A culture device capable of monitoring activity of methanogens in situ in real time and a monitoring method thereof

Abstract

This invention discloses a culture device and method for in-situ real-time monitoring of methanogenic bacteria activity, relating to the field of hydrogen-consuming microbial culture technology. The device comprises a cap and container body connected in an airtight manner to form a pressure-resistant, sealed anaerobic culture chamber; a methane monitoring component continuously detects the concentration of gaseous methane; a temperature control component and a pressure monitoring component are used to regulate temperature and monitor pressure; an integrated gas injection and sampling component is used to adjust the gas composition and obtain gaseous samples; a liquid phase sampling component utilizes dual airtight valves and a quantitative buffer chamber to achieve isolation, depressurization, and aseptic sampling of high-pressure liquid phase samples; and a data acquisition and processing component combines gas phase monitoring data and liquid phase detection results to evaluate methanogenic bacteria activity. This invention can simulate an underground hydrogen storage environment, enabling in-situ continuous monitoring of the methanogenesis process and low-disturbance gas-liquid phase sampling, improving the real-time performance and accuracy of the evaluation.

Description

Technical Field

[0001] This invention relates to the field of hydrogen-consuming microbial culture technology, and in particular to a culture device and monitoring method for in-situ real-time monitoring of methanogenic bacteria activity; applicable to microbial simulation culture of underground hydrogen storage reservoirs, evaluation of methanogenic bacteria activity, hydrogen consumption risk research, screening of culture conditions, and related anaerobic microbial experiments. Background Technology

[0002] Hydrogen energy is low-carbon, highly efficient, and has broad application prospects, making it an important carrier of my country's new energy system. Geological bodies such as underground salt caverns and depleted oil and gas reservoirs possess large reserves and excellent peak-shaving capabilities, making them core scenarios for large-scale, long-term hydrogen storage. However, underground hydrogen storage environments are characterized by high pressure, anaerobic conditions, and constant temperature. Methanogenic bacteria within the reservoir can utilize hydrogen and carbon dioxide to metabolize and produce methane, easily leading to hydrogen loss, decreased gas purity, and alterations in reservoir pressure and gas composition. This severely impacts the operational safety and stability of hydrogen storage facilities, necessitating precise and continuous monitoring of methanogenic bacteria activity.

[0003] Existing detection technologies have significant drawbacks. Offline gas chromatography suffers from data lag, and methods like qPCR require opening the container for sampling, which can disrupt the anaerobic environment. Furthermore, conventional culture devices cannot accurately simulate the complex environment of underground hydrogen storage, and various parameters cannot generate continuous time-series data, making it difficult to distinguish the causes of methane increments. High-pressure sampling also easily disturbs the culture system and can clog pipelines. Therefore, this invention proposes a dedicated culture device that can simulate underground hydrogen storage conditions, enabling in-situ monitoring of methane generation and safe, closed-loop sampling, and joint evaluation of gas-liquid phase indicators. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of insufficient real-time methane monitoring, disruption of anaerobic conditions by opening the lid for sampling, risk of ejection and depressurization by high-pressure liquid phase sampling, inability to comprehensively evaluate methanogenic bacteria activity by a single gas phase index, and insufficient ability to simulate underground hydrogen storage environments in the prior art. The invention provides a culture device and monitoring method for in-situ real-time monitoring of methanogenic bacteria activity.

[0005] This invention integrates a methane monitoring component, a constant temperature control component, a pressure monitoring component, a gas injection and sampling integrated component, a data acquisition and processing component, and a liquid phase sampling component into a pressure-resistant, sealed anaerobic culture chamber. This enables the establishment of a culture environment, continuous detection of gaseous methane, gas replenishment and sampling, low-disturbance acquisition of liquid phase samples, and joint evaluation of gas and liquid phase data.

[0006] Its technical solution is:

[0007] A culture device for in-situ real-time monitoring of methanogenic bacteria activity includes an anaerobic culture container body, an airtight sealing cover, a methane monitoring component, a constant temperature control component, a pressure monitoring component, an integrated gas injection and sampling component, a data acquisition and processing component, and a liquid phase sampling component.

[0008] The anaerobic culture container body is a pressure-resistant container structure. The airtight sealing cover is detachably set at the opening end of the anaerobic culture container body and is airtightly fitted with the anaerobic culture container body to form a pressure-resistant and airtight anaerobic culture chamber inside the anaerobic culture container body for containing culture medium, methanogenic bacteria samples, simulated strata samples and gas phase space.

[0009] The methane monitoring component includes at least one set of miniature methane sensors. The miniature methane sensors are airtightly installed on the body of the anaerobic culture container or the airtight sealed cover through a sealing joint. The detection probe of the miniature methane sensor extends into the gas phase space of the pressure-resistant and airtight anaerobic culture chamber to continuously detect the methane concentration without opening the airtight sealed cover.

