In-situ on-line analysis of rhizosphere oxygen fluctuation abiotic methane production device and method

Abstract

The application discloses a device and a method for in-situ online analysis of rhizosphere oxygen fluctuation abiotic methane production, which comprises an electrolytic cell composed of a working electrode chamber and a counter electrode chamber separated by a proton exchange membrane. Sterilized nutrient solution is arranged in the counter electrode chamber and the working electrode chamber. The working electrode chamber also contains an electron mediator. A working electrode and a reference electrode are arranged in the working electrode chamber, and a counter electrode is arranged in the counter electrode chamber. The liquid in the working chamber contains the roots of living aquatic plants, which are provided with an iron film and are subjected to sterilization treatment. The working electrode chamber is connected with a gas detection device working electrode chamber in sequence through a gas circulation pipeline, and the working electrode chamber is provided with airtightness. The device and the method are suitable for in-situ quantitative determination of rhizosphere abiotic aerobic methane production of aquatic plants, and can effectively analyze the amount of rhizosphere abiotic methane production in the normal growth process of the plant roots. The device is simple in structure, the method is simple, and the environmental requirements are low.

Description

Technical Field

[0001] This invention belongs to the field of environmental geochemistry, specifically relating to an apparatus and method for analyzing the abiotic generation of methane driven by redox fluctuations induced by the rhizosphere rhythmic oxygen secretion (ROL) of aquatic plants, which can provide technical support for the study of the iron-carbon cycle in the field of environmental geochemistry. Background Technology

[0002] Methane is a critical greenhouse gas, and changes in its atmospheric concentration have a significant impact on global climate change. Traditionally, it was believed that methane was produced only by methanogenic archaea in strictly anaerobic environments through the decomposition of organic matter. However, research over the past two decades has shown that various aerobic organisms (such as plants, fungi, and cyanobacteria) can also release methane under aerobic conditions. Simultaneously, reactive oxygen species (ROS) generated by abiotic processes such as photochemical and thermal reactions can also degrade organic matter and produce methane under aerobic conditions, further complicating global methane sources. Therefore, elucidating the mechanisms and contributions of abiotic methanogenesis under aerobic conditions is of significant scientific importance.

[0003] The rhizosphere of aquatic plants exhibits significant redox dynamics. Rhythmic root oxygen secretion (ROL) induces periodic redox fluctuations and generates ROS, potentially driving abiotic degradation of organic matter and producing methane. However, distinguishing between abiotic and microbial sources of rhizosphere methane in situ while maintaining normal root physiological activity remains a challenging technical problem. Existing methods often measure total methane through rhizosphere pore water or headspace gas, or involve ex-situ analysis after stripping and sterilizing rhizosphere soil. Both methods disrupt the true rhizosphere microenvironment and fail to reflect the in-situ processes at the oxygen fluctuation interface. Therefore, a novel technique is urgently needed to analyze and quantify abiotic methane production in situ under natural rhizosphere conditions. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an apparatus and method for in-situ online analysis of rhizosphere redox fluctuations-driven abiotic methane production. This apparatus and method are suitable for in-situ quantitative determination of abiotic aerobic methane production in the rhizosphere of aquatic plants, enabling accurate and effective analysis of the amount of abiotic methane produced in the rhizosphere during normal root growth.

[0005] The above-mentioned objective of the present invention is achieved as follows: An in-situ online device for analyzing rhizosphere oxygen fluctuations and abiotic methanogenesis includes an electrolytic cell, a gas circulation pipeline 13, and a gas detection device 12. The electrolytic cell includes a working electrode chamber 14 and a counter electrode chamber 15 separated by a proton exchange membrane. The counter electrode chamber 15 contains a sterile nutrient solution, and the working electrode chamber 14 contains an electrolyte, which is a sterile nutrient solution containing electron mediators. The counter electrode chamber 15 is equipped with a counter electrode 5, and the working electrode chamber 14 is equipped with a working electrode 4 and a reference electrode 6. The working electrolyte also contains the roots 2 of a live aquatic plant, the surface of which is covered with an iron film, and the roots have undergone surface sterilization treatment. The working electrode chamber 14 also has a circulating gas outlet 7 and a circulating gas inlet 8. The circulating gas outlet 7 is connected to the gas detection device 12 and the circulating gas inlet 8 in sequence through the gas circulation pipeline 13. The working electrode chamber 14 and the gas circulation pipeline 13 are airtight.

