An automated system and method for simultaneous measurement of N2O and N2 emission rates in lakes

Abstract

This invention discloses an automated simultaneous measurement system and method for N₂O and N₂ emission rates in lakes, belonging to the technical field of gas emission rate measurement. The method involves acquiring the properties of lake samples and selecting a suitable culture mode, setting experimental parameters based on experimental requirements to form experimental and control groups; constructing a pollution-proof sampling pipeline according to the selected sampling mode, and setting the water bath temperature and headspace gas displacement within the culture vessel based on the culture parameters; controlling the composition and concentration of headspace gas by monitoring the real-time gas pressure and displacement parameters within the culture vessel; after a predetermined culture time, quantitatively collecting gas samples from the culture vessel according to the measurement parameters and transferring them to independent sample vials for measurement. This invention, through its pollution-proof closed-loop design, precise pressure control, and automated, standardized, and collaborative approach, comprehensively upgrades the accuracy, efficiency, and scenario adaptability of lake N₂ emission rate measurement, providing more realistic, reliable, and comparable technical support for nitrogen cycle research in lake ecosystems.

Description

Technical Field

[0001] This invention belongs to the technical field of gas emission rate measurement, and in particular relates to an automated synchronous measurement system and method for N2O and N2 emission rates in lakes. Background Technology

[0002] In lake ecosystem nitrogen cycle research, nitrogen (N2) emission rate is a key indicator reflecting nitrogen biogeochemical processes, and its accurate measurement is of great significance for revealing lake nitrogen balance mechanisms and assessing eutrophication risks. However, existing commercially available nitrogen emission rate measurement equipment and methods have significant shortcomings: Firstly, the bottle opening lacks protection against nitrogen contamination. During the measurement process, frequent sampling and measurement using a micro-syringe are required, which easily leads to nitrogen leakage, especially significantly interfering with samples with low nitrogen emission rates. Existing equipment lacks a dedicated bottle opening anti-contamination device, failing to effectively isolate external nitrogen and causing sample contamination and distorted results. Furthermore, these devices are difficult to directly couple with a gas chromatograph, requiring additional manual intervention, further increasing the risk of contamination and sources of error.

[0003] Secondly, there is a lack of pressure control and monitoring in the sampling bottles. During the experiment, operations such as gas replacement, gas replenishment, and sample injection in the sample bottles all require the internal pressure as a basic parameter. The stability and uniformity of the pressure directly affect the accuracy of gas concentration measurement. However, existing commercial equipment lacks both real-time detection capabilities for the internal pressure of the sampling bottles and precise pressure control methods. This leads to differences in pressure within different sampling bottles, and the pressure of the same sampling bottle cannot be standardized at different operational stages, ultimately causing deviations in the calculation of nitrogen emission rates. For example, pressure fluctuations can alter the distribution equilibrium of gas in the liquid and gas phases, causing nitrogen of the same concentration to exhibit different detection signals under different pressure conditions, severely affecting the reliability and comparability of the data. Summary of the Invention

[0004] To address the technical problems existing in the background art, the present invention provides an automated synchronous measurement system and method for N2O and N2 emission rates in lakes.

[0005] The present invention adopts the following technical solution: an automated synchronous measurement system for N2O and N2 emission rates in lakes, comprising: an automatic culture device, a sampling and injection device installed on the automatic culture device, and a GC analysis device adapted to the sampling and injection device; The automatic culture device includes a constant temperature water bath, which is divided into a culture bottle culture area, a transfer area, and a sample injection bottle culture area; a culture tray is provided in the culture bottle culture area, and a temporary storage tray is provided in the transfer area. The sampling and injection device includes: a three-axis robotic arm and a pin assembly mounted on the three-axis robotic arm; the pin assembly has guiding and blowing functions.

[0006] In a further embodiment, the ejector pin assembly includes: The carrier is assembled at the output end of the three-axis robotic arm; A positioning plate is installed at the bottom of the carrier; a through hole is provided at the center of the positioning plate. The injection needle is assembled inside the carrier; the injection needle has a vertical degree of freedom of movement, the top end of the injection needle is used to connect to the tubing, and the bottom end is aligned with the perforation; A pressure monitor is mounted on a carrier; the pressure monitor is connected to the injection needle, vacuum pump and high-frequency solenoid valve.

