Culture device and method for carrying out excitation effect research by alkali liquor tracking method

Abstract

The invention relates to the technical field of ecological environment monitoring, and discloses a culture device and a culture method for performing excitation effect research by an alkali liquor tracking method, which can remarkably improve experimental precision, repeatability and long-term stability. The system comprises a culture container containing soil, a collection bottle containing alkali liquor, an air inlet module, an air outlet module and an adding module. According to the culture device and method, the alkali liquor collecting bottle is externally arranged, and the culture container is of a wide-mouth structure and is divided into the upper part and the lower part, so that soil samples are more convenient to load; the gas inlet module is provided with a high-sensitivity online CO2 / O2 sensor and an automatic flow controller, and gas flow can be adjusted in real time under the condition that the system is not closed so as to maintain an ideal culture environment; the adding module is integrated on the bottle cap and comprises a micro injection pump for adding the 13C isotope into the culture container, the 13C isotope added into the culture container is uniformly dispersed through the dispersing unit, the cap does not need to be opened, soil is not touched, operation is efficient, and a culture system is not damaged.

Description

Technical Field

[0001] This invention relates to the field of ecological environment monitoring technology, specifically to a culture device and method for studying the excitation effect using the alkaline solution tracking method. Background Technology

[0002] The carbon cycle is a fundamental process in the function of terrestrial ecosystems, determining their productivity maintenance, climate regulation capacity, and long-term ecological sustainability. Through photosynthesis, respiration, decomposition, and the formation of long-term carbon pools, carbon is constantly exchanged and transformed among the atmosphere, organisms, soil, and water. Photosynthesis, as a key pathway for fixing inorganic carbon into organic carbon, is the primary source of carbon input for ecosystems. However, this natural cycle is being disturbed like never before. Human activities such as fossil fuel combustion and land-use change have significantly increased atmospheric CO2 concentrations, disrupting the balance between terrestrial carbon sinks and sources and exacerbating global warming. Against the backdrop of continuously rising temperatures and CO2, the carbon input, release, and storage processes of ecosystems may undergo profound changes, thus feeding back into the climate system. Elucidating the carbon cycle mechanisms within ecosystems and their responses to climate change pressures is not only a scientific prerequisite for understanding global change processes but also a necessary foundation for accurately assessing the carbon sink function of terrestrial ecosystems, predicting future climate trajectories, and developing mitigation strategies.

[0003] The activating effect is a core component of global terrestrial carbon cycle research. It refers to the phenomenon where the input of exogenous organic carbon (such as readily decomposable low-molecular-weight carbon substrates, root exudates, and plant residues) alters the rate of decomposition of native soil organic matter by soil microbial communities, either in the short or long term. This effect is categorized into positive and negative activation. It directly determines whether exogenous carbon input leads to additional soil organic matter loss, thus affecting the soil's functional role as a carbon sink or source, and is crucial for terrestrial carbon balance and climate model parameterization. Simultaneously, the activating effect, accompanying changes in organic matter mineralization or solidification processes, influences the release and plant availability of nutrients such as nitrogen and phosphorus, ultimately affecting plant growth and community dynamics. Clarifying the mechanism of the activating effect in scenarios such as agricultural and forestry fertilization, residue return, wetland management, and ecological restoration can provide scientific guidance for optimizing organic matter input strategies and preventing unintended soil organic matter loss.

[0004] The alkaline solution tracing method is a traditional chemical method for determining soil CO2 release (or cumulative release). Its core principle is to use an alkaline solution (commonly NaOH solution) in a closed culture system to absorb CO2 released from the soil and fix it into carbonates or bicarbonates. The amount of carbon absorbed in the alkaline solution is then measured. 13Carbon isotope abundance (e.g., acid titration, inorganic carbon analyzer determination) can be used to infer soil CO2 release during cultivation. This method is suitable for long-term incubation, intermittent sampling experiments, including indoor incubation, rhizosphere experiments, and some field co-op experiments. When combined with isotope tracing techniques (e.g., adding...) 13 When C-labeled glucose is used, the isotopic composition of CO2 captured by alkali solution can be analyzed by mass spectrometry. With the help of isotopic mass spectrometry / elemental analysis and isotopic balance formula, the total CO2 can be separated into substrate-derived CO2 and soil native organic matter (SOM)-derived CO2, thereby directly quantifying the excitation effect (i.e. the difference in native SOM-derived CO2 between the exogenous carbon treatment group and the control group).

[0005] Currently, when the alkali tracking method is used to study the excitation effect, it commonly employs directly purchased glass bottles as culture containers, with small plastic bottles containing alkali solution placed inside the open container. Tweezers are used to handle the alkali solution bottle to complete the experimental operation. This traditional setup has several drawbacks: 1. The narrow opening of the culture flask makes it inconvenient to handle soil loading and alkali solution handling. Traditional glass culture flasks have narrow openings and deep interiors, making soil loading, mixing, and subsequent operations extremely difficult. Handling alkali solution bottles requires repeated manipulation with tweezers in a confined space, which is not only inefficient and susceptible to human disturbance, but also prone to tipping over, colliding with soil, or contaminating the bottle walls, thus reducing the standardization of experiments. 2. The culture bottle is quite tall, with the soil at the bottom. 13 Adding C isotope labels is difficult and results in uneven distribution, easily triggering localized excitation effects and affecting experimental reliability. The culture soil is usually located at the bottom of the flask, making it difficult to add... 13 C-labeled substrates (such as...) 13 When C-labeled glucose is used, it is difficult to spread evenly due to the limited space, especially in deep bottle structures where thorough mixing is even more challenging. This often results in abnormally high local substrate concentrations, triggering a "hotspot" excitation effect that deviates from the true dynamics of soil carbon processes, thus reducing the ecological representativeness and interpretability of the data. Third, the transparent body of the bottle cannot effectively block light, causing light disturbance and biological contamination. In order to facilitate observation of the internal operation, the culture bottle should be made of colorless and transparent material and additional artificial light protection is required. However, during the long culture period, if the light protection is not thorough, algae or plant germination can easily grow inside the bottle, changing the carbon source distribution, oxygen concentration and microbial metabolic environment, seriously interfering with the excitation process and distorting the experimental results.

