Soil anaerobic ammonia oxidation microorganism nitrogen isotope labeling device

Abstract

The utility model discloses a nitrogen isotope labeling device for soil anaerobic ammonia oxidation microorganisms, and relates to the technical field of soil anaerobic ammonia oxidation. A first liquid adding structure of the nitrogen isotope labeling device for the soil anaerobic ammonia oxidation microorganisms is communicated with a first reagent bottle and a sample bottle respectively; the second liquid adding structure is respectively communicated with the second reagent bottle, the third reagent bottle, the fourth reagent bottle, the vacuum structure and the sample bottle. According to the invention, test errors caused by manual operation can be reduced, and the pretreatment efficiency of soil anaerobic ammonia oxidation activity determination and the quality of detection data are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of soil anaerobic ammonium oxidation, and particularly to a device for microbial nitrogen isotope labeling of soil anaerobic ammonium oxidation. Background Art

[0002] The discovery of the anaerobic ammonium oxidation reaction is one of the major breakthroughs in the environmental field this century, and it is a nitrogen sink process in terrestrial ecosystems driven by anaerobic ammonium oxidizing bacteria. The anaerobic ammonium oxidation reaction is a process in which under anaerobic conditions, with NO 2 as the electron acceptor, NH 4 is oxidized to N 2 . As a result, the nitrogen in terrestrial ecosystems is converted into N 2 and released into the atmosphere, adding new content to the global nitrogen cycle. With the discovery of this process, researchers' understanding of the nitrogen cycle has undergone a qualitative change. Recent research has found that the anaerobic ammonium oxidation process in paddy soil may account for 4%-37% of soil nitrogen loss. Anaerobic ammonium oxidation activity is an important indicator characterizing the anaerobic ammonium oxidation process, which is the nitrogen gas produced by unit mass of soil in unit time for this process, and it reflects the intensity of anaerobic ammonium oxidation in soil.

[0003] Anaerobic ammonium oxidation is a process in which ammonium reacts directly with nitrite to generate nitrogen gas under anaerobic conditions. The specific reaction process is: NH 4 + + NO 2 - → N 2 + 2H 2 O (1).

[0004] Under anaerobic conditions, the denitrification process also occurs in soil. The denitrification reaction is a process in which denitrifying bacteria reduce NO 3 - to N 2 O and N 2 . The reaction process is: NO 3 - → NO 2 - → NO → N 2 O → N 2 (2).

[0005] Under anaerobic conditions, both reactions (1) and (2) produce nitrogen gas, but their reaction mechanisms are different. The N 2 produced by the anaerobic ammonium oxidation reaction is provided with one nitrogen atom by NH 4 + and NO 2 - respectively, while the nitrogen gas produced by denitrification is produced by a series of intermediate reactions from nitrate nitrogen and all comes from NO 3 -Therefore, the nitrogen isotope tracing method can be used to distinguish nitrogen gas from different sources. The measurement process of the nitrogen isotope tracing method is cumbersome, and there is no supporting pretreatment device on the market at present. Summary of the Invention

[0006] The object of the present invention is to provide a device for nitrogen isotope labeling of soil anaerobic ammonium oxidation microorganisms, standardize the sample pretreatment process, reduce the test errors introduced by manual operation, improve the pretreatment efficiency and the quality of detection data before the determination of soil anaerobic ammonium oxidation activity, and provide technical support for the research on the soil anaerobic ammonium oxidation process and soil nitrogen sink.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a device for nitrogen isotope labeling of soil anaerobic ammonium oxidation microorganisms, including: an anaerobic box and a sample bottle, a reagent bottle and a liquid adding structure arranged in the anaerobic box;

[0009] The anaerobic box is used to provide an anaerobic environment;

[0010] The reagent bottle includes a first reagent bottle, a second reagent bottle, a third reagent bottle and a fourth reagent bottle. The first reagent bottle is used to contain pure water, the second reagent bottle is used to contain ammonium nitrogen, the third reagent bottle is used to contain nitrate nitrogen, and the fourth reagent bottle is used to contain zinc chloride.

[0011] The liquid adding structure includes a first liquid adding structure and a second liquid adding structure. The first liquid adding structure is respectively communicated with the first reagent bottle and the sample bottle, and the second liquid adding structure is respectively communicated with the second reagent bottle, the third reagent bottle, the fourth reagent bottle, a vacuum structure and the sample bottle.