[0010] The thermostatic control component is located on the outer periphery or bottom of the anaerobic culture container body to regulate and maintain the culture temperature in the pressure-resistant and sealed anaerobic culture chamber;

[0011] The pressure monitoring component is installed on the airtight sealed cover or the body of the anaerobic culture container through a sealed interface and is connected to the pressure-resistant sealed anaerobic culture chamber for real-time monitoring of the pressure inside the pressure-resistant sealed anaerobic culture chamber.

[0012] The integrated gas injection and sampling assembly is airtightly installed on the airtight sealing cover and connected to the pressure-resistant sealed anaerobic culture chamber. The integrated gas injection and sampling assembly includes a gas injection branch, a gas phase sampling branch, and independent airtight valve cores that control the on / off of the gas injection branch and the gas phase sampling branch respectively.

[0013] The liquid phase sampling component is airtightly installed on the airtight sealed cover or the body of the anaerobic culture container through a sealed structure. The liquid inlet end of the liquid phase sampling component extends into the liquid phase area of ​​the pressure-resistant sealed anaerobic culture chamber, which is used to obtain liquid phase samples without opening the airtight sealed cover.

[0014] The data acquisition and processing component is connected to the miniature methane sensor, the temperature sensor in the thermostat component, and the pressure monitoring component to synchronously acquire data on methane concentration, culture temperature, and culture pressure, and to obtain the evaluation results of methanogenic bacteria activity based on the change of methane concentration over time.

[0015] Furthermore, a sealing gasket is provided between the airtight sealing cover and the anaerobic culture container body, and a compression seal is achieved through threaded connection, snap-fit ​​connection, flange bolt connection or a combination thereof.

[0016] Furthermore, the anaerobic culture container body is made of 316L stainless steel, pressure-resistant transparent polycarbonate, or pressure-resistant glass; when the anaerobic culture container body is made of opaque pressure-resistant material, a pressure-resistant transparent observation window is provided on the anaerobic culture container body; the rated pressure resistance of the pressure-resistant sealed anaerobic culture chamber is not less than 10 MPa.

[0017] Furthermore, the miniature methane sensor is an infrared absorption methane sensor, a semiconductor methane sensor, or an electrochemical methane sensor; the outer side of the detection probe is provided with a sensor protective cover with gas diffusion holes, and the gas inlet end of the detection probe is provided with a hydrophobic and breathable membrane or an anti-condensation structure.

[0018] Furthermore, the constant temperature control component includes at least one of annular heating jacket, flexible heating film, constant temperature water bath jacket, and semiconductor heating and cooling component, as well as a temperature control unit and a temperature sensor that are matched thereto; the detection end of the temperature sensor extends into the pressure-resistant sealed anaerobic culture chamber or is attached to the outer wall of the anaerobic culture container body.

[0019] Furthermore, the pressure monitoring component is a digital pressure gauge, pressure sensor, or pressure transmitter; the airtight sealing cover or the anaerobic culture container body is also equipped with a safety pressure relief component that communicates with the pressure-resistant sealed anaerobic culture chamber. The safety pressure relief component is used to release pressure when the pressure in the pressure-resistant sealed anaerobic culture chamber exceeds a preset safety threshold.

[0020] Furthermore, the gas injection branch is used to introduce nitrogen, argon, hydrogen, carbon dioxide, methane, or a mixture thereof into the pressure-resistant sealed anaerobic culture chamber to establish an anaerobic environment and regulate the gas composition and pressure within the pressure-resistant sealed anaerobic culture chamber; the gas phase sampling branch is used to extract gas samples without opening the airtight sealed cover and is connected to a gas bag, syringe, gas chromatograph injection device, or gas analysis equipment.

[0021] Furthermore, the liquid phase sampling assembly includes a sampling tube, a first airtight valve, a quantitative buffer chamber, a second airtight valve, and a sterile sampling interface;

[0022] The inlet end of the sampling tube extends into the liquid phase region of the pressure-resistant, sealed anaerobic culture chamber, and the outlet end of the sampling tube is connected to the quantitative buffer chamber through the first airtight valve. The quantitative buffer chamber is connected to the sterile sampling interface through the second airtight valve.

[0023] The effective volume of the quantitative buffer chamber is 1 to 20 mL, and the quantitative buffer chamber is equipped with a scale or liquid level observation structure for displaying the volume of the liquid sample entering the quantitative buffer chamber.