[0007] In a further embodiment, it further includes: an adapter interface; the adapter interface includes: A body adapted to the temporary storage tray; the body has a connecting end and a collecting end, the connecting end being used to connect to a culture column, and the collecting end having a rubber stopper.

[0008] In a further embodiment, the ejector pin assembly further includes: A pressure ring is fixed to the bottom surface of the positioning plate; the through hole is located inside the pressure ring; A protective gas inlet is provided on one side of the pressure ring; the bottom of the pressure ring has a serrated structure to form a gas displacement port inside the pressure ring.

[0009] Using the lake as described above The measurement method of the automated synchronous measurement system for N2 emission rates includes the following steps: The properties of lake samples were obtained and a suitable culture mode was selected. Experimental parameters were set based on experimental requirements to form experimental and control groups. The experimental parameters included culture parameters and measurement parameters. The sampling mode is determined based on the culture mode, and the sampling injection device performs the following steps based on the selected sampling mode: Contamination-proof sampling pipelines were constructed based on the selected sampling mode, and the water bath temperature and headspace gas in the culture vessel were set based on the culture parameters. The composition and concentration of the headspace gas were controlled by monitoring the real-time gas pressure value and replacement parameters in the culture vessel. After the predetermined incubation time, gas samples are quantitatively collected from the incubator according to the measurement parameters and transported to an independent sample bottle to enter the measurement stage. This process was repeated to achieve sample culture, sample transport, and emission rate determination for both the experimental and control groups.

[0010] In a further embodiment, the measurement phase includes the following steps: Continuously monitor the contents of the sample vials at predetermined time intervals. Concentration and Concentration, based on Concentration and The linear relationship between concentration and time was calculated, and the corresponding values ​​were obtained by combining the culture parameters. Emission rate and Emission rate; After completing one measurement of the gas sample in the current injection bottle, the sampling and injection device first performs self-cleaning of its own sampling pipeline and sampling components, and then extracts the gas sample from the injection bottle.

[0011] In a further embodiment, the culture mode includes at least: static culture based on culture flasks and dynamic culture based on culture columns.

[0012] In a further embodiment, when the culture mode is static culture based on culture bottles, the culture bottles are placed directly in the water bath culture tank and positioned by the culture tray, and the caps of the culture bottles are rubber stoppers; the rubber stoppers are adapted to the sampling and injection device to form a contamination-proof sampling pipeline.

[0013] In a further embodiment, the method for constructing the anti-pollution sampling pipeline is as follows: When the culture mode is dynamic culture based on culture column, the culture column is placed externally for water bath culture and connected to the adapter interface through pipeline; a peristaltic pump is installed on the pipeline. The adapter is positioned by a temporary storage tray, and the output end of the adapter has a rubber plug; the rubber plug is adapted to the sampling and injection device to form a contamination-proof sampling pipeline.

[0014] In a further embodiment, the working process of the sampling and injection device is as follows: During cultivation: The sampling and injection device adjusts its position through the three-axis degrees of freedom so that its sampling needle is aligned with the rubber stopper of the culture vessel; the gas replacement function is activated, the culture vessel is evacuated by a vacuum pump, and then the preset headspace gas is introduced. The evacuation-induction operation is repeated several times; at the same time, the built-in blowing component blows protective gas into the contact gap between the rubber stopper and the sampling needle to form an air curtain barrier to prevent external nitrogen contamination. During transfer: After the culture is completed, the pin of the sampling and injection device penetrates the rubber stopper of the culture vessel to directly collect the gas sample. Then, with the help of the three-axis degrees of freedom, the pin moves to the injection bottle and injects the sample into the injection bottle. During sample injection: After the sample is injected, the ejector pin is withdrawn and the tubing cleaning program is started, using high-purity He gas to flush the ejector pin and tubing.

[0015] The beneficial effects of this invention are as follows: Through pollution prevention closed-loop design, precise pressure control, and automated and standardized collaboration, this invention comprehensively upgrades the accuracy, efficiency, and scenario adaptability of lake N2 emission rate measurement, providing more realistic, reliable, and comparable technical support for nitrogen cycle research in lake ecosystems, helping to accurately assess water nitrogen balance and eutrophication risks, and promoting scientific decision-making for lake ecological protection and governance.