[0012] Preferably, a plurality of the sample bottles are arranged on a sample tray, an oscillator is arranged at the bottom of the sample tray, and the oscillator is electrically connected with a controller.

[0013] Preferably, the liquid adding structure realizes movement through a driving structure. The driving structure includes a motor and a lead screw. The motor is electrically connected with the controller. The power output end of the motor is in transmission connection with the lead screw. The liquid adding structure is arranged on the lead screw and is in threaded connection with the lead screw. The motor drives the lead screw to rotate, thereby driving the liquid adding structure to perform linear motion.

[0014] Preferably, the first reagent bottle, the second reagent bottle, the third reagent bottle and the fourth reagent bottle are also respectively connected with an external gas source. The external gas source is nitrogen. Solenoid valves and pressure valves are arranged on the pipelines between the first reagent bottle, the second reagent bottle, the third reagent bottle and the fourth reagent bottle and the external gas source. The solenoid valves and the pressure valves are respectively electrically connected with the controller.

[0015] Preferably, a first liquid addition valve is provided on the pipeline between the first reagent bottle and the first liquid addition structure. The first liquid addition valve is electrically connected to the controller. Second liquid addition valves are respectively provided on the pipelines between the second reagent bottle, the third reagent bottle, and the fourth reagent bottle and the second liquid addition structure. The second liquid addition valves are electrically connected to the controller;

[0016] The first reagent bottle is connected to the air outlet pipe, and an air vent valve is provided on the air outlet pipe. The air vent valve is electrically connected to the controller.

[0017] Preferably, the vacuum structure is located outside the anaerobic chamber. A pressure relief valve, a vacuum pump solenoid valve, and a pneumatic valve are provided on the pipeline connecting the vacuum structure and the second liquid addition structure. The pressure relief valve, the vacuum pump solenoid valve, and the pneumatic valve are respectively electrically connected to the controller.

[0018] Preferably, the first liquid addition structure includes a first liquid addition driving structure and a grooved side hole needle. The first liquid addition driving structure is electrically connected to the controller. The grooved side hole needle is provided at the telescopic end of the first liquid addition driving structure. The telescopic movement of the first liquid addition driving structure drives the linear movement of the grooved side hole needle. A first channel is provided inside the grooved side hole needle. Grooves are formed on the outer surface of the grooved side hole needle. The grooves extend along the length direction of the grooved side hole needle. The first channel is respectively communicated with the sample bottle and the first reagent bottle.

[0019] Preferably, the second liquid addition structure includes a second liquid addition driving structure and a double side hole needle. The second liquid addition driving structure is electrically connected to the controller. The double side hole needle is provided at the telescopic end of the second liquid addition driving structure. The telescopic movement of the second liquid addition driving structure drives the linear movement of the double side hole needle. A second channel and a third channel are provided inside the double side hole needle. The second channel is respectively communicated with the sample bottle and the vacuum structure. The third channel is respectively communicated with the sample bottle, the second reagent bottle, the third reagent bottle, and the fourth reagent bottle.

[0020] Preferably, a heating structure and a deoxidation structure are provided inside the anaerobic chamber. The heating structure and the deoxidation structure are respectively electrically connected to the controller.

[0021] The present invention has achieved the following technical effects compared with the prior art:

[0022] The present invention can realize processes such as the preparation of aerated water, soil pre-(anaerobic) culture, isotope addition, and gas sample preparation during the pretreatment for the determination of soil anaerobic ammonium oxidation activity; it can standardize the sample pretreatment process, reduce test errors introduced by manual operation, improve the pretreatment efficiency for the determination of soil anaerobic ammonium oxidation activity and the quality of detection data, and provide technical support for research on soil anaerobic ammonium oxidation processes and soil nitrogen sinks. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is a schematic diagram of the soil anaerobic ammonium oxidation microbial nitrogen isotope labeling device of the present invention;