[0024] The quantitative buffer chamber is connected to a pressure regulating branch, which is used to introduce inert gas into the quantitative buffer chamber, slowly depressurize the quantitative buffer chamber, or flush the liquid phase sampling component; the pressure regulating branch is equipped with at least one of a pressure regulating valve, a needle valve, a check valve, or a filter.

[0025] The liquid phase sampling assembly also includes a pressure balance branch, which is connected to the gas injection branch or an external compensation gas source. This branch is used to replenish inert gas, hydrogen, carbon dioxide, or a mixture of gases into the pressure-resistant, sealed anaerobic culture chamber after liquid phase sampling to compensate for the pressure changes caused by the liquid phase sampling.

[0026] The liquid inlet of the sampling tube is equipped with a detachable anti-clogging cover or a coarse filter structure; the sampling tube is a telescopic sampling tube with adjustable sampling depth, or the liquid phase sampling assembly includes multiple sampling tubes that extend to different liquid phase heights.

[0027] Furthermore, the bottom of the anaerobic culture container is provided with a detachable sample carrying area, which is used to carry at least one of the following: culture medium, formation water, core particles, mineral particles, and reservoir sediments.

[0028] Furthermore, the data acquisition and processing components include a signal acquisition module, a data processing module, a data storage module, and a wireless transmission module;

[0029] The signal acquisition module is electrically connected to the miniature methane sensor, temperature sensor, and pressure monitoring component, respectively.

[0030] The data processing module is used to generate time-series data on methane concentration, culture temperature and culture pressure, and to calculate methane production rate, methane production per unit time or relative methane production activity index.

[0031] The wireless transmission module is used to transmit monitoring data and processing results to the terminal device.

[0032] The data acquisition and processing component is also used to record the liquid phase sampling time, liquid phase sampling volume, type and amount of compensation gas, and to receive external detection results of the liquid phase sample; the data processing module is used to time-match the gas phase methane generation data with the concentration, metabolic activity or proliferation data of methanogens in the liquid phase sample to obtain the overall activity of methanogens, methanogen production activity per unit cell or risk assessment results of the culture system.

[0033] The data processing module is used to calculate the amount of gaseous methane based on the effective gas volume, absolute pressure, culture temperature, and methane volume fraction of the pressure-resistant sealed anaerobic culture chamber, and to correct the amount of gaseous methane based on the gas injection volume, gas phase sampling volume, volume change caused by liquid phase sampling, and blank control data.

[0034] The data acquisition and processing component also includes an alarm module and a controller. When the methane concentration, methane generation rate, culture temperature, or culture pressure exceeds a preset threshold, the alarm module issues an alarm signal, and / or the controller controls the gas injection branch to stop gas supply and controls the safety pressure relief component to release pressure.

[0035] Furthermore, the cultivation device includes multiple parallel and independent pressure-resistant sealed anaerobic culture chambers, each of which is equipped with a methane monitoring component, a pressure monitoring component, an integrated gas injection and sampling component, and a liquid phase sampling component.

[0036] The monitoring method for this culture device capable of in-situ real-time monitoring of methanogenic bacteria activity includes the following steps:

[0037] Culture medium, methanogenic bacteria samples, and simulated formation samples were added to the anaerobic culture container under anaerobic conditions, and the liquid volume and effective gas volume were recorded.

[0038] The airtight sealing cover is airtightly connected to the anaerobic culture container body to form a pressure-resistant and airtight anaerobic culture chamber.

[0039] Inert gas is introduced into the pressure-resistant sealed anaerobic culture chamber through the gas injection branch to replace the residual oxygen, and then the target gas containing hydrogen and carbon dioxide is introduced, and the pressure in the pressure-resistant sealed anaerobic culture chamber is adjusted to the preset value.

[0040] The culture temperature is maintained within a preset range by a constant temperature control component, and the culture pressure is continuously monitored by a pressure monitoring component.

[0041] The methane concentration in the gas phase space of the pressure-resistant, sealed anaerobic culture chamber is continuously detected by a miniature methane sensor, and the methane concentration, culture temperature and culture pressure data are collected simultaneously by the data acquisition and processing component.

[0042] A predetermined volume of liquid phase sample is collected using a liquid phase sampling component at a preset incubation time point, and the changes in incubation pressure caused by liquid phase sampling are compensated.

[0043] Analyze liquid samples to obtain data on methanogen concentration, metabolic activity, or proliferation.

[0044] The amount and rate of methane production are calculated based on methane concentration, culture pressure, culture temperature, and effective gas phase volume. The amount or rate of methane production is then time-matched with the detection results of liquid phase samples to obtain the evaluation results of methanogenic bacteria activity.

[0045] The simulated formation sample includes at least one of formation water, core particles, mineral particles, and reservoir sediments; the inert gas is nitrogen or argon.

