way valve I (22) is sleeved with an injection hose (23), the other end of the injection hose (23) is sleeved with a two-way valve II (24), and the bottom end of the two-way valve II (24) is fixedly connected with the male connector (12) in communication with the inner cavity.

2. The apparatus for determining the rate of biological nitrogen fixation of plants by the modified acetylene reduction method as claimed in claim 1, wherein: The inner wall bottom of the gas circulation box (1) is fixedly installed with a mounting square frame (14), the inner cavity of the mounting square frame (14) is fixedly installed with a fan (141), the power supply input end of the fan (141) is electrically connected with a power supply lead wire (142), and the other end of the power supply lead wire (142) penetrates to the top of the gas circulation box (1) and is electrically connected with the power supply output end of the storage battery (13).

3. The apparatus for determining the rate of biological nitrogen fixation of plants by the modified ethylene reduction method according to claim 1, characterized in that: The detection mechanism (3) comprises a portable ethylene detector (31), the top output end of the portable ethylene detector (31) is sleeved with an air outlet hose (33), and the front input end of the portable ethylene detector (31) is sleeved with an air inlet hose (32).

4. The apparatus for determining the rate of biological nitrogen fixation of a plant by the modified acetylene reduction method as claimed in claim 3, wherein: The top left side of the gas circulation box (1) is provided with an air suction hole (15) penetrating through the inner cavity, the top right side of the gas circulation box (1) is provided with an air exhaust hole (16) penetrating through the inner cavity, the inner rings of the air suction hole (15) and the air exhaust hole (16) are both fixedly installed with a Teflon pipe (17), and the top ends of the two Teflon pipes (17) are both sleeved with a connecting hose (171).

5. The apparatus for determining the rate of biological nitrogen fixation of a plant by the modified acetylene reduction method as claimed in claim 4, wherein: The end of the air inlet hose (32) away from the portable ethylene detector (31) is fixedly sleeved with the left connecting hose (171), and the end of the air outlet hose (33) away from the portable ethylene detector (31) is fixedly sleeved with the right connecting hose (171).

6. A method for determining the rate of biological nitrogen fixation in plants by the modified acetylene reduction method, characterized by: The steps include: S1: cover the gas circulation box (1) with the plant to be measured and seal it, and ensure that the air volume in the box is fixed; S2: extract a predetermined volume of air in the inner cavity of the gas circulation box (1) through the injection mechanism (2), and inject an equal volume of acetylene gas; S3: start the fan (141) in the gas circulation box (1) to fully mix the gas in the box; S4: turn on the portable ethylene detector (31) in the detection mechanism (3), make the gas in the box enter the air inlet hose (32) through the air suction hole (15), and return to the box through the air exhaust hole (16) after detection, to form gas circulation; S5: collect and record ethylene concentration data multiple times within a predetermined time interval, and monitor the temperature in the box. S6: Calculate the ethylene production flux according to the slope of the change of ethylene concentration over time, and then calculate the biological nitrogen fixation rate.

7. The method for determining the rate of biological nitrogen fixation of a plant by the modified ethylene reduction method according to claim 6, characterized in that: The sealing method in S1 includes inserting the gas circulation box (1) into the soil or water body, or fixing it through the water tank base, and the volume of the extracted air in S2 is 10% of the total volume of the air in the cavity of the gas circulation box (1).

8. The method for determining the rate of biological nitrogen fixation of a plant by the modified ethylene reduction method according to claim 6, characterized in that: The flow rate of the gas circulation in S4 is controlled by the built-in circulation pump of the portable ethylene detector (31), and the flow rate is 0.5-2 L / min, the preset time interval in S5 is 1-10 minutes, and the total detection time is within 1 hour.

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