[0025] In the figure: 1 - anaerobic chamber, 2 - sample bottle, 3 - first reagent bottle, 4 - second reagent bottle, 5 - third reagent bottle, 6 - fourth reagent bottle, 7 - vacuum structure, 8 - sample tray, 9 - oscillator, 10 - motor, 11 - lead screw, 12 - nitrogen cylinder, 13 - nitrogen pressure controller, 14 - solenoid valve, 15 - pressure valve, 16 - first liquid addition valve, 17 - second liquid addition valve, 18 - gas outlet pipe, 19 - ventilation valve, 20 - pressure relief valve, 21 - vacuum pump solenoid valve, 22 - pneumatic valve, 23 - first liquid addition drive structure, 24 - groove side hole needle, 25 - second liquid addition drive structure, 26 - double side hole needle, 27 - heating structure, 28 - deoxidation structure, 29 - temperature controller, 30 - controller, 31 - compressed air cylinder, 32 - air pressure controller. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0027] The purpose of the present invention is to provide a soil anaerobic ammonium oxidation microbial nitrogen isotope labeling device, which standardizes the sample pretreatment process, reduces test errors introduced by manual operation, improves the pretreatment efficiency for the determination of soil anaerobic ammonium oxidation activity and the quality of detection data, and provides technical support for research on soil anaerobic ammonium oxidation processes and soil nitrogen sinks.

[0028] To make the above objects, features, and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] As Figure 1 shown, this embodiment provides a soil anaerobic ammonium oxidation microbial nitrogen isotope labeling device, including: an anaerobic chamber 1 and a sample bottle 2, reagent bottles, and a liquid adding structure arranged inside the anaerobic chamber 1;

[0030] The anaerobic chamber 1 is used to provide an anaerobic environment; the sample bottle 2 is used to hold soil samples;

[0031] The reagent bottles include a first reagent bottle 3, a second reagent bottle 4, a third reagent bottle 5, and a fourth reagent bottle 6. The first reagent bottle 3 is used to hold pure water, and the second reagent bottle 4 is used to hold ammonium nitrogen ( 15 N-NH 4 + ), the third reagent bottle 5 is used to hold nitrate nitrogen ( 15 N-NO 3 - ), and the fourth reagent bottle 6 is used to hold zinc chloride;

[0032] The liquid adding structure includes a first liquid adding structure and a second liquid adding structure. The first liquid adding structure is respectively connected to the first reagent bottle 3 and the sample bottle 2, and the second liquid adding structure is respectively connected to the second reagent bottle 4, the third reagent bottle 5, the fourth reagent bottle 6, a vacuum structure 7, and the sample bottle 2. The vacuum structure 7 is a vacuum pump, and the vacuum structure 7 is electrically connected to a controller 30.

[0033] Specifically, in this embodiment, the anaerobic chamber 1 is made of acrylic plates, with a volume ≥ 5L. Through holes for pipelines or wires to pass through are provided on the side walls of the anaerobic chamber 1, and tetrafluoro gaskets are arranged at each through hole to achieve sealing. A heating structure 27, a temperature controller 29, and a deoxidation structure 28 are arranged inside the anaerobic chamber 1. The heating structure 27, the temperature controller 29, and the deoxidation structure 28 are respectively electrically connected to the controller 30. The temperature controller 29 is used to control the temperature of the heating pipe. The deoxidation structure 28 is a deoxidation tube, and both ends of the deoxidation structure 28 are connected to a circulation pump. The oxygen content in the anaerobic chamber 1 < 0.1%; the anaerobic chamber 1 is heated by an air bath, with a temperature control range of room temperature to 60°C and a temperature control accuracy of ±1°C.

[0034] In this embodiment, the first reagent bottle 3, the second reagent bottle 4, the third reagent bottle 5, and the fourth reagent bottle 6 are also respectively connected to an external gas source. The external gas source is nitrogen, which is stored in a nitrogen cylinder 12 located outside the anaerobic chamber 1. A nitrogen pressure controller 13 is provided on the pipeline between the nitrogen cylinder 12 and the first reagent bottle 3, the second reagent bottle 4, the third reagent bottle 5, and the fourth reagent bottle 6. Solenoid valves 14 and pressure valves 15 are provided on the pipelines between the first reagent bottle 3, the second reagent bottle 4, the third reagent bottle 5, and the fourth reagent bottle 6 and the external gas source. The solenoid valves 14 and the pressure valves 15 are respectively electrically connected to the controller 30.