[0016] Furthermore, this invention constructs a fully enclosed process from cultivation to detection through a contamination-proof sampling pipeline, a gas curtain protection system for the sampling needle assembly, and a sealed delivery vial for independent sample injection. Before the sampling needle is punctured, a protective gas is purged to form a local inert gas curtain, blocking external nitrogen and water vapor contamination, reducing the probability of external nitrogen contamination by over 90%. The entire process requires no opening of the cap, and compared to traditional manual connection, the loss rate of nitrogen components (N2, N2O) is ≤5%, solving the problem of data distortion caused by contamination in existing equipment and ensuring the authenticity of low-emission-rate sample measurements.

[0017] This invention also takes into account the different culture methods that may be used in actual cultivation. Therefore, for static culture bottles, the rubber stopper is precisely matched and sealed with the sampling and injection device; for dynamic culture columns, the adapter interface works in conjunction with the temporary storage tray, and the rubber stopper at the output end ensures sealing. Both modes effectively prevent contamination, covering different culture scenarios for lake samples, and ensuring stable and controllable sample purity under complex conditions.

[0018] Based on experimental requirements, parameters such as intra-bottle pressure and gas volume for the experimental and control groups are precisely preset. Through automated control of the sampling and injection device, it is ensured that the pressure is controlled within a uniform standard (e.g., ±0.5 kPa) and the sampling volume error is ≤0.1 mL in different groups and batches of experiments. This standardizes experimental parameters, and the data deviation rate between different laboratories and batches is ≤5%, breaking through the arbitrariness of existing technologies and constructing a reproducible and comparable standardized system. Attached Figure Description

[0019] Figure 1 This is a diagram showing the layout of a system for measuring the nitrogen emission rate of a lake.

[0020] Figure 2 This is a structural diagram of an automated culture device.

[0021] Figure 3 This is a structural diagram of the ejector pin assembly.

[0022] Figure 4 This is a diagram showing the connection of the anti-contamination sampling pipeline during dynamic culture based on a culture column.

[0023] Figure 5 This is a flowchart of a method for the automated simultaneous measurement of N2O and N2 emission rates in lakes.

[0024] Figures 1 to 4 The components are labeled as follows: 1. Automatic culture device; 2. Sampling and injection device; 3. GC analysis device; 4. Adapter interface; 101. Constant temperature water bath; 102. Culture tray; 201. Three-axis robotic arm; 202. Pin assembly; 202-a. Carrier; 202-b. Positioning plate; 202-c. Injection needle; 202-d. Pressure monitor; 202-e. Pressure ring; 202-f. Protective gas inlet; 401. Peristaltic pump; 402. Culture column; 403. Culture flask. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] Example 1 like Figure 1 As shown in the figure, this embodiment discloses an automated simultaneous measurement system for N2O and N2 emission rates in lakes, including: an automatic culture device 1, a sampling and injection device 2 installed on the automatic culture device 1, and a GC analysis device 3 adapted to the sampling and injection device 2. It should be noted that the GC analysis device 3 in this embodiment includes: a gas chromatograph, a thermal conductivity detector, and an electron capture detector.

[0027] Combination Figure 2 The automatic culture device 1 described in this embodiment has a constant temperature water bath 101, which is divided into a culture bottle culture area, a transfer area and a sample injection bottle culture area.

[0028] Furthermore, the constant temperature water bath 101 is constructed with a double-layer structure and is equipped with a temperature sensor and an insulation box. Constant temperature water is injected into the constant temperature water bath 101 through the right-side inlet. The water temperature is monitored in real time by a temperature probe. When the water level in the bath reaches the experimental requirements, it is drained from the drain outlet for circulation, ensuring a constant water temperature within the bath. The interior of the bath is equipped with a sample bottle placement rack that can hold 30 sample bottles, which can be arranged in an orderly manner according to experimental requirements to form a simple and clear control group. In this embodiment, the constant temperature water bath 101 uses a stainless steel inner liner and a temperature control system employing a high-performance CPU processing chip and a highly sensitive, high-precision platinum resistance sensor, making temperature control more accurate and operation more convenient.

[0029] The sampling and injection device 2 in this embodiment includes: a three-axis robotic arm 201 and a pin assembly 202 mounted on the three-axis robotic arm 201; the pin assembly 202 has a guiding function and an air blowing function.

[0030] For specific references Figure 3The ejector assembly 202 includes a carrier 202-a mounted on the output end of the three-axis robotic arm 201. It should be noted that the three axes in this embodiment are the X-axis, Y-axis, and Z-axis, and the three-axis robotic arm 201 is a robotic arm with X-axis, Y-axis, and Z-axis sliding joints, which can be achieved using existing technology and will not be described in detail here.