[0006] Fourth, the alkali solution bottle is in direct contact with the soil, and some soil is easily carried away when it is replaced, which interferes with the culture substrate. Traditional methods require the alkali solution bottle to be placed directly on or even in contact with the soil. When the alkali solution is removed or replaced, soil particles are inevitably brought out, interfering with the cultured soil sample. This disturbance effect can accumulate and be amplified, especially in sampling designs with multiple time points in a cycle, which will produce systematic biases in the study of excitation effect kinetics. Fifth, the enclosed space of the culture bottle leads to oxygen consumption by the soil during respiration. Long-term cultivation or the later stages of cultivation can easily result in an anaerobic environment, inhibiting the activation effect and causing experimental deviations. Soil respiration continuously consumes O2, but traditional sealed culture bottles lack oxygen compensation or gas exchange control mechanisms. In the later stages of cultivation, insufficient oxygen or excessive CO2 accumulation can easily occur within the bottle, inhibiting microbial activity and the normal occurrence of the activation effect, thus significantly underestimating the true soil carbon decomposition potential. VI. Traditional apparatus and methods rely solely on natural diffusion to achieve CO2 migration to the alkaline solution surface. This results in an unclear gas flow path, poor stability, and significant susceptibility to bottle structure and operational disturbances. The unstable CO2 absorption efficiency hinders accurate calculation of time-series carbon mineralization flux, particularly in low-flux or short-timescale experiments where the bias is more pronounced.

[0007] While these traditional devices and methods, with their simple structures and lack of control and standardized design, met early experimental needs to some extent, they have become key bottlenecks restricting the accuracy, efficiency, and scalability of the alkaline tracking method in the current context of increased demands for quantifying excitation effects, strong dependence on isotope accuracy, and higher requirements for device stability and repeatability. Developing a novel culture device with a reasonable structure, user-friendly operation, and gas and isotope control capabilities has urgent practical significance and significant technological innovation value. Summary of the Invention

[0008] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a culture device and method for studying the excitation effect using the alkali tracking method, thereby solving the problems mentioned in the background art.

[0009] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a culture device for studying the excitation effect using the alkali tracking method, comprising: a culture container filled with soil, a collection bottle filled with alkali solution, an air inlet module, an air outlet module, and an addition module; The dosing module includes adding substances to the interior of the culture container. 13 A micro-injection pump for C isotopes, which delivers the solution to the culture vessel via a dispersion unit. 13 C isotopes are uniformly dispersed; The air intake module includes an air intake manifold for injecting clean air into the culture container, and the bottom end of the air intake manifold is inserted into the soil. The air intake manifold is equipped with a desorption unit for removing CO2 from the clean air source. The gas outlet module includes a main gas outlet pipe that discharges the gas generated in the culture container to a collection bottle for CO2 collection, and one end of the main gas outlet pipe is inserted into the alkaline solution in the collection bottle.

[0010] Preferably, the culture container is a wide-mouth plastic amber bottle, which is divided into upper and lower parts, and the two parts are installed in a detachable manner; The top of the culture container is detachably fitted with a main sealing plug, which is used to seal the air inlet module, air outlet module, and dosing module to the inside of the culture container.

[0011] Preferably, the intake end of the main intake pipe is equipped with a microporous filter with a pore size of 0.2μm and a humidifier. The humidifier is used to humidify the injected clean air source, and the desorption unit is located between the microporous filter and the humidifier.

[0012] Preferably, the intake manifold is also equipped with a main switching valve, an online sensor group, and a flow controller; The online sensor group includes a miniature optical CO2 sensor and an O2 sensor, and is connected in series with the flow controller for high-frequency monitoring of the air source and automatic adjustment of the air flow rate.

[0013] Preferably, the desorption unit includes two desorption cylinders installed on the intake manifold, the two desorption cylinders are equipped with CO2 absorption columns for absorbing CO2 in the air source, and the two desorption cylinders are connected to the intake manifold in parallel. The intake manifold is equipped with a control device for controlling the direction of airflow.

[0014] Preferably, the control component includes a conversion cylinder that is fixedly connected to the intake manifold. Both sides of the conversion cylinder are connected to two desorption cylinders through input connection pipes, and the output ends of the two desorption cylinders are reconnected to the intake manifold through output connection pipes. Both input connection pipes and both output connection pipes are telescopic flexible hoses. The valve plate is rotatably connected to the inside of the conversion cylinder via a rotating shaft, and the valve plate is set at a 45-degree angle. One end of the rotating shaft extends to the outside of the conversion cylinder and is fixedly connected to a transmission bracket. The transmission bracket is perpendicular to the valve plate. The bottom of the conversion cylinder is rotatably connected to an elastic telescopic rod via a bracket, and the telescopic end of the elastic telescopic rod is hinged to one end of the transmission bracket. One end of the transmission bracket is fixedly connected to a connecting rope, and the connecting rope is detachably connected to one of the conversion cylinders.