[0046] Liquid phase samples are collected using liquid phase sampling components, including:

[0047] Keep the second airtight valve closed and open the first airtight valve to allow the liquid sample to enter the quantitative buffer chamber under the pressure inside the pressure-resistant and airtight anaerobic culture chamber.

[0048] Once the liquid sample entering the quantitative buffer chamber reaches the predetermined volume, the first airtight valve is closed to isolate the quantitative buffer chamber from the pressure-resistant, airtight anaerobic culture chamber.

[0049] The pressure in the quantitative buffer chamber is slowly reduced by the pressure regulating branch;

[0050] Connect the sterile sample container to the sterile sampling interface, open the second airtight valve and output the liquid sample;

[0051] The target gas is supplied to the pressure-resistant, sealed anaerobic culture chamber through the pressure balancing branch, so that the culture pressure is restored to the preset range.

[0052] The detection of liquid samples includes at least one of the following: microscopic counting, flow cytometry, qPCR, RT-qPCR, ATP detection, coenzyme F420 fluorescence detection, live / dead staining, metabolite analysis, and detection of physicochemical parameters of the culture medium.

[0053] The data acquisition and processing component calculates the amount of gaseous methane using the following formula:

[0054] in, This represents the amount of gaseous methane. This represents the volume fraction of methane. The absolute pressure of the culture chamber; This represents the effective volume of the gas phase. It is the gas compressibility factor; It is the gas constant; This refers to absolute temperature.

[0055] The data acquisition and processing component calculates the methane generation rate based on the difference in the amount of gaseous methane at two adjacent detection time points, and calculates the methane production rate per unit cell based on the number of methanogenic bacteria cells obtained from the detection of liquid phase samples at the corresponding time points.

[0056] During the monitoring process, gas samples are extracted through the gas phase sampling branch and verified using gas chromatography or external gas analysis equipment. The gas-tight seal is kept closed during the gas sample extraction process.

[0057] The terminal device can be a computer, tablet computer, mobile terminal, or laboratory monitoring platform. The data acquisition and processing component may also include an alarm module. When the methane concentration, methane generation rate, culture temperature, or culture pressure exceeds a preset threshold, the alarm module issues an alarm signal, or controls the gas injection branch to stop gas supply and controls the safety pressure relief component to perform pressure relief.

[0058] The advantages of this invention compared to the prior art are as follows:

[0059] This invention incorporates a methane monitoring component within a pressure-resistant, airtight anaerobic culture chamber, enabling continuous acquisition of methane concentration change data without opening the airtight lid. This avoids data lag and anaerobic environment disruption caused by repeated opening of the lid and offline monitoring.

[0060] This invention, through structures such as constant temperature control, pressure monitoring, target gas injection, and safety pressure relief, can simulate the constant temperature, high pressure, and specific gas composition conditions of underground hydrogen storage strata, thereby improving the scenario relevance of methanogenic bacteria cultivation and hydrogen consumption risk assessment.

[0061] The liquid phase sampling assembly adopts a hierarchical isolation structure consisting of a liquid sampling tube, a first airtight valve, a quantitative buffer chamber, a second airtight valve, and a sterile sampling interface. This allows the high-pressure culture medium to first enter the quantitative buffer chamber and then be slowly depressurized after being isolated from the culture chamber, which can reduce the risks of liquid spraying, sudden depressurization of the culture chamber, and backflow of external air.

[0062] Compensating for pressure changes caused by liquid phase sampling by using a pressure balancing branch or a gas injection branch can reduce the disturbance of sampling to the culture chamber pressure, gas-liquid ratio, and gas composition, which is beneficial for continuous culture and comparison of data at different time points.

[0063] The liquid inlet end of the sampling tube is equipped with an anti-clogging cover or a coarse filter structure, which can adapt to culture systems containing rock fragments, mineral particles or biofilms; the adjustable sampling depth or multi-height sampling structure can obtain liquid phase samples from different locations, improving the representativeness of liquid phase detection.

[0064] This invention can jointly analyze the dynamics of gas-phase methane generation with the concentration, metabolic activity, and proliferation of methanogens in the liquid phase, which helps to distinguish between changes in cell number and changes in metabolic capacity per cell, thereby improving the accuracy and interpretability of methanogen activity evaluation.

[0065] The device has a compact structure and a high degree of modularity. It can conduct parallel experiments under different temperature, pressure, gas ratio, reservoir sample or inhibitor conditions by connecting multiple culture chambers in parallel, thereby improving experimental efficiency and reducing systematic errors. Attached Figure Description

[0066] Figure 1 This is a schematic diagram of the overall structure of a culture device for in-situ real-time monitoring of methanogenic bacteria activity according to the present invention.