[0031] It also includes: a positioning plate 202-b installed at the bottom of the carrier 202-a, wherein a through hole is provided at the center of the positioning plate 202-b. A sample injection needle 202-c is assembled inside the carrier 202-a. The sample injection needle 202-c has a vertical degree of freedom of movement. The top end of the sample injection needle 202-c is used to connect to the tubing, and the bottom end is aligned with the through hole.

[0032] To monitor the pressure inside the bottle in real time and ensure data consistency, a pressure monitor 202-d is also installed on the carrier 202-a. The pressure monitor 202-d is connected to the injection needle 202-c, the vacuum pump, and the high-frequency solenoid valve.

[0033] In use, the three-axis robotic arm 201 moves the carrier 202-a and the bottom positioning plate 202-b, aligning the central perforation of the positioning plate 202-b with the rubber stopper of the incubator (culture flask 403 or adapter interface 4). The positioning plate 202-b ensures that the injection needle 202-c is aligned with the puncture point of the rubber stopper. The injection needle 202-c inside the carrier 202-a can move vertically, with its top end connected to tubing (including gas delivery, vacuum, and cleaning pathways), and its bottom end aligned with the rubber stopper through the perforation.

[0034] When gas replacement is initiated, the injection needle 202-c, driven by the Z-axis, presses against the rubber stopper. The pressure monitor 202-d connects to the inside of the incubator through the injection needle 202-c. A vacuum pump is used to evacuate the incubator (300s), and a high-frequency solenoid valve controls the introduction of headspace gas. This vacuum-to-gas cycle is repeated, for example, 6 times. During the process, the pressure monitor 202-d provides real-time feedback on the pressure inside the incubator to ensure the replacement effect, such as a vacuum degree ≤ -0.09MPa and gas filling to 0.02MPa.

[0035] During sampling, the injection needle 202-c vertically penetrates the rubber stopper, while the pressure monitor 202-d simultaneously monitors the pressure stability inside the bottle. Gas is quantitatively collected through the tubing. After sampling, the three-axis robotic arm 201 moves the injection needle 202-c to the sample bottle, repeating the puncture action to complete sample transfer. Throughout the process, the pressure monitor 202-d is linked with the high-frequency solenoid valve to ensure pressure consistency during gas replacement and sampling, reducing errors caused by pressure fluctuations.

[0036] Furthermore, to prevent external gas contamination when the injection needle 202-c punctures the rubber stopper, the ejector assembly 202 in this embodiment further includes a pressure ring 202-e fixed to the bottom surface of the positioning plate 202-b, with the perforation located inside the pressure ring 202-e. A protective gas inlet 202-f is provided on one side of the pressure ring 202-e, and the bottom of the pressure ring 202-e has a serrated structure to form a gas replacement port inside the pressure ring 202-e.

[0037] Before puncture, He gas is continuously purged onto the surface of the rubber stopper at a rate of 50-80 mL / min through the serrated structure to remove any adhering air (containing N2). During puncture, the tight engagement between the serrations and the rubber stopper, combined with the positive pressure of He gas, prevents external gases from seeping into the culture vessel through the puncture hole. After sampling, He gas continues to purge when the needle is withdrawn to prevent the rubber stopper from drawing in external gases when it rebounds, and to avoid gas leakage from the culture vessel contaminating the tubing.

[0038] In addition, the serrated structure of the pressure ring 202-e can also assist in positioning. When the pressure ring 202-e presses against the rubber stopper, the embedding depth of the serrations can be confirmed by the Z-axis feedback of the three-axis robotic arm 201, ensuring that the perpendicularity and depth of each puncture are consistent, reducing sampling deviation caused by rubber stopper deformation, and further ensuring the accuracy of headspace gas composition determination.

[0039] In another embodiment, to ensure the stability of the culture bottle 403 or the adapter interface 4 during cultivation and sampling, a culture tray 102 is configured in the culture area of ​​the culture bottle 403 in this embodiment, and a temporary storage tray is configured in the transfer area. To adapt to different cultivation methods of the culture bottle 403, the culture tray 102 in this embodiment has slots set at predetermined intervals and in predetermined quantities. Each slot matches the top curvature of the culture bottle 403, ensuring that the bottle body is vertically fixed in the water bath culture tank and the rubber stopper is exposed outside the culture tray 102.