[0015] Preferably, a secondary sealing plug is detachably installed on the top of the collection bottle, and the secondary sealing plug is used for sealing connection between the gas outlet module and the inside of the collection bottle; The main outlet pipe is fixedly connected to the extraction pipe via a bracket, and one end of the extraction pipe is inserted into the collection bottle through a secondary sealing plug, with the end positioned above the surface of the alkali solution. Both the main outlet pipe and the extraction pipe are equipped with secondary switching valves, and a miniature diaphragm pump is installed on the extraction pipe.

[0016] Preferably, the dispersion unit includes a plastic mesh plate installed in a culture container, the plastic mesh plate being used to uniformly integrate the isotopes added to the culture container into the cultured soil sample; The plastic mesh is installed between the upper and lower parts of the culture container and is located above the culture soil.

[0017] Preferably, the dispersing unit further includes a rotary nozzle rotatably installed in the dosing tube of the micro injection pump, the top of the rotary nozzle being fixedly connected to a blade holder via a connecting shaft, and a flywheel block being fixed below the blade holder; The dosing tube of the micro-injection pump has a constricted section inside, and the constricted section is located directly above the blade holder.

[0018] A culture method for studying the excitation effect using the alkali tracking method includes the following steps: S1. Weigh 50g of the soil sample to be cultured and spread it evenly at the bottom of the culture container, ensuring that the soil surface is flat and avoiding local accumulation; tighten the screws on the upper and lower parts of the culture container to ensure that the plastic mesh plate is stable and does not come into direct contact with the soil; seal the assembled main sealing plug to the culture container; open the air inlet and outlet valves, confirm that the desorption unit in the air inlet module is in standby mode, the low flow rate mass flow controller is set to zero, and the isotope addition module is in standby mode, thus completing the construction of the entire culture system; S2. Start the isotope dosing module, set the flow rate of the small multi-channel micro-injection pump to 0.05-0.5 ml / min, and add... 13 The C isotope labeled solution was slowly injected into the plastic mesh plate in the center of the culture container, while the dispersion unit was turned on to allow the labeled solution to be evenly sprinkled onto the soil surface through the holes, simulating the "small amount, multiple times" secretion characteristics of root exudates under natural conditions; the addition operation was repeated in time periods according to the experimental design to avoid local excitation effects caused by a single addition; S3. Place the entire culture system in a constant temperature environment of 22℃ to maintain a constant culture temperature; start the low flow rate mass flow controller, and combine it with the high-frequency monitoring data of the online sensor group to automatically adjust the air intake flow rate to ensure that clean air is continuously introduced into the culture container, and that the main air intake pipe extends below the soil surface to maintain sufficient oxygen and a stable gas phase environment in the bottle. S4. Replace the alkali collection bottle according to the time gradient of the experimental design: Close the gas inlet valve of the culture container and the gas outlet valve of the alkali collection bottle, disconnect the connection between the alkali collection bottle and the gas outlet main pipe, replace with a new collection bottle containing 15ml of 1M NaOH solution, reconnect and open the valve to continue culturing; there is no need to open the culture container during the replacement process to avoid disturbing the soil. S5. Send the replaced alkali solution collection bottle to the laboratory, determine the absorbed carbon content in the NaOH solution using acid titration, and measure the δ-carbon content using isotope mass spectrometry. 13 The C-value, combined with the isotope balance formula, decomposes the total CO2 into substrate-derived CO2 and soil native organic matter-derived CO2, quantifying the excitation effect. After the culture is completed, the culture container is opened, and soil samples are taken out for subsequent determination of physicochemical properties such as carbon and nitrogen content, microbial community structure, and enzyme activity.

[0019] (III) Beneficial Effects Compared with existing technologies, this invention addresses the systemic shortcomings of traditional alkaline tracking methods in terms of carbon flux monitoring, isotope labeling accuracy, culture system stability, and operational controllability. It provides a culture device and method for studying excitation effects using the alkaline tracking method, offering the following advantages: I. This invention achieves "zero-disturbance" carbon capture through an external alkaline collection bottle design coupled with valve control. In this system, the culture bottle does not need to be opened during replacement, avoiding disruption of the gas composition of the culture environment; it also eliminates soil removal during replacement, preventing soil sample interference and errors; and it achieves complete isolation between internal and external gas paths, realizing truly high-precision, continuous carbon sink collection.

[0020] 2. The culture container adopts a wide-mouth structure and is divided into upper and lower parts, which facilitates rapid and uniform filling and lightweight operation. The internal flat-bottom short-path structure can shorten the diffusion path of the labeling solution, avoiding deviations in the excitation effect caused by the bottle structure from the source.

[0021] Third, the isotope dosing module is integrated into the bottle cap, allowing isotope labeling to be added without contact with the soil, without opening the bottle, and without damaging the culture system. This makes the operation highly efficient and does not disrupt the culture system. It enables precise adjustment of the single dosing amount by a micro-controller, delivering the product to the soil interface via micro-spraying, enhancing diffusion uniformity, and eliminating the need for shaking or stirring, thus avoiding disturbance to the microbial-soil interface structure.