[0006] Preferably, the walls of the working electrode chamber 14 are opaque.

[0007] Preferably, the device further includes a plant planting box 1, in which the aquatic plant 16 is planted. A root outlet is provided on the wall of the plant planting box 1, which is connected to the working electrode chamber 14. The root 2 extends from the plant planting box 1 into the working electrode chamber 14 through the root outlet.

[0008] Preferably, a root connection port is provided on the side of the working electrode chamber, and the root outlet is connected to the root connection port through an opaque silicone tube 3. The root connection port is lower than the liquid level of the electrolyte or the planting liquid level of the aquatic plant. The airtightness of the root connection port is achieved by liquid sealing.

[0009] Preferably, the nutrient solution has the following composition: NH4NO3 1.43 mM, CaCl2 1.00 mM, NaH2PO4 0.32 mM, K2SO4 0.51 mM, MgSO4 1.64 mM, MnCl2 7.58 μM, H3BO3 15.11 μM, CuSO4 0.12 μM, (NH4)6Mo7O 24 0.06 μM, ZnSO4 0.12 μM, MOPS (pH = 7.0) 1 mM.

[0010] Preferably, the working electrode is a glassy carbon foam electrode, the counter electrode is a platinum mesh electrode, and the reference electrode is an Ag / AgCl electrode.

[0011] Preferably, the gas detection device is a membrane mass spectrometer.

[0012] Preferably, a pump is also provided in the gas circulation pipeline to drive the circulation of gas in the gas circulation pipeline and the gas detection device.

[0013] Preferably, the aquatic plant is Vallisneria natans, rice, reeds, or sedge.

[0014] Preferably, the electron mediator is ethyl viologen dibromide (EtV). The electron mediator can enhance the electron transfer efficiency between the electrode and the solid iron film.

[0015] The second objective of this invention is to provide a method for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis using the aforementioned device, comprising the following steps: (1) After surface sterilization treatment of the roots of live aquatic plants with iron film, the roots are introduced into the working electrode chamber to complete the setup of the device and the airtight setup of the working electrode chamber and the gas circulation pipeline. (2) Apply a natural or simulated circadian rhythm to the aquatic plant. At night, apply a reduction potential to the working electrode. During the day, turn off the reduction potential of the working electrode and start the gas detection device to detect methane.

[0016] At night, the reduction potential applied by the working electrode reduces EtV, thereby driving the conversion of ferric iron to ferrous iron in the root iron film. During the day, after the reduction potential is turned off, aquatic plants rely on sunlight for photosynthesis, causing the roots to secrete oxygen, which in turn oxidizes the ferrous iron in the iron film and produces reactive oxygen species (ROS). These ROS can further oxidize dissolved organic matter (NOM) on the iron film and induce methane formation. During the reaction, the methane and oxygen produced in the solution are sent to a gas detection device through a gas circulation pipeline for qualitative or quantitative analysis.

[0017] Preferably, the reduction potential is -0.45 V relative to a standard hydrogen electrode. The beneficial effects of this invention are: This invention is the first to couple electrochemical reduction with the plant's oxygen secretion and oxidation process, enabling the natural diurnal redox cycle in the rhizosphere, thereby achieving in-situ online measurement of rhizosphere abiotic methane production in a real rhizosphere environment.