[0040] In another embodiment, the temporary storage tray also has several matrix-distributed grooves adapted to the adapter interface 4. Therefore, the adapter interface 4 in this embodiment includes: a body adapted to the temporary storage tray; the body has a connecting end and a collecting end, the connecting end being used to connect to the culture column 402, and the collecting end having a rubber stopper. The connecting end is connected to the culture column 402 via a pipeline, and a peristaltic pump 401 is installed on the pipeline, which mixes the gas in the pipeline with the gas in the culture device uniformly.

[0041] Example 2 like Figure 5 As shown, based on the lake provided in Example 1 This embodiment provides a corresponding measurement method for the automated synchronous measurement system for N2 emission rates, including the following procedures: The properties of lake samples are obtained, and a suitable culture mode is selected. Experimental parameters are set based on experimental requirements to form experimental and control groups. These experimental parameters include culture parameters and measurement parameters. In this embodiment, culture parameters may include: simulated environment, culture components, culture temperature, culture time, and headspace gas composition during culture, etc. Measurement parameters may include: measurement intervals and measurement quantities for gas samples, etc. A sampling mode is determined based on the culture mode, and the sampling and injection device performs the following steps based on the selected sampling mode: Contamination-proof sampling pipelines were constructed based on the selected sampling mode, and the water bath temperature and headspace gas in the culture vessel were set based on the culture parameters. The composition and concentration of the headspace gas were controlled by monitoring the real-time gas pressure value and replacement parameters in the culture vessel. After the predetermined incubation time, gas samples are quantitatively collected from the incubator according to the measurement parameters and transported to an independent sample bottle to enter the measurement stage. This process was repeated to achieve sample culture, sample transport, and emission rate determination for both the experimental and control groups.

[0042] Based on the above culture process, the determination stage includes the following steps: Continuously monitor the contents of the sample vials at predetermined time intervals. Concentration and Concentration, based on Concentration and The linear relationship between concentration and time was calculated, and the corresponding values ​​were obtained by combining the culture parameters. Emission rate and Emission rate; After completing one measurement of the gas sample in the current injection bottle, the sampling and injection device first performs self-cleaning of its own sampling pipeline and sampling components, and then extracts the gas sample from the injection bottle.

[0043] Furthermore, the experimental group is the lake sample group to be studied (multiple parallel samples can be set according to variables such as sampling point, water depth, and pollution level), while the control group is the baseline lake sample group with stable environment and no variable interference. The experimental group and the control group have the same number of parallel samples to ensure that the experimental variables are unique and the data can be compared.

[0044] For example, the experimental group in this embodiment could be the effect of different locations or depths within the same river or lake on the nitrogen emission rate. The control group, on the other hand, serves as a benchmark reference, such as selecting a typical location or depth within the same lake where the environment is stable and disturbances are minimal.

[0045] Different culture modes are selected based on the region and experiment during actual water sample collection. For example, if the water sample is from a relatively enclosed area with weak water flow in a simulated lake (e.g., bottom still water or shallow nearshore water), static culture based on culture bottles is suitable, meaning the culture bottles are relatively small, such as 100ml or 50ml. However, for areas with strong water flow in a simulated lake (e.g., lake inlets or surface convection zones), dynamic water circulation is achieved using peristaltic pumps; therefore, dynamic culture based on culture columns is chosen. In this case, the culture column has a relatively large volume and occupies more space.

[0046] For ease of understanding, the following embodiments are described The formula for calculating emission rate is as follows: ; for Emission rate, in units , for Slope of the concentration-time linear fit, in units of .

[0047] It should be noted that if the emission rate results need to be converted to a mass concentration unit in nitrogen (N), The above calculations are obtained The results were multiplied by the molar mass of nitrogen. This will complete the unit conversion.

[0048] Furthermore, the embodiments described herein The formula for calculating emission rate is as follows: ; in, for Emission rate, in units , The gas volume inside the incubator is preset in the culture parameters, in units of... ; for Slope of the concentration-time linear fit, in units of ; The absolute pressure of the culture system preset in the culture parameters, in units of ; The sample liquid volume is the preset value in the culture parameters, in units of... ; The gas constant is taken as 8.314 in this embodiment. ; The absolute humidity of the experiment is expressed in units of . , Water bath temperature (°C).