[0022] Fourth, the desorption unit employs a dual-desorption cylinder automatic switching design. When one adsorption column becomes saturated, the gas path automatically switches to the other. The saturated column is regenerated during the interruption of connection, ensuring continuous removal of CO2 from the inlet gas and avoiding interference with experimental data. This design enables stable removal of CO2 from the inlet gas during continuous cultivation, eliminating interference from exogenous carbon at its source.

[0023] Fifth, an online sensor array is integrated into the air intake module in series with a flow controller. This allows for high-frequency monitoring of CO2 and O2 concentrations and automatic adjustment of airflow speed to maintain a stable gaseous environment within the culture vessel, reducing experimental errors. This design enables continuous maintenance of sufficient O2 within the culture system while preventing CO2 accumulation. It also allows for precise control of available O2 levels for soil respiration, preventing the inhibition of the excitation effect due to later hypoxia. This helps obtain dynamic and continuous gas data, more accurately reflecting the rate and process of the excitation effect.

[0024] six, 13 The carbon isotope is uniformly dispersed through dispersion units, and the method of adding it in small amounts multiple times avoids local excitation effects and ensures the uniformity of the reaction between the labeling solution and the soil. The isotope addition module constructed in this invention can significantly improve the uniformity of the label in the soil, the contact efficiency between the label and microorganisms, and the control of the spatial heterogeneity of the excitation effect. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2 This is a cross-sectional view of the structure for cultivating aeration according to the present invention; Figure 3 This is a schematic diagram of the structure of the rotating nozzle of the present invention; Figure 4 This is a cross-sectional view of the structure of the collection bottle of the present invention; Figure 5 This is a schematic diagram of the air outlet module of the present invention; Figure 6 This is a schematic diagram of the intake module of the present invention; Figure 7 This is a schematic diagram of the structure of the control component of the present invention; Figure 8 (a) is an initial state diagram of the control component of the present invention; Figure 8 (b) is a diagram showing the state of the control component after conversion according to the present invention.

[0026] In the diagram: 10. Culture container; 11. Main sealing plug; 20. Collection bottle; 21. Secondary sealing plug; 30. Intake module; 31. Intake manifold; 32. Microfiltration filter; 33. Humidifier; 34. Online sensor array; 35. Flow controller; 36. Desorption unit; 361. Desorption cylinder; 362. Conversion cylinder; 363. Valve plate; 364. Transmission bracket; 365. Elastic telescopic rod; 366. Connecting rope; 40. Exhaust module; 41. Main exhaust pipe; 42. Suction pipe; 43. Miniature diaphragm pump; 50. Dosing module; 51. Miniature injection pump; 52. Plastic mesh plate; 53. Rotary nozzle; 54. Blade holder; 55. Flywheel block. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: See attached document Figures 1 to 8 A culture device for studying the excitation effect using the alkali tracking method includes: a culture container 10 containing soil, a collection bottle 20 containing alkali solution, an air inlet module 30, an air outlet module 40, and an addition module 50. The dosing module 50 includes dosing into the culture container 10. 13 A micro-injection pump 51 for C isotopes, which dispenses the substance into the culture container 10 via a dispersion unit. 13 C isotopes are uniformly dispersed; 13 C isotopes are sprayed and added in small amounts multiple times; The dosing module 50 is located on top of the culture vessel 10 and is combined with a small multi-channel micro-injection pump 51 located on the cap (main sealing plug 11) to deliver the solution at extremely low flow rates (e.g., 0.05–0.5 ml / min). -1 The labeling solution is injected slowly and repeatedly. This method makes the "addition a process" rather than a one-time impact, better simulating the characteristics of slow, repeated root exudation; it also allows for... 13 The carbon isotope is continuously injected into the soil being cultivated in small amounts and multiple times, thereby achieving better simulation and cultivation results.

[0029] The air intake module 30 includes an air intake manifold 31 for injecting clean air into the culture container 10, and the bottom end of the air intake manifold 31 is inserted into the soil. The air intake manifold 31 is provided with a desorption unit 36 ​​for removing CO2 from the clean air source. The desorption unit 36 ​​includes two adsorption columns, which can automatically switch to the backup column when one column is saturated, thereby enabling the replacement or regeneration of the front column without shutting down the system.

[0030] The gas outlet module 40 includes a gas outlet main pipe 41 that discharges the gas generated in the culture container 10 to the collection bottle 20 for CO2 collection, and one end of the gas outlet main pipe 41 is inserted into the alkaline solution in the collection bottle 20.

[0031] By designing the alkali collection bottle 20 independently, it is convenient to collect and replace the alkali solution without disturbing the soil during the replacement process, thus avoiding interference with the cultured soil sample. Because the operations inside the bottle (such as replacing the alkali solution) are moved out of the bottle body, the reliance on the visibility inside the culture bottle is reduced.

[0032] Brown bottles were chosen as the culture container 10 to protect the plant from light during cultivation and prevent unexpected plant growth from interfering with the experiment; and additional... 13 C isotope labeling solutions are very convenient and homogeneous, avoiding localized excitation caused by uneven addition.

[0033] This invention offers advantages such as easy replication, washability and disinfection, compatibility with isotope sampling, safety, and controllable cost.