[0067] Figure 2 This is a schematic diagram of the connection structure between the airtight sealing cover and the anaerobic culture container body in this invention.

[0068] Figure 3 This is a schematic diagram showing the arrangement of the methane monitoring component, pressure monitoring component, and integrated gas injection and sampling component in this invention.

[0069] Figure 4This is a schematic diagram of the liquid phase sampling component in this invention.

[0070] Figure 5 This is a schematic diagram of the module connections of the data acquisition and processing component in this invention.

[0071] Figure 6 This is a schematic diagram of the workflow of the present invention for monitoring the activity of methanogens.

[0072] The reference numerals in the attached drawings are explained as follows: 1. Anaerobic culture container body; 2. Airtight sealing cover; 3. Sealing gasket; 4. Pressure-resistant sealed anaerobic culture chamber; 5. Miniature methane sensor; 6. Sealing joint; 7. Detection probe; 8. Thermostatic control component; 9. Temperature sensor; 10. Pressure monitoring component; 11. Integrated gas injection and sampling component; 12. Gas injection branch; 13. Gas phase sampling branch; 14. Independent airtight valve core; 15. Signal acquisition module; 16. Wireless transmission module; 17. Terminal equipment; 18. Safety pressure relief component; 19. Sample carrying area; 20. Observation window; 21. Liquid phase sampling component; 22. Liquid sampling tube; 23. First airtight valve; 24. Quantitative buffer chamber; 25. Second airtight valve; 26. Aseptic sampling interface; 27. Pressure regulation branch; 28. Anti-clogging cover; 29. ​​Pressure balancing branch; 30. Data processing module; 31. Data storage module; 32. Controller. Detailed Implementation

[0073] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the described embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the embodiments and technical features in the embodiments of the present invention can be combined with each other.

[0074] Example 1: Overall Structure of the Cultivation Device

[0075] like Figures 1 to 3 As shown, this embodiment provides a culture device for in-situ real-time monitoring of methanogenic bacteria activity, including an anaerobic culture container body 1, an airtight sealed cover 2, a methane monitoring component, a constant temperature control component 8, a pressure monitoring component 10, an integrated gas injection and sampling component 11, a data acquisition and processing component, and a liquid phase sampling component 21.

[0076] The anaerobic culture container body 1 is made of 316L stainless steel, and its interior forms a pressure-resistant, sealed anaerobic culture chamber 4 with a rated working pressure of 0–10 MPa. A pressure-resistant transparent observation window 20 is provided on the front of the anaerobic culture container body 1, and a removable sample carrying area 19 is provided at the bottom. The sample carrying area 19 is used to place culture medium, formation water, core particles, mineral particles, or reservoir sediments.

[0077] The airtight sealing cover 2 is connected to the anaerobic culture container body 1 via flange bolts, with a fluororubber sealing gasket 3 placed between them. After tightening the flange bolts, the airtight sealing cover 2 presses against the sealing gasket 3 to form a pressure-resistant, airtight anaerobic culture chamber 4. Depending on the size of the device and the pressure rating, threaded connections, snap-fit ​​connections, or a combination of threaded and snap-fit ​​connections can also be used.

[0078] A miniature methane sensor 5 is mounted on an airtight sealed cover 2 via a sealing connector 6, and a detection probe 7 extends into the gas phase space of a pressure-resistant, sealed anaerobic culture chamber 4. A protective cover with gas diffusion holes is provided on the outside of the detection probe 7, and a hydrophobic and breathable membrane is provided at the gas inlet end. The miniature methane sensor 5 outputs a methane concentration signal at a preset sampling period.

[0079] The thermostatic control component 8 uses a ring-shaped flexible heating sleeve, which is fitted around the outer periphery of the anaerobic culture container body 1. The detection end of the temperature sensor 9 extends into the culture medium, and the temperature control unit adjusts the heating power according to the feedback signal from the temperature sensor 9 to maintain the culture temperature within the preset range.

[0080] The pressure monitoring component 10 uses a pressure transmitter and is connected to the pressure-resistant, sealed anaerobic culture chamber 4 through a sealed interface. The safety pressure relief component 18 is installed on the airtight sealing cover 2 and automatically releases pressure when the pressure in the culture chamber exceeds a preset threshold.

[0081] The integrated gas injection and sampling assembly 11 is mounted on the airtight sealing cover 2 and includes a gas injection branch 12, a gas phase sampling branch 13, and an independent airtight valve core 14. The gas injection branch 12 is connected to an external high-pressure gas source or a gas mixing device, and the gas phase sampling branch 13 is connected to a gas bag, syringe, or gas chromatography injection device.