[0049] To adapt to the nitrogen emission rate measurement requirements of different water environments in lakes, the culture mode described in this embodiment includes at least: static culture based on culture bottles and dynamic culture based on culture columns. Static culture is suitable for samples from areas with weak water flow, such as the bottom still water and shallow near-shore water areas of lakes, while dynamic culture is suitable for samples from areas with strong water flow, such as the lake inlet and surface convection zone.

[0050] By setting up two different culture modes, the technical problems of a single culture mode being unable to fully simulate the different water environment characteristics of lakes and the large deviation between the measurement results and the actual nitrogen emission of lakes are solved, so as to achieve accurate measurement of nitrogen emission rate of water samples of different types of lakes.

[0051] Therefore, new problems arise under the two different culture modes: the sampling area of ​​the sampling and injection device in this embodiment is a fixed area, and its automated operation trajectory and working range are adapted to the setting of this fixed area. However, the culture column used for dynamic culture is larger in volume and requires a matching water circulation component. It adopts an external water bath culture method and is difficult to place directly in this fixed sampling area. Therefore, it is difficult to achieve standardized culture, sampling and detection operations for samples of the two culture modes in the same fixed sampling area, and it is impossible to guarantee the consistency of experimental operations and the comparability of measurement data under the two culture modes.

[0052] Therefore, in a further embodiment, the method for constructing the anti-contamination sampling pipeline includes two types: one is for static culture based on culture bottles, and the other is for dynamic culture based on culture columns. Further, when the culture mode is static culture based on culture bottles, the culture bottle is placed directly in a water bath and positioned using a culture tray. The cap of the culture bottle is a rubber stopper; the rubber stopper is adapted to the sampling and injection device to form an anti-contamination sampling pipeline. For example, a 100mL serum bottle is used as the culture bottle, and its cap is made of chemically resistant butyl rubber (good elasticity and strong sealing performance), which fits tightly against the bottle opening to form a seal.

[0053] During cultivation, 30 culture flasks are positioned using a cultivation tray (made of 304 stainless steel). The tray is designed with 3×10 arrayed slots, each matching the curvature of the top of the culture flask to ensure the flask is vertically fixed in the water bath and the rubber stopper is exposed outside the tray. The constant temperature water bath is insulated with a double-layer structure. Circulating water enters from the right inlet and flows out from the left. As the water flows through the hollow area at the bottom of the tray, it creates a uniform flush, allowing the water temperature of each culture flask to quickly reach the set value. The sampling needle of the sampling device is inserted into the butyl rubber stopper, and the cleanliness of the sampling pipeline is ensured by a multi-layered anti-contamination structure (He gas purging, pipeline cleaning, and protective cover isolation). Combined with the precise positioning of the cultivation tray and the uniform temperature control of the water bath, the stability and accuracy of nitrogen emission rate measurement under static cultivation are achieved.

[0054] In another embodiment, when the culture mode is dynamic culture based on a culture column, the culture column is placed externally for water bath culture and connected to the adapter interface through a pipeline; a peristaltic pump is provided on the pipeline; the adapter interface is positioned by a temporary storage tray, and the output end of the adapter interface has a rubber plug; the rubber plug is adapted to the sampling injection device to form a contamination-proof sampling pipeline.

[0055] For example, a 500mL cylindrical glass column (with a built-in porous sieve plate to simulate water stratification) is used for the culture column. An external water bath culture is adopted. A double-layer constant temperature sleeve is installed on the outside of the column. The sleeve is connected to an independent constant temperature circulating water bath through a pipeline. The water temperature inside the sleeve is monitored in real time by a temperature probe to ensure that the temperature of the water sample inside the column deviates from the temperature of the in-situ sampling point in the lake by ≤1℃.

[0056] To facilitate the setting of culture parameters and sampling by the sampling and injection device within its fixed area, the culture column and the adapter are connected by a polytetrafluoroethylene (PTFE) pipeline. A peristaltic pump is connected in series on the pipeline, and the pump speed is controlled by programming to achieve dynamic water circulation: the water sample in the column flows out from the bottom outlet through the pipeline, is pumped into the top inlet by the peristaltic pump to form a closed loop, simulating the natural convection of water flow in a lake. At the same time, the pump body adopts a buffer mechanism to avoid the water flow impact causing violent disturbance of the headspace gas in the column.