[0035] In this embodiment, a first liquid addition valve 16 is provided on the pipeline between the first reagent bottle 3 and the first liquid addition structure. The first liquid addition valve 16 is electrically connected to the controller 30. Second liquid addition valves 17 are provided on the pipelines between the second reagent bottle 4, the third reagent bottle 5, and the fourth reagent bottle 6 and the second liquid addition structure respectively. The second liquid addition valves 17 are electrically connected to the controller 30. The first reagent bottle 3 is connected to the air outlet pipe 18, and an air vent valve 19 is provided on the air outlet pipe 18. The air vent valve 19 is electrically connected to the controller 30.

[0036] In this embodiment, the first reagent bottle 3, the second reagent bottle 4, the third reagent bottle 5, and the fourth reagent bottle 6 are all glass screw - mouth bottles with a volume of 100 mL. The first reagent bottle 3, the second reagent bottle 4, the third reagent bottle 5, and the fourth reagent bottle 6 are all equipped with flat - bottom screw caps, and a PTFE gasket is provided inside the flat - bottom screw caps for sealing. Three holes are opened on the flat - bottom screw cap of the first reagent bottle 3, and sealing gaskets are provided at each hole. The three holes on the flat - bottom screw cap of the first reagent bottle 3 are respectively connected to the air vent valve 19, the solenoid valve 14, the pressure valve 15, and the first liquid addition valve 16 to control the opening and closing of the corresponding pipelines. Two holes are opened on the flat - bottom screw caps of the second reagent bottle 4, the third reagent bottle 5, and the fourth reagent bottle 6, and sealing gaskets are provided at each hole. The two holes on the flat - bottom screw caps of the second reagent bottle 4, the third reagent bottle 5, and the fourth reagent bottle 6 are respectively connected to the pressure valve 15 and the second liquid addition valve 17.

[0037] In this embodiment, the gas pressure control accuracy is ≤0.1 psi, and the liquid injection pressure control accuracy is 0.1 psi.

[0038] In this embodiment, the sample bottle 2 is a glass screw - mouth bottle with a volume of 12 mL. A number of sample bottles 2 are arranged on the sample tray 8. The number of sample bottles 2 is preferably twenty. An oscillator 9 is provided at the bottom of the sample tray 8. The oscillator 9 is electrically connected to the controller 30. The oscillator 9 is a gyratory oscillator with an amplitude of 20 mm and a maximum vibration speed of 200 revolutions per minute.

[0039] In this embodiment, the liquid adding structure is moved by a driving structure. The driving structure includes a motor 10 and a lead screw 11. The motor 10 is electrically connected to a controller 30. The motor 10 is preferably a stepping motor. The power output end of the motor 10 is in transmission connection with the lead screw 11. The liquid adding structure is arranged on the lead screw 11 and is in threaded connection with the lead screw 11. The motor 10 drives the lead screw 11 to rotate, thereby driving the liquid adding structure to perform a linear motion. The driving structure can move the first liquid adding structure or the second liquid adding structure above each sample bottle 2.

[0040] In this embodiment, the first liquid adding structure includes a first liquid adding driving structure 23 and a groove side hole needle 24. The first liquid adding driving structure 23 is electrically connected to the controller 30. The first liquid adding driving structure 23 is preferably a cylinder. The cylinder body of the cylinder is connected to a compressed air bottle 31 outside the anaerobic box 1. An air pressure controller 32 and a valve are arranged on the pipeline between the compressed air bottle 31 and the cylinder body of the cylinder. The valve is electrically connected to the controller 30. The groove side hole needle 24 is arranged at the telescopic end of the first liquid adding driving structure 23. The first liquid adding driving structure 23 expands and contracts to drive the linear motion of the groove side hole needle 24. A first channel is arranged inside the groove side hole needle 24. A groove is formed on the outer surface of the groove side hole needle 24. The groove extends along the length direction of the groove side hole needle 24. The first channel is respectively connected to the sample bottle 2 and the first reagent bottle 3. When the groove side hole needle 24 conveys aerated water into the sample bottle 2, one end of the groove is communicated with the sample bottle 2, and the other end of the groove is located outside the sample bottle 2.