[0082] Example 2: High-Pressure Anaerobic Liquid Phase Sampling Component

[0083] like Figure 4 As shown, the liquid phase sampling assembly 21 is installed on the airtight sealing cover 2 and includes a liquid sampling tube 22, a first airtight valve 23, a quantitative buffer chamber 24, a second airtight valve 25, a sterile sampling interface 26, a pressure regulating branch 27, an anti-clogging cover 28, and a pressure balancing branch 29.

[0084] The lower end of the sampling tube 22 extends into the liquid phase region of the pressure-resistant, sealed anaerobic culture chamber 4, preferably below the culture medium surface and avoiding the sample carrying area 19. The upper end of the sampling tube 22 is connected to the quantitative buffer chamber 24 via the first airtight valve 23. The effective volume of the quantitative buffer chamber 24 can be 1–20 mL, and it is equipped with an external scale or liquid level observation structure to control the volume of a single sampling.

[0085] The quantitative buffer chamber 24 is connected to the sterile sampling interface 26 via the second airtight valve 25. The sterile sampling interface 26 can be a Luer connector, a sterile diaphragm interface, or a quick connector, used to connect sterile sample tubes, syringes, or external liquid chromatography devices.

[0086] The pressure regulating branch 27 is connected to the quantitative buffer chamber 24, and is equipped with a needle valve, a pressure regulating valve, and a sterile filter. Before sampling, nitrogen or argon gas can be introduced into the quantitative buffer chamber 24 through the pressure regulating branch 27 to replace the residual air; after sampling, the pressure in the quantitative buffer chamber 24 can be slowly released through the pressure regulating branch 27.

[0087] The anti-clogging cover 28 is detachably installed at the inlet end of the liquid collection tube 22. The anti-clogging cover 28 has multiple through holes that allow the culture medium and methanogens to pass through. The diameter of the through holes is larger than the typical cell size of methanogens and smaller than the size of rock chips or mineral particles that can easily cause blockages in the tube.

[0088] The pressure balancing branch 29 is connected to the gas injection branch 12. Based on the pressure change fed back by the pressure monitoring component 10, the controller 32 replenishes a predetermined amount of target gas to the pressure-resistant sealed anaerobic culture chamber 4 through the pressure balancing branch 29 after liquid phase sampling, so that the pressure of the culture chamber is restored to the preset range.

[0089] The liquid phase sampling operation in this embodiment includes: closing the second airtight valve 25 and opening the first airtight valve 23, allowing the culture medium to enter the quantitative buffer chamber 24 under the pressure of the culture chamber; closing the first airtight valve 23 after reaching a predetermined volume; slowly reducing the pressure of the quantitative buffer chamber 24 to a safe range through the pressure regulating branch 27; connecting the sterile sample container to the sterile sampling interface 26 and opening the second airtight valve 25 to discharge the liquid phase sample; closing the second airtight valve 25 and compensating the culture chamber pressure through the pressure balancing branch 29 according to the pressure monitoring results. In other embodiments, the sampling tube can be set as a telescopic sampling tube with adjustable sampling depth, or multiple sampling tubes can be set to extend to different liquid phase heights to obtain samples at different liquid phase positions. After the liquid phase sampling is completed, the target gas can be replenished to the pressure-resistant sealed anaerobic culture chamber through the pressure balancing branch to compensate for the pressure change caused by the liquid phase sampling.

[0090] Example 3: In-situ monitoring method for methanogenic bacteria activity

[0091] like Figure 5 and Figure 6 As shown, the method for in-situ monitoring of methanogenic bacteria activity using the above-mentioned culture device includes the following steps:

[0092] S1. Clean and sterilize the anaerobic culture container body 1, the airtight sealing cover 2, the sealing gasket 3, the sample carrying area 19, the liquid collection tube 22, and the parts that come into contact with the culture medium.

[0093] S2. Under anaerobic conditions, add culture medium, methanogenic bacteria sample and simulated formation sample into the anaerobic culture container body 1, and record the liquid phase volume and effective gas phase volume.

[0094] S3. Connect the airtight sealing cover 2 to the anaerobic culture container body 1 in an airtight manner to form a pressure-resistant and airtight anaerobic culture chamber 4; introduce nitrogen or argon through the gas injection branch 12 to replace the residual oxygen.

[0095] S4. Introduce the target gas containing hydrogen and carbon dioxide through the gas injection branch 12, and adjust the pressure of the culture chamber to a preset value to simulate the gas composition and pressure conditions of the underground hydrogen storage strata.

[0096] S5. Turn on the constant temperature control component 8 to maintain the culture temperature within the preset range based on the feedback signal from the temperature sensor 9; turn on the pressure monitoring component 10 to continuously record the pressure in the culture chamber.