[0034] See attached document Figure 1 and Figure 2 The culture container 10 is a wide-mouthed, opaque plastic amber bottle, divided into upper and lower parts that are detachably connected by a screw-tightening mechanism. The lower part is used to hold the soil sample to be cultured, and the upper part is connected to the lid (main sealing plug 11). The 1L volume of the culture container 10 is suitable for culturing approximately 50g of soil sample. If more soil sample is to be cultured or the culture material is to be changed, the size of the culture container 10 can be adjusted according to the actual situation. The main sealing plug 11 is detachably installed on the top of the culture container 10. The main sealing plug 11 is used for sealing the air inlet module 30, air outlet module 40, and dosing module 50 with the inside of the culture container 10. The main sealing plug 11 contains a three-way flange, which can fit the culture container 10 well.

[0035] See attached document Figure 6 The intake manifold 31 is equipped with a microporous filter 32 with a pore size of 0.2 μm and a humidifier 33. The humidifier 33 is used to humidify the injected clean air source, restoring the actual humid environment of the soil and making the microbial activity closer to the natural state. The desorption unit 36 ​​is located between the microporous filter 32 and the humidifier 33. The intake manifold 31 is also equipped with a main switch valve, an online sensor group 34, and a flow controller 35. The online sensor group 34 includes a miniature optical CO2 sensor and an O2 sensor, and the online sensor group 34 is connected in series with the flow controller 35 for high-frequency monitoring of the air source and automatic adjustment of the air flow rate. The injected air source passes through a 0.2μm microporous filter 32, then through a CO2 desorption unit 36 ​​(an alkaline chemical absorption column, such as one containing a solid CO2 absorbent), and then through a humidifier 33 before passing through a main switch valve (open state; additional valves are added to open or close the passage as needed). This air is then connected to the air inlet on the main sealing plug 11 of the culture container 10. A small-range, low-flow-rate mass flow controller 35 is connected in the air inlet passage to precisely supply air to each bottle. The low-flow-rate mass flow controller 35 displays the flow rate digitally and can be used for data logging. The flow controller 35 automatically adjusts the airflow rate based on the CO2 and O2 values ​​within the culture container 10 from the online sensor group 34, maintaining a stable gaseous environment, continuous airflow, and minimizing the benefits of the CO2 absorbent within the culture container 10.

[0036] By adding a low-flow-rate mass flow controller 35 in series with the intake passage system, and combining it with an online sensor group 34 to monitor CO2 and O2 concentrations, the intake air flow rate can be automatically adjusted, and CO2 removal agent can be saved.

[0037] See attached document Figures 6 to 8 The desorption unit 36 ​​includes two desorption cylinders 361 installed on the intake manifold 31. The interior of the two desorption cylinders 361 is equipped with CO2 absorption columns for absorbing CO2 in the air source, and the two desorption cylinders 361 are connected to the intake manifold 31 in parallel. The intake manifold 31 is equipped with a control component for controlling the air source flow direction. Two desorption cylinders 361 are installed in parallel and controlled by a control unit, allowing one cylinder 361 to be in normal use while the other is on standby. The control unit switches between the two, ensuring that if one cylinder 361 becomes saturated, the standby cylinder 361 immediately starts operating. This prevents the inability to remove CO2 from the air source in time when a single cylinder 361 becomes saturated, thus avoiding disruption to subsequent excitation effect studies. The control unit includes, but is not limited to, instruments such as solenoid valves that change the airflow output direction. The CO2 absorption column includes, but is not limited to, materials such as NaOH, which absorbs and desorbs CO2 and whose mass changes after adsorption saturation.

[0038] See attached document Figure 4 and Figure 5 The top of the collection bottle 20 is detachably equipped with a secondary sealing plug 21, which is used for sealing the connection between the gas outlet module 40 and the inside of the collection bottle 20. The main gas outlet pipe 41 is fixedly connected to the suction pipe 42 through the bracket, and one end of the suction pipe 42 is inserted into the collection bottle 20 through the secondary sealing plug 21, with the end located above the surface of the alkaline solution. A secondary switch valve is installed on both the main gas outlet pipe 41 and the suction pipe 42, and a micro diaphragm pump 43 is installed on the suction pipe 42. The collection bottle 20 is preferably 50ml in volume and contains 15ml of alkaline solution (1M NaOH solution). The air inlet pipe on the cap (secondary sealing plug 21) of the collection bottle 20 is connected to the air outlet system of the culture container 10. Specifically, a connecting pipe is installed on the secondary sealing plug 21, which connects to the main air outlet pipe 41 and the extraction pipe 42. This connecting pipe is detachably connected to the main air outlet pipe 41 and the extraction pipe 42, facilitating subsequent replacement of the collection bottle 20 by operators, or direct separation of the collection bottle 20 from the secondary sealing plug 21 to achieve the purpose of collecting CO2 in stages. During the culture process, the purpose of collecting CO2 in stages is achieved by replacing the collection bottle 20 while retaining the secondary sealing plug 21. The secondary switch valve is a manual switch valve used to temporarily cut off the airflow when replacing the collection bottle 20 during the culture process. A micro diaphragm pump 43 provides the suction power to exhaust the CO2-removed waste gas from the collection bottle 20.