[0041] In this embodiment, the second liquid adding structure includes a second liquid adding driving structure 25 and a double side hole needle 26. The second liquid adding driving structure 25 is electrically connected to the controller 30. The double side hole needle 26 is arranged at the telescopic end of the second liquid adding driving structure 25. The second liquid adding driving structure 25 expands and contracts to drive the linear motion of the double side hole needle 26. A second channel and a third channel are arranged inside the double side hole needle 26. The second channel is respectively connected to the sample bottle 2 and the vacuum structure 7. The third channel is respectively connected to the sample bottle 2, the second reagent bottle 4, the third reagent bottle 5 and the fourth reagent bottle 6.

[0042] In this embodiment, the vacuum structure 7 is located outside the anaerobic box 1. A pressure relief valve 20, a vacuum pump solenoid valve 21 and a pneumatic valve 22 are arranged on the pipeline connecting the vacuum pump and the second liquid adding structure. The pressure relief valve 20, the vacuum pump solenoid valve 21 and the pneumatic valve 22 are respectively electrically connected to the controller 30.

[0043] In this embodiment, the controller 30 is a PLC controller. This embodiment aims at the improvement of the device structure, and the control process of the controller 30 is the prior art.

[0044] The soil anaerobic ammonium oxidation microbial nitrogen isotope labeling device of this embodiment can be used for the automated processing of steps such as soil pre-(anaerobic) cultivation, isotope addition, and gas sample preparation during the pretreatment process for measuring soil anaerobic ammonium oxidation activity. It not only saves a large amount of labor and time costs required for sample pretreatment, but also standardizes the sample pretreatment operation, ensuring the accuracy and reliability of the detection data, and providing key technical support for in-depth exploration of the influencing factors and action mechanisms of key processes such as soil anaerobic ammonium oxidation, and for research on soil nitrogen sinks and greenhouse gas emissions reduction.

[0045] This embodiment relates to a soil anaerobic ammonium oxidation microbial nitrogen isotope labeling device, which can be used as an automated pretreatment device for studying soil anaerobic ammonium oxidation activity, and can achieve in-situ anaerobic isotope labeling and temperature-controlled cultivation, including the automation of multiple steps such as in-situ automatic liquid addition, vacuum pumping, gas addition, and exhaust. As a front-end device, it is used in combination with a stable isotope analyzer, and the anaerobic ammonium oxidation activity can be obtained through data analysis, which can more truly reflect the anaerobic ammonium oxidation activity of paddy soil under in-situ conditions, and is suitable for the research on soil anaerobic ammonium oxidation microbial activity by scientific research and environmental detection departments.

[0046] The production materials of the soil anaerobic ammonium oxidation microbial nitrogen isotope labeling device of this embodiment are cheap and the cost is relatively low. The use of anti-corrosion solenoid valves and polytetrafluoroethylene pipes reduces reagent corrosion and is durable.

[0047] This embodiment provides an isotope labeling method using a soil anaerobic ammonium oxidation microbial nitrogen isotope labeling device, including:

[0048] Step 1: Put pure water in the first reagent bottle 3, put ammonium nitrogen in the second reagent bottle 4, put nitrate nitrogen in the second reagent bottle 4, put zinc chloride in the fourth reagent bottle 6, and put soil samples in each sample bottle 2. Each sample bottle 2 is placed on the sample tray 8;

[0049] Step 2: Open the solenoid valve 14 and the ventilation valve 19, fill nitrogen into the first reagent bottle 3, the pressure is controlled by the pressure gauge, the pressure < 1.0 MPa, displace the air in the first reagent bottle 3, and the air is discharged to the atmosphere through the air outlet pipe 18. Fill with nitrogen for about 15 min until nitrogen saturation, and then close the solenoid valve 14 and the ventilation valve 19 to complete the preparation of aerated water;