[0018] The device and method of this invention are simple to set up and have low environmental requirements. It can be widely used for research on the non-biological contribution of methane sources in ecosystems such as wetlands, paddy fields, and lakeshores. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the device for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis as described in Embodiment 1 of the present invention. In the diagram, 1 is a plant growing box, 2 is a root, 3 is a silicone tube, 4 is a working electrode, 5 is a counter electrode, 6 is a reference electrode, 7 is a circulating gas outlet, 8 is a circulating gas inlet, 9 is a cold trap, 10 is a pump, 11 is the sample inlet of the gas detection device, and 12 is the gas detection device. 13 is a gas circulation pipeline, 14 is the working electrode chamber, 15 is the counter electrode chamber, and 16 is an aquatic plant.

[0020] Figure 2This is a schematic diagram illustrating the day and night working principle of the electrolytic cell in Embodiment 1 of the present invention.

[0021] Figure 3 This is a graph showing the daytime and nighttime rhizosphere dissolved methane and dissolved oxygen data measured by a membrane sample mass spectrometer in Example 1 of this invention.

[0022] Figure 4 This is a graph showing the diurnal variation of ferrous solubility in the electrolyte in Example 1 of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. The scope of protection of the present invention is not limited to the specific embodiments, but is defined by the claims.

[0024] Example 1 (1) The rhizosphere box is filled with air-dried and sieved soil, and aquatic plants (any one or more of Vallisneria natans, rice, reeds or sedges) are planted in it and kept submerged. Small holes are made in the side wall of the rhizosphere box, and silicone tubes are connected to the outside of the small holes to guide some of the plant roots with iron film to the working electrode chamber.

[0025] (2) The roots introduced into the working electrode chamber were surface sterilized with 70% ethanol and 1% sodium hypochlorite to eliminate microbial interference.

[0026] (3) The H-type electrolytic cell consists of a working electrode chamber and a counter electrode chamber, which are separated by a proton exchange membrane. The working electrode chamber is connected to the rhizosphere chamber through a silicone tube, allowing the root system with the iron film to extend into the working electrode chamber. The silicone tube and the working electrode chamber are wrapped with aluminum foil to protect them from light.

[0027] (4) The working electrode is a glassy carbon foam electrode, the counter electrode is a platinum mesh electrode, and the reference electrode is an Ag / AgCl electrode. The electrodes are connected to an electrochemical workstation to apply and control the potential.

[0028] (5) Add nutrient solution sterilized by high temperature and high pressure to the working electrode chamber, and add 200 μL of ethyl viologen dibromide (EtV) as an electron mediator.

[0029] The nutrient salt formula is as follows: NH4NO3 1.43 mM, CaCl2 1.00 mM, NaH2PO4 0.32 mM, K2SO4 0.51 mM, MgSO4 1.64 mM, MnCl2 7.58 μM, H3BO3 15.11 μM, CuSO4 0.12 μM, (NH4)6Mo7O 24 0.06 μM, ZnSO4 0.12 μM, MOPS (pH = 7.0) 1 mM.

[0030] (6) Electrochemical reduction stage (night): The working electrode potential is set to −0.45 V (relative to the standard hydrogen electrode). The reduction potential is applied under dark conditions to reduce EtV in the solution, thereby promoting the conversion of Fe(III) to Fe(II) in the root iron film.

[0031] (7) Light-driven oxidation stage (daytime): The applied potential is turned off, and the system is placed under natural light conditions. The plant releases oxygen into the rhizosphere through photosynthesis, which oxidizes Fe(II) in the iron film and produces reactive oxygen species (ROS).

[0032] (8) The generated reactive oxygen species further oxidize the dissolved organic matter (NOM) on the iron film, inducing the generation of non-biological methane.

[0033] (9) The gas generated in the working electrode chamber is continuously pumped into the membrane sample mass spectrometer by a peristaltic pump to quantitatively monitor methane and oxygen.