[0097] S6. Turn on the miniature methane sensor 5, and the detection probe 7 continuously detects the methane concentration in the gas phase space; the signal acquisition module 15 simultaneously acquires the methane concentration, culture temperature and culture pressure.

[0098] S7. At the preset incubation time point, a predetermined volume of liquid phase sample is collected through the liquid phase sampling component 21, and the pressure change caused by sampling is compensated through the pressure balance branch 29.

[0099] S8. Perform at least one of the following on the liquid sample: microscopic counting, flow cytometry, qPCR, RT-qPCR, ATP detection, coenzyme F420 fluorescence detection, live / dead staining, metabolite analysis, or physicochemical parameter detection of the culture medium, to obtain the concentration, activity, or proliferation status of methanogens.

[0100] S9, Data Processing Module 30 calculates the amount and rate of methane production based on methane concentration, pressure, temperature and effective gas phase volume, and calculates the unit cell methane production activity or comprehensive activity evaluation result in combination with liquid phase detection results.

[0101] S10 and terminal equipment 17 display methane concentration curve, pressure curve, temperature curve, methanogen concentration change curve and activity evaluation results; when offline verification is required, gas samples are extracted through gas sampling branch 13 for gas chromatography analysis.

[0102] In one specific calculation method, the data processing module 30 calculates the methane generation rate based on the difference in the amount of gaseous methane at two adjacent time points, and calculates the methane production rate per unit cell based on the number of methanogenic bacterial cells obtained from liquid phase detection at the corresponding time point. This method allows for the differentiation between the contribution of increased bacterial count and enhanced single-cell metabolism to the overall methane generation.

[0103] The beneficial effects of this invention are as follows: The culture device provided by this invention can simulate the underground hydrogen storage stratum environment under pressure-resistant, sealed anaerobic conditions, continuously monitor methane concentration, culture temperature, and culture pressure, and safely obtain liquid phase samples through a liquid phase sampling component with a quantitative buffer chamber and a double airtight valve structure. This device can be used for risk assessment of underground hydrogen storage microorganisms, screening of methanogenic bacteria culture conditions, evaluation of inhibitor effects, comparison of reservoir samples, and related anaerobic microorganism research, and has clear industrial applicability.

Claims

1. A culture device for in-situ real-time monitoring of methanogenic bacteria activity, characterized in that, It includes an anaerobic culture container body (1), an airtight sealing cover (2), a methane monitoring component, a constant temperature control component (8), a pressure monitoring component (10), an integrated gas injection and sampling component (11), a data acquisition and processing component, and a liquid phase sampling component (21). The anaerobic culture container body (1) is a pressure-resistant container structure. The airtight sealing cover (2) is detachably set at the opening end of the anaerobic culture container body (1) and airtightly fits with the anaerobic culture container body (1) to form a pressure-resistant and airtight anaerobic culture chamber (4) inside the anaerobic culture container body (1) for containing culture medium, methanogenic bacteria samples, simulated strata samples and gas phase space. The methane monitoring component includes at least one set of miniature methane sensors (5). The miniature methane sensors (5) are airtightly installed on the anaerobic culture container body (1) or the airtight sealed cover (2) through a sealing joint (6). The detection probe (7) of the miniature methane sensor (5) extends into the gas phase space of the pressure-resistant sealed anaerobic culture chamber (4) to continuously detect the methane concentration without opening the airtight sealed cover (2). The thermostat (8) is located on the outer periphery or bottom of the anaerobic culture container body (1) to regulate and maintain the culture temperature in the pressure-resistant and sealed anaerobic culture chamber (4); The pressure monitoring component (10) is installed on the airtight sealing cover (2) or the anaerobic culture container body (1) through a sealed interface and is connected to the pressure-resistant sealed anaerobic culture chamber (4) for real-time monitoring of the pressure inside the pressure-resistant sealed anaerobic culture chamber (4); The integrated gas injection and sampling assembly (11) is airtightly installed on the airtight sealing cover (2) and connected to the pressure-resistant sealed anaerobic culture chamber (4). The integrated gas injection and sampling assembly (11) includes a gas injection branch (12), a gas phase sampling branch (13), and independent airtight valve cores (14) that control the opening and closing of the gas injection branch (12) and the gas phase sampling branch (13) respectively. The liquid phase sampling component (21) is airtightly installed on the airtight sealed cover (2) or the anaerobic culture container body (1) through a sealed structure. The liquid inlet end of the liquid phase sampling component (21) extends into the liquid phase area of ​​the pressure-resistant sealed anaerobic culture chamber (4) to obtain liquid phase samples without opening the airtight sealed cover (2). The data acquisition and processing component is connected to the temperature sensor (9) in the micro methane sensor (5), the temperature control component (8), and the pressure monitoring component (10) to synchronously acquire data on methane concentration, culture temperature, and culture pressure, and to obtain the evaluation results of methanogenic bacteria activity based on the change of methane concentration over time.