[0050] Step 3: The motor 10 drives the lead screw 11 to rotate. The second liquid addition structure moves above the sample bottle 2. Open the valve between the compressed air bottle 31 and the second liquid addition structure, so that the second liquid addition driving structure 25 drives the double-side hole needle 26 to extend into the sample bottle 2. Open the vacuum structure 7, the vacuum pump solenoid valve 21 and the pneumatic valve 22, and evacuate the air from each sample bottle 2 through the second liquid addition structure. After 10 s, close the vacuum structure 7, the vacuum pump solenoid valve 21 and the pneumatic valve 22. Open the pressure relief valve 20 for 5 s and then close it. Open the valve between the compressed air bottle 31 and the second liquid addition structure, so that the second liquid addition driving structure 25 drives the double-side hole needle 26 to withdraw from the sample bottle 2, and the vacuum cleaning program ends; The motor 10 drives the lead screw 11 to rotate. The first liquid addition structure moves above the sample bottle 2. Open the valve between the compressed air bottle 31 and the first liquid addition structure, so that the first liquid addition driving structure 23 drives the groove side hole needle 24 to extend into the sample bottle 2. Open the solenoid valve 14, the pressure regulating valve 15 and the first liquid addition valve 16 between the nitrogen bottle 12 and the first reagent bottle 3, and press the aerated water in the first reagent bottle 3 into the sample bottle 2. Control the delivery flow rate of the aerated water by accurately controlling the pressure of nitrogen, and control the amount of the aerated water by accurately controlling the liquid addition time, so as to ensure that when the aerated water in the sample bottle 2 enters the groove through the first channel and flows out of the sample bottle 2 from the groove, the addition of the aerated water ends. Open the valve between the compressed air bottle 31 and the first liquid addition structure, so that the first liquid addition driving structure 23 drives the groove side hole needle 24 to withdraw from the sample bottle 2;

[0051] Step 4: After the addition of the aerated water is completed, anaerobic cultivation is carried out. Start the oscillator 9, the temperature controller 29 and the deoxidation structure 28, and set the rotation speed of the oscillator 9 and the heating temperature of the heating structure 27 until the nitrate in the soil sample is consumed;

[0052] Step 5: The motor 10 drives the lead screw 11 to rotate. The second liquid addition structure moves above the sample bottle 2. Open the valve between the compressed air bottle 31 and the second liquid addition structure, so that the second liquid addition driving structure 25 drives the double-side hole needle 26 to extend into the sample bottle 2. Open the solenoid valve 14, the pressure regulating valve 15 and the second liquid addition valve 17 between the nitrogen bottle 12 and the second reagent bottle 4, and add the ammonium nitrogen in the second reagent bottle 4 to each sample bottle 2 through the second liquid addition structure; Open the solenoid valve 14, the pressure regulating valve 15 and the second liquid addition valve 17 between the nitrogen bottle 12 and the third reagent bottle 5, and add the nitrate nitrogen in the third reagent bottle 5 to each sample bottle 2 through the second liquid addition structure; Control the delivery flow rate of the ammonium nitrogen or nitrate nitrogen by accurately controlling the pressure of nitrogen, and control the amount of the ammonium nitrogen or nitrate nitrogen by accurately controlling the liquid addition time; Complete the addition of the ammonium nitrogen or nitrate nitrogen;

[0053] Step 6, perform anaerobic cultivation: Start the oscillator 9, temperature controller 29 and deoxygenation structure 28, set the rotation speed of the oscillator 9 and the heating temperature of the heating structure 27, and perform anaerobic cultivation for different times;

[0054] Step 7, the motor 10 drives the lead screw 11 to rotate, the second liquid addition structure moves above the sample bottle 2, open the valve between the compressed air bottle 31 and the second liquid addition structure, so that the second liquid addition driving structure 25 drives the double-side hole needle 26 to extend into the sample bottle 2, open the solenoid valve 14, pressure valve 15 between the nitrogen bottle 12 and the fourth reagent bottle 6, and the second liquid addition valve 17 between the fourth reagent bottle 6 and the second liquid addition structure, and add zinc chloride in the fourth reagent bottle 6 into each sample bottle 2 through the second liquid addition structure to terminate the reaction.

[0055] The above steps realize the automatic processing of in-situ water preparation by high-purity nitrogen aeration, soil pre-(anaerobic) cultivation, isotope addition, and gas sample preparation in the pretreatment process of soil anaerobic ammonium oxidation activity determination.

[0056] In the present invention, specific examples are used to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation manner and application scope. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A soil anaerobic ammonium oxidizing microbial nitrogen isotope labeling device, characterized in that: include: An anaerobic box and a sample bottle, a reagent bottle and a liquid adding structure arranged in the anaerobic box; The anaerobic box is used to provide an anaerobic environment; The reagent bottles include a first reagent bottle, a second reagent bottle, a third reagent bottle and a fourth reagent bottle, wherein the first reagent bottle is used to hold pure water, the second reagent bottle is used to hold ammonium nitrogen, the third reagent bottle is used to hold nitrate nitrogen, and the fourth reagent bottle is used to hold zinc chloride; The liquid adding structure includes a first liquid adding structure and a second liquid adding structure, the first liquid adding structure is respectively connected to the first reagent bottle and the sample bottle, and the second liquid adding structure is respectively connected to the second reagent bottle, the third reagent bottle, the fourth reagent bottle, a vacuum structure and the sample bottle.