[0057] As the core connecting component between the culture column and the sampling injection device, the adapter interface is fixed in position by a temporary storage tray to ensure that the interface output end is precisely aligned with the trajectory of the sampling injection device.

[0058] Based on the above description, the workflow of the sampling and injection device disclosed in this embodiment is as follows: During cultivation: The sampling and injection device adjusts its position using three-axis freedom to align the sampling needle with the rubber stopper of the culture vessel; the gas replacement function is activated, a vacuum pump evacuates the culture vessel, and then a preset headspace gas (such as a He / O2 mixture or high-purity He gas) is introduced, repeating the vacuum-injection cycle several times; simultaneously, a protective gas is blown into the contact gap between the rubber stopper and the sampling needle through the built-in blowing component, forming an air curtain barrier to prevent external nitrogen contamination. For example, at a flow rate of 60... Ml High-purity He gas is blown at a rate of / min to form an air curtain barrier, preventing external nitrogen gas from seeping in through gaps. This can reduce the risk of contamination to below the detection limit, especially for control group samples with low nitrogen emissions.

[0059] During transfer: After the culture is completed, the pin of the sampling and injection device penetrates the rubber stopper of the culture vessel to directly collect the gas sample. Then, by moving with the three degrees of freedom in the three-axis direction, the pin is aligned with the injection bottle and the sample is injected into the injection bottle. During sample injection: After sample insertion, the ejector pin is withdrawn and the tubing cleaning program is initiated, flushing the ejector pin and tubing with high-purity He gas. Specifically, high-purity He gas is used to flush the ejector pin and connecting tubing at a flow rate of 120 mL / min for 15 seconds. The waste gas generated during flushing is discharged through a dedicated channel, ensuring that the amount of residual sample in the tubing is ≤0.1%, preventing cross-contamination of the next sample. During the cleaning process, the triaxial system resets to its initial position, preparing for the next round of sampling. The entire process relies on automated control of the equipment to achieve unattended operation, adapting to the continuous measurement needs of multiple sample sets.

[0060] Based on the above description, the specific measurement stages are given below: First pre-culture: Add 30g of air-dried soil and 3mL of distilled water to a 120mL serum bottle and pre-culture for 24h, using a culture bottle 407 without soil as a control. Make 15 culture bottles 407 for each type, for a total of 30 culture bottles 407.

[0061] After the second pre-incubation: Add 5 mL of carbon and nitrogen substrate solutions of 5 different concentrations, then seal with rubber septa and aluminum caps. Make 3 samples of each different concentration, placed in parallel, for a total of 30 culture flasks (407).

[0062] After adding the substrate solution, the headspace in the flask is first replaced: first, a vacuum is drawn for 300 seconds, then the flask is filled with He (99.999% purity, the same below), and this operation is repeated 6 times. 2 mL of distilled water (with the water layer sealed to prevent atmospheric exchange) is inserted into the flask to balance the pressure.

[0063] Fifth, the concentrations of N2 and N2O in the bottle were monitored online (samples were taken at 0, 16, 23h, 1 day, 3 days, 6 days, 10 days, and 14 days). The emission rates of N2 and N2O (both expressed as N) were calculated based on the linear relationship between the concentrations of N2 and N2O in the bottle and the change over time.

Claims

1. An automated synchronous measurement system for N2O and N2 emission rates in lakes, characterized in that, include: An automated culture device, a sampling and injection device installed on the automated culture device, and a GC analysis device adapted to the sampling and injection device; The automatic culture device includes a constant temperature water bath, which is divided into a culture bottle culture area, a transfer area, and a sample injection bottle culture area; a culture tray is provided in the culture bottle culture area, and a temporary storage tray is provided in the transfer area. The sampling and injection device includes: a three-axis robotic arm and a pin assembly mounted on the three-axis robotic arm; the pin assembly has guiding and blowing functions.

2. The automated synchronous measurement system for lake N2O and N2 emission rates according to claim 1, characterized in that, The ejector pin assembly includes: The carrier is assembled at the output end of the three-axis robotic arm; A positioning plate is installed at the bottom of the carrier; a through hole is provided at the center of the positioning plate. The injection needle is assembled inside the carrier; the injection needle has a vertical degree of freedom of movement, the top end of the injection needle is used to connect to the tubing, and the bottom end is aligned with the perforation; A pressure monitor is mounted on a carrier; the pressure monitor is connected to the injection needle, vacuum pump and high-frequency solenoid valve.