[0039] Example 2: The difference from Example 1 is that; See attached document Figures 6 to 8 The control unit includes a converter cylinder 362 that is fixedly connected to the intake manifold 31. Both sides of the converter cylinder 362 are connected to two desorption cylinders 361 through input connection pipes, and the output ends of the two desorption cylinders 361 are reconnected to the intake manifold 31 through output connection pipes. Both input connection pipes and both output connection pipes are telescopic hoses. The intake manifold 31 is connected to two desorption cylinders 361 via two output connecting pipes, allowing the air source inside the intake manifold 31 to enter the desorption cylinder 361. The CO2 absorber column then removes CO2 from the air source. Both output connecting pipes are flexible hoses, allowing the CO2 absorber column in one of the desorption cylinders 361 to continuously absorb CO2, resulting in increased mass. This causes the desorption cylinder 361 to sink, ultimately triggering the control terminal of the control unit to switch the gas input position. One-way valves are installed on both output connecting pipes to prevent gas from flowing back into the other spare desorption cylinder 361. The desorption cylinder 361 consists of a cylinder body and a cover, facilitating future replacement of the CO2 absorber column. A valve plate 363 is rotatably connected inside the converter cylinder 362 via a rotating shaft, and the valve plate 363 is set at a 45-degree angle. One end of the rotating shaft extends to the outside of the converter cylinder 362 and is fixedly connected to a transmission bracket 364. The transmission bracket 364 is perpendicular to the valve plate 363. An elastic telescopic rod 365 is rotatably connected to the bottom of the converter cylinder 362 via a bracket, and the telescopic end of the elastic telescopic rod 365 is hinged to one end of the transmission bracket 364. A connecting rope 366 is fixedly connected to one end of the transmission bracket 364, and the connecting rope 366 is detachably connected to one of the converter cylinders 362. The device is connected to one of the desorption cylinders 361 via a connecting rope 366. When the adsorption column inside the desorption cylinder 361 becomes saturated with CO2 from the air source, its mass increases. This causes the desorption cylinder 361 to move downwards due to gravity, which in turn pulls the transmission bracket 364 via the connecting rope 366, causing the transmission bracket 364 to rotate 90 degrees. This indirectly drives the valve plate 363 to rotate 90 degrees, resulting in a change in the exhaust position inside the conversion cylinder 362. This fully utilizes the mass change caused by the adsorption column becoming saturated with CO2, triggering the control unit and providing an automatic switching function. It eliminates the need for precision sensors, reducing device costs and improving the convenience of later maintenance. The device is connected via a flexible telescopic rod 365 using a rotating and hinged method, with the bottom of the elastic telescopic rod 365 located at the center of the conversion cylinder 362. This ensures the stability of the valve plate 363 before and after rotation.

[0040] Example 3: The difference from Example 1 is that; See attached document Figure 2 and Figure 3 The dispersion unit includes a plastic mesh plate 52 installed in the culture container 10. The plastic mesh plate 52 is used to uniformly integrate the isotopes added to the culture container 10 into the cultured soil sample. The plastic mesh plate 52 is installed between the upper and lower parts of the culture container 10 and is located above the cultured soil. The dispersion unit also includes a rotary nozzle 53 rotatably installed in the dosing tube of the micro-injection pump 51. The top of the rotary nozzle 53 is fixedly connected to a blade holder 54 through a connecting shaft, and a flywheel block 55 is fixed below the blade holder 54. The dosing tube of the micro-injection pump 51 is provided with a constriction section inside, and the constriction section is located directly above the blade holder 54. The isotope liquid directly injected by the dosing module 50 is evenly dispersed into the cultured soil sample through several mesh holes on the plastic mesh plate 52, which can better simulate the characteristics of slow and multiple secretions from roots. The plastic mesh plate 52 is installed between the upper and lower parts of the culture container 10, allowing for simultaneous assembly and disassembly of the plastic mesh plate 52 when the culture container 10 is disassembled, thus improving operational convenience. The rotating nozzle 53 is rotatably connected to the dosing tube, and the blade holder 54 receives the liquid from the dosing tube. 13The pressure of the carbon isotope causes the blade holder 54 to drive the rotary nozzle 53 to rotate. This increases the spraying range through centrifugal force, effectively utilizing the driving force of the isotope liquid. No electric power source is required, reducing the cost and maintenance difficulty of the device. Furthermore, the flywheel block 55 stores the rotational force of the blade holder 54, allowing it to continue driving the rotary nozzle 53 to rotate even when the micro-injection pump 51 stops adding isotopes. This ensures that residual liquid within the rotary nozzle 53 is effectively discharged, preventing waste. The constricted nozzle further increases the pressure injected into the rotary nozzle 53 through the dosing pipe, thus ensuring the orderly rotation of the rotary nozzle 53 and creating a rotary spraying operation.