2. The soil anaerobic ammonium oxidizing microorganism nitrogen isotope labeling device according to claim 1, characterized in that: A plurality of sample bottles are arranged on a sample tray, an oscillator is arranged at the bottom of the sample tray, and the oscillator is electrically connected to a controller.

3. The soil anaerobic ammonium oxidizing microorganism nitrogen isotope labeling device according to claim 1, characterized in that: The liquid adding structure is moved by a driving structure, and the driving structure includes a motor and a lead screw. The motor is electrically connected to a controller, and a power output end of the motor is transmission-connected to the lead screw. The liquid adding structure is arranged on the lead screw and is threadedly connected to the lead screw. The motor drives the lead screw to rotate, thereby driving the liquid adding structure to perform linear motion.

4. The soil anaerobic ammonium oxidizing microorganism nitrogen isotope labeling device according to claim 1, characterized in that: The first reagent bottle, the second reagent bottle, the third reagent bottle and the fourth reagent bottle are also connected to an external gas source, respectively. The external gas source is nitrogen. Solenoid valves and pressure valves are arranged on the pipelines between the first reagent bottle, the second reagent bottle, the third reagent bottle and the fourth reagent bottle and the external gas source. The solenoid valves and the pressure valves are electrically connected to the controller, respectively.

5. The soil anaerobic ammonium oxidizing microorganism nitrogen isotope labeling device according to claim 1, characterized in that: A first liquid adding valve is provided on the pipeline between the first reagent bottle and the first liquid adding structure, and the first liquid adding valve is electrically connected to the controller; a second liquid adding valve is provided on the pipeline between the second reagent bottle, the third reagent bottle and the fourth reagent bottle and the second liquid adding structure, respectively, and the second liquid adding valve is electrically connected to the controller; The first reagent bottle is connected to an air outlet pipe, a vent valve is provided on the air outlet pipe, and the vent valve is electrically connected to a controller.

6. The soil anaerobic ammonium oxidizing microorganism nitrogen isotope labeling device according to claim 1, characterized in that: The vacuum structure is located outside the anaerobic box. A pressure relief valve, a vacuum pump solenoid valve and a pneumatic valve are arranged on the pipeline connecting the vacuum structure and the second liquid adding structure. The pressure relief valve, the vacuum pump solenoid valve and the pneumatic valve are electrically connected to the controller respectively.

7. The soil anaerobic ammonium oxidizing microorganism nitrogen isotope labeling device according to claim 1, characterized in that: The first liquid-adding structure includes a first liquid-adding drive structure and a groove side hole needle. The first liquid-adding drive structure is electrically connected to the controller. The groove side hole needle is arranged at the telescopic end of the first liquid-adding drive structure. The telescopic movement of the first liquid-adding drive structure drives the linear movement of the groove side hole needle. A first channel is arranged inside the groove side hole needle. A groove is formed on the outer surface of the groove side hole needle. The groove extends along the length direction of the groove side hole needle. The first channel is respectively connected to the sample bottle and the first reagent bottle.

8. The soil anaerobic ammonium oxidizing microorganism nitrogen isotope labeling device according to claim 1, characterized in that: The second liquid-adding structure includes a second liquid-adding drive structure and a double-sided hole needle. The second liquid-adding drive structure is electrically connected to the controller. The double-sided hole needle is arranged at the telescopic end of the second liquid-adding drive structure. The telescopic movement of the second liquid-adding drive structure drives the linear movement of the double-sided hole needle. A second channel and a third channel are arranged in the double-sided hole needle. The second channel is respectively connected to the sample bottle and the vacuum structure, and the third channel is respectively connected to the sample bottle, the second reagent bottle, the third reagent bottle and the fourth reagent bottle.

9. The soil anaerobic ammonium oxidizing microorganism nitrogen isotope labeling device according to claim 1, characterized in that: A heating structure and a deoxidation structure are arranged in the anaerobic box, and the heating structure and the deoxidation structure are electrically connected to a controller respectively.

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