3. The automated synchronous measurement system for lake N2O and N2 emission rates according to claim 1, characterized in that, It also includes: an adapter interface; the adapter interface includes: A body adapted to the temporary storage tray; the body has a connecting end and a collecting end, the connecting end being used to connect to a culture column, and the collecting end having a rubber stopper.

4. The automated synchronous measurement system for lake N2O and N2 emission rates according to claim 2, characterized in that, The ejector pin assembly also includes: A pressure ring is fixed to the bottom surface of the positioning plate; the through hole is located inside the pressure ring; A protective gas inlet is provided on one side of the pressure ring; the bottom of the pressure ring has a serrated structure to form a gas displacement port inside the pressure ring.

5. A measurement method using the automated synchronous measurement system for lake N2O and N2 emission rates as described in any one of claims 1 to 4, characterized in that, Includes the following steps: The properties of lake samples were obtained and a suitable culture mode was selected. Experimental parameters were set based on experimental requirements to form experimental and control groups. The experimental parameters included culture parameters and measurement parameters. The sampling mode is determined based on the culture mode, and the sampling injection device performs the following steps based on the selected sampling mode: Contamination-proof sampling pipelines were constructed based on the selected sampling mode, and the water bath temperature and headspace gas in the culture vessel were set based on the culture parameters. The composition and concentration of the headspace gas were controlled by monitoring the real-time gas pressure value and replacement parameters in the culture vessel. After the predetermined incubation time, gas samples are quantitatively collected from the incubator according to the measurement parameters and transported to an independent sample bottle to enter the measurement stage. This process was repeated to achieve sample culture, sample transport, and emission rate determination for both the experimental and control groups.

6. The automated synchronous measurement method for lake N2O and N2 emission rates according to claim 5, characterized in that, The measurement phase includes the following steps: Continuously monitor the contents of the sample vials at predetermined time intervals. Concentration and N2O concentration, based on The linear relationships between the concentrations of nitrogen and oxygen (N2O) and time were calculated, and the corresponding values ​​were obtained by combining these with the culture parameters. Emission rate and Emission rate; After completing one measurement of the gas sample in the current injection bottle, the sampling and injection device first performs self-cleaning of its own sampling pipeline and sampling components, and then extracts the gas sample from the injection bottle.

7. The automated synchronous measurement method for lake N2O and N2 emission rates according to claim 5, characterized in that, The culture modes include at least: static culture based on culture flasks and dynamic culture based on culture columns.

8. The automated synchronous measurement method for lake N2O and N2 emission rates according to claim 5, characterized in that, When the culture mode is static culture based on culture bottles, the culture bottles are placed directly in the water bath culture tank and positioned by the culture tray. The caps of the culture bottles are rubber stoppers. The rubber stoppers are adapted to the sampling and injection device to form a contamination-proof sampling pipeline.

9. The automated synchronous measurement method for lake N2O and N2 emission rates according to claim 5, characterized in that, The method for constructing the pollution-proof sampling pipeline is as follows: When the culture mode is dynamic culture based on culture column, the culture column is placed externally for water bath culture and connected to the adapter interface through pipeline; a peristaltic pump is installed on the pipeline. The adapter is positioned by a temporary storage tray, and the output end of the adapter has a rubber plug; the rubber plug is adapted to the sampling and injection device to form a contamination-proof sampling pipeline.

10. The automated synchronous measurement method for lake N2O and N2 emission rates according to claim 5, characterized in that, The working process of the sampling and injection device is as follows: During cultivation: The sampling and injection device adjusts its position through the three-axis degrees of freedom so that its sampling needle is aligned with the rubber stopper of the culture vessel; the gas replacement function is activated, the culture vessel is evacuated by a vacuum pump, and then the preset headspace gas is introduced. The evacuation-induction operation is repeated several times; at the same time, the built-in blowing component blows protective gas into the contact gap between the rubber stopper and the sampling needle to form an air curtain barrier to prevent external nitrogen contamination. During transfer: After the culture is completed, the pin of the sampling and injection device penetrates the rubber stopper of the incubator to directly collect the gas sample. Then, with the help of the three-axis degrees of freedom, the pin moves to aim at the injection bottle and injects the sample into the injection bottle. During sample injection: After the sample is injected, the ejector pin is withdrawn and the tubing cleaning program is started, using high-purity He gas to flush the ejector pin and tubing.

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