[0041] A culture method for studying the excitation effect using the alkali tracking method includes the following steps: S1. Weigh 50g of the soil sample to be cultured and spread it evenly on the lower part of the culture container 10, ensuring that the soil surface is flat and avoiding local accumulation; tighten the upper and lower parts of the culture container 10 with screws to ensure that the position of the plastic mesh plate 52 is stable and does not come into direct contact with the soil; seal the assembled main sealing plug 11 to the culture container 10; open the air inlet and outlet valves, confirm that the desorption unit 36 ​​in the air inlet module 30 is in standby mode, the low flow rate mass flow controller 35 is set to zero, and the isotope addition module 50 is in standby mode, thus completing the construction of the entire culture system; S2. Start the isotope dosing module 50 and set the flow rate of the small multi-channel micro-injection pump 51 to 0.05-0.5 ml / min. –1 ,Will 13 The C isotope labeled solution was slowly injected into the plastic mesh plate 52 in the center of the culture container 10. At the same time, the dispersion unit was turned on so that the labeled solution was evenly sprinkled into the soil surface through the holes, simulating the "small amount and multiple times" secretion characteristics of root exudates under natural conditions. According to the experimental design, the addition operation was repeated in time periods to avoid the local excitation effect caused by a single addition. S3. Place the entire culture system in a constant temperature environment of 22℃ to maintain a constant culture temperature; start the low flow rate mass flow controller 35, and combine it with the high frequency monitoring data of the online sensor group 34 to read the CO2 and O2 concentration in the culture bottle in real time, and automatically adjust the air intake flow rate to ensure that clean air is continuously introduced into the culture container 10 after being filtered by 0.2μm, CO2 removal and humidification treatment, and the main air intake pipe 31 extends below the soil surface to maintain sufficient oxygen and a stable gas phase environment in the bottle; S4. Replace the alkali collection bottle 20 according to the experimental design time gradient (e.g., day 1, day 2, day 4, day 8, day 16, day 32, day 64 of cultivation): Close the air inlet valve of the cultivation container 10 and the air outlet valve of the alkali collection bottle 20, disconnect the connection between the alkali collection bottle 20 and the main air outlet pipe 41, replace it with a new collection bottle 20 containing 15ml of 1M NaOH solution, reconnect it, open the valve, and continue cultivation; there is no need to open the cultivation container 10 during the replacement process to avoid disturbing the soil; S5. The replaced alkali solution collection bottle 20 was sent to the laboratory, where the carbon content absorbed in the NaOH solution was determined by acid titration, and the δ-carbon content was determined by isotope mass spectrometry (IRMS). 13 The C value, combined with the isotope balance formula, decomposes the total CO2 into substrate-derived CO2 and soil native organic matter (SOM-derived CO2) to quantify the excitation effect. After the culture is completed, the culture container 10 is opened and soil samples are taken out for subsequent determination of physicochemical properties such as carbon and nitrogen content, microbial community structure, and enzyme activity.

[0042] S6. Dilute the waste NaOH solution with distilled water, neutralize it with acid solution until neutral, and then discharge it in an environmentally friendly manner. Disassemble all components of the culture system, rinse the culture container 10, pipelines, lids, and isotope dosing system components with distilled water to remove residual soil and reagents, dry them, and store them properly for reuse in subsequent experiments. Compile the flow data from the low-flow-rate mass flow controller 35, the monitoring data from the CO2 / O2 sensor, the isotope dosing amount and time records, the alkali analysis data, and the soil physicochemical index data to form a complete experimental report, providing data support for the study of the evoked effect.

[0043] Finally, it should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture device for studying the excitation effect using the alkali tracking method, characterized in that, include: A culture container (10) containing soil, a collection bottle (20) containing alkali solution, an air inlet module (30), an air outlet module (40), and a dosing module (50). The dosing module (50) includes dosing into the culture container (10). 13 A micro-injection pump (51) for C isotopes, and via a dispersion unit, injects the solution into the culture container (10). 13 C isotopes are uniformly dispersed; The air intake module (30) includes an air intake manifold (31) for injecting clean air into the culture container (10), and the bottom end of the air intake manifold (31) is inserted into the soil. The air intake manifold (31) is provided with a desorption unit (36) for removing CO2 from the clean air source. The gas outlet module (40) includes a gas outlet manifold (41) that discharges the gas generated in the culture container (10) to the collection bottle (20) for CO2 collection, and one end of the gas outlet manifold (41) is inserted into the alkaline solution in the collection bottle (20).

2. The culture device for studying the excitation effect using the alkali tracking method according to claim 1, characterized in that: The culture container (10) is a wide-mouth plastic amber bottle, which is divided into upper and lower parts, and the two parts are installed in a detachable manner; The top of the culture container (10) is detachably fitted with a main sealing plug (11), which is used to seal the air inlet module (30), the air outlet module (40) and the dosing module (50) with the inside of the culture container (10).

3. The culture device for studying the excitation effect using the alkali tracking method according to claim 2, characterized in that: The intake end of the intake manifold (31) is equipped with a microporous filter (32) with a pore size of 0.2 μm and a humidifier (33). The humidifier (33) is used to humidify the injected clean air source. The desorption unit (36) is located between the microporous filter (32) and the humidifier (33).

4. The culture device for studying the excitation effect using the alkali tracking method according to claim 3, characterized in that: The intake manifold (31) is also equipped with a main switch valve, an online sensor group (34) and a flow controller (35). The online sensor group (34) includes a miniature optical CO2 sensor and an O2 sensor, and the online sensor group (34) is connected in series with the flow controller (35) for high-frequency monitoring of the air source and automatic adjustment of the air flow rate.

5. The culture device for studying the excitation effect using the alkali tracking method according to claim 1, characterized in that: The desorption unit (36) includes two desorption cylinders (361) installed on the intake manifold (31). The two desorption cylinders (361) are equipped with CO2 absorption columns for absorbing CO2 in the air source, and the two desorption cylinders (361) are connected to the intake manifold (31) in parallel. The intake manifold (31) is equipped with a control device for controlling the direction of air flow.