[0034] (10) Data on dissolved methane and dissolved oxygen produced in situ from the rhizosphere are as follows: Figure 3 As shown like Figure 3 As shown, during the reaction, the methane and oxygen generated in the solution are extracted online by a peristaltic pump and sent to a membrane mass spectrometer for quantitative analysis in real time.

[0035] like Figure 4 As shown, the changes in dissolved ferrous iron in the electrolyte during the day-night reaction process indicate that the reduction process at night did indeed cause a reduction reaction in the iron film, and that the oxygen secreted by the roots during the day did oxidize the dissolved ferrous iron.

Claims

1. A device for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis, characterized in that, It includes an electrolytic cell, a gas circulation pipeline (13), and a gas detection device (12). The electrolytic cell includes a working electrode chamber (14) and a counter electrode chamber (15) separated by a proton exchange membrane. The counter electrode chamber (15) contains a sterile nutrient solution, and the working electrode chamber (14) contains an electrolyte, which is a sterile nutrient solution containing an electron mediator. The counter electrode chamber (15) is provided with a counter electrode (5), and the working electrode chamber (14) is provided with a working electrode (4) and a reference electrode (6). The working electrolyte also contains the roots (2) of a live aquatic plant (16), the surface of which is covered with an iron film, and the roots have undergone surface sterilization treatment. The working electrode chamber (14) also has a circulating gas outlet (7) and a circulating gas inlet (8). The circulating gas outlet (7) is connected to the gas detection device (12) and the circulating gas inlet (8) in sequence through the gas circulation pipeline (13). The working electrode chamber (14) and the gas circulation pipeline (13) are airtight.

2. The apparatus for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis according to claim 1, characterized in that, The walls of the working electrode chamber (14) are opaque.

3. The apparatus for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis according to claim 1, characterized in that, The device also includes a plant planting box (1), in which the aquatic plant (16) is planted. A root outlet is provided on the wall of the plant planting box (1), which is connected to the working electrode chamber (14). The root (2) extends from the plant planting box (1) into the working electrode chamber (14) through the root outlet.

4. The apparatus for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis according to claim 3, characterized in that, The working electrode chamber is provided with a root connection port on its side. The root outlet is connected to the root connection port through an opaque silicone tube (3). The root connection port is lower than the liquid surface of the electrolyte or the planting liquid surface of the aquatic plant.

5. The apparatus for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis according to claim 1, characterized in that, The nutrient solution consists of 1.43 mM NH4NO3, 1.00 mM CaCl2, 0.32 mM NaH2PO4, 0.51 mM K2SO4, 1.64 mM MgSO4, 7.58 μM MnCl2, 15.11 μM H3BO3, 0.12 μM CuSO4, and (NH4)6Mo7O 24 0.06 μM, ZnSO4 0.12 μM, MOPS 1 mM at pH = 7.

0.

6. The apparatus for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis according to claim 1, characterized in that, The working electrode is a glassy carbon foam electrode, the counter electrode is a platinum mesh electrode, and the reference electrode is an Ag / AgCl electrode.

7. The apparatus for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis according to claim 1, characterized in that, The gas detection device (12) is a membrane mass spectrometer.

8. The apparatus for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis according to claim 1, characterized in that, The aquatic plants (16) are Vallisneria natans, rice, reeds or sedges.

9. The apparatus for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis according to claim 1, characterized in that, The electronic mediator is ethyl viologen dibromide.

10. A method for in-situ online analysis of rhizosphere oxygen fluctuations and abiotic methanogenesis using the apparatus described in any one of claims 1-9, characterized in that, Includes the following steps: (1) After surface sterilization treatment of the roots of live aquatic plants with iron film, the roots are introduced into the working electrode chamber to complete the setup of the device and the airtight setup of the working electrode chamber and the gas circulation pipeline. (2) Apply a natural or simulated circadian rhythm to the aquatic plant. At night, apply a reduction potential to the working electrode. During the day, turn off the reduction potential of the working electrode and start the gas detection device to detect methane.

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