6. The culture device for studying the excitation effect using the alkali tracking method according to claim 5, characterized in that: The control unit includes a converter cylinder (362) that is fixedly connected to the intake manifold (31). Both sides of the converter cylinder (362) are connected to two desorption cylinders (361) through input connecting pipes, and the output ends of the two desorption cylinders (361) are reconnected to the intake manifold (31) through output connecting pipes. Both input connecting pipes and both output connecting pipes are telescopic hoses. The inside of the conversion cylinder (362) is rotatably connected to a valve plate (363) via a rotating shaft, and the valve plate (363) is set at a 45-degree angle. One end of the rotating shaft extends to the outside of the conversion cylinder (362) and is fixedly connected to a transmission bracket (364). The transmission bracket (364) is perpendicular to the valve plate (363). The bottom of the conversion cylinder (362) is rotatably connected to an elastic telescopic rod (365) via a bracket, and the telescopic end of the elastic telescopic rod (365) is hinged to one end of the transmission bracket (364). One end of the transmission bracket (364) is fixedly connected to a connecting rope (366), and the connecting rope (366) is detachably connected to one of the conversion cylinders (362).

7. The culture device for studying the excitation effect using the alkali tracking method according to claim 1, characterized in that: The top of the collection bottle (20) is detachably fitted with a secondary sealing plug (21), which is used to seal the connection between the gas outlet module (40) and the inside of the collection bottle (20). The main outlet pipe (41) is fixedly connected to the extraction pipe (42) by a bracket, and one end of the extraction pipe (42) is inserted into the collection bottle (20) through the secondary sealing plug (21), and the end is located above the liquid surface of the alkali solution. A secondary switch valve is installed on both the main outlet pipe (41) and the extraction pipe (42), and a micro diaphragm pump (43) is installed on the extraction pipe (42).

8. The culture device for studying the excitation effect using the alkali tracking method according to claim 2, characterized in that: The dispersion unit includes a plastic mesh plate (52) installed in the culture container (10), the plastic mesh plate (52) being used to uniformly integrate the isotopes added to the culture container (10) into the cultured soil sample; The plastic mesh (52) is installed between the upper and lower parts of the culture container (10) and is located above the culture soil.

9. A culture device for studying the excitation effect using the alkali tracking method according to claim 8, characterized in that: The dispersion unit also includes a rotary nozzle (53) that is rotatably installed in the dosing tube of the micro injection pump (51). The top of the rotary nozzle (53) is fixedly connected to a blade holder (54) via a connecting shaft, and a flywheel block (55) is fixed below the blade holder (54). The micro-injection pump (51) has a constricted section inside the injection tube, and the constricted section is located directly above the blade holder (54).

10. A cultivation method for studying the excitation effect using the alkali tracking method as described in any one of claims 1-9, characterized in that, Includes the following steps: S1. Weigh 50g of soil sample to be cultured and spread it evenly on the bottom of the culture container (10) to ensure that the soil surface is flat and avoid local accumulation. Tighten the upper and lower parts of the culture container (10) with screws to ensure that the position of the plastic mesh plate (52) is stable and does not come into direct contact with the soil. Seal the assembled main sealing plug (11) to the culture container (10). Open the air inlet and outlet valves, confirm that the desorption unit (36) in the air inlet module (30) is in standby mode, the low flow rate mass flow controller (35) is zeroed, and the isotope addition module (50) is in standby mode to complete the construction of the entire culture system. S2. Start the isotope dosing module (50), set the flow rate of the small multi-channel micro-injection pump (51) to 0.05-0.5 ml / min, and add... 13 The C isotope labeling solution was slowly injected into the plastic mesh plate (52) in the center of the culture container (10), and the dispersion unit was turned on at the same time to allow the labeling solution to be evenly sprinkled into the soil surface through the holes, simulating the "small amount and multiple times" secretion characteristics of root exudates under natural conditions; the addition operation was repeated in time periods according to the experimental design to avoid the local excitation effect caused by a one-time addition. S3. Place the entire culture system in a constant temperature environment of 22℃ to maintain a constant culture temperature; start the low flow rate mass flow controller (35), and combine the high frequency monitoring data of the online sensor group (34) to automatically adjust the air flow rate to ensure that clean air is continuously introduced into the culture container (10), and the main air inlet pipe (31) extends below the soil surface to maintain sufficient oxygen and a stable gas phase environment in the bottle; S4. Replace the alkaline solution collection bottle (20) according to the time gradient of the experimental design: Close the air inlet valve of the culture container (10) and the air outlet valve of the alkaline solution collection bottle (20), disconnect the alkaline solution collection bottle (20) from the main air outlet pipe (41), replace it with a new collection bottle (20) containing 15ml of 1M NaOH solution, reconnect it and open the valve to continue the culture; there is no need to open the culture container (10) during the replacement process to avoid disturbing the soil; S5. The replaced alkaline solution collection bottle (20) is sent to the laboratory, and the carbon content absorbed in the NaOH solution is determined by acid titration. The δ is measured by isotope mass spectrometry. 13 The C value, combined with the isotope balance formula, decomposes the total CO2 into substrate-derived CO2 and soil native organic matter-derived CO2 to quantify the excitation effect; after the culture is completed, open the culture container (10), take out the soil sample, and use it for subsequent determination of physicochemical properties such as carbon and nitrogen content, microbial community structure, and enzyme activity.

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