2. The culture device for in-situ real-time monitoring of methanogenic bacteria activity according to claim 1, characterized in that, The gas injection branch (12) is used to introduce nitrogen, argon, hydrogen, carbon dioxide, methane or a mixture thereof into the pressure-resistant sealed anaerobic culture chamber (4) to establish an anaerobic environment and adjust the gas composition and pressure in the pressure-resistant sealed anaerobic culture chamber (4); the gas sampling branch (13) is used to extract gas samples without opening the airtight sealing cover (2) and is connected to a gas bag, syringe, gas chromatograph injection device or gas analysis equipment.

3. The culture device for in-situ real-time monitoring of methanogenic bacteria activity according to claim 1, characterized in that, The liquid phase sampling assembly (21) includes a liquid sampling tube (22), a first airtight valve (23), a quantitative buffer chamber (24), a second airtight valve (25), and a sterile sampling interface (26). The inlet end of the liquid collection tube (22) extends into the liquid phase region of the pressure-resistant sealed anaerobic culture chamber (4), and the outlet end of the liquid collection tube (22) is connected to the quantitative buffer chamber (24) through the first airtight valve (23). The quantitative buffer chamber (24) is connected to the sterile sampling interface (26) through the second airtight valve (25).

4. The culture device for in-situ real-time monitoring of methanogenic bacteria activity according to claim 3, characterized in that, The liquid phase sampling assembly (21) also includes a pressure balance branch (29), which is connected to the gas injection branch (12) or an external compensation gas source to supplement inert gas, hydrogen, carbon dioxide or mixed gas into the pressure-resistant sealed anaerobic culture chamber (4) after liquid phase sampling to compensate for the pressure change caused by liquid phase sampling.

5. The culture device for in-situ real-time monitoring of methanogenic bacteria activity according to claim 3, characterized in that, The quantitative buffer chamber (24) is connected to a pressure regulating branch (27), which is used to introduce inert gas into the quantitative buffer chamber (24), slowly depressurize the quantitative buffer chamber (24), or flush the liquid phase sampling assembly (21). The pressure regulating branch (27) is equipped with at least one of a pressure regulating valve, a needle valve, a check valve, or a filter.

6. The culture device for in-situ real-time monitoring of methanogenic bacteria activity according to claim 1, characterized in that, The cultivation device includes multiple pressure-resistant and airtight anaerobic culture chambers (4) that are arranged in parallel and independent of each other. Each pressure-resistant and airtight anaerobic culture chamber (4) is equipped with a methane monitoring component, a pressure monitoring component (10), an integrated gas injection and sampling component (11), and a liquid phase sampling component (21).

7. The culture device for in-situ real-time monitoring of methanogenic bacteria activity according to claim 1, characterized in that, The data acquisition and processing components include a signal acquisition module (15), a data processing module (30), a data storage module (31), and a wireless transmission module (16). The signal acquisition module (15) is electrically connected to the miniature methane sensor (5), the temperature sensor (9), and the pressure monitoring component (10), respectively; The data processing module (30) is used to generate time series data of methane concentration, culture temperature and culture pressure, and to calculate methane generation rate, methane production per unit time or relative methane production activity index; The wireless transmission module (16) is used to transmit monitoring data and processing results to the terminal device (17). The data acquisition and processing component is also used to record the liquid phase sampling time, liquid phase sampling volume, type of compensation gas and compensation amount, and to receive the external detection results of the liquid phase sample; the data processing module (30) is used to time match the gas phase methane generation data with the concentration, metabolic activity or proliferation data of methanogens in the liquid phase sample to obtain the overall activity of methanogens, methanogen production activity per unit cell or risk assessment results of the culture system. The data processing module (30) is used to calculate the amount of gaseous methane based on the effective gas volume, absolute pressure, culture temperature and methane volume fraction of the pressure-resistant sealed anaerobic culture chamber (4), and to correct the amount of gaseous methane based on the gas injection volume, gas sampling volume, volume change caused by liquid sampling and blank control data. The data acquisition and processing component also includes an alarm module and a controller (32); when the methane concentration, methane generation rate, culture temperature or culture pressure exceed the preset threshold, the alarm module issues an alarm signal, and / or the controller (32) controls the gas injection branch (12) to stop the gas supply and controls the safety pressure relief component (18) to release pressure.

8. A method for in-situ real-time monitoring of methanogenic bacteria activity, characterized in that, A culture device for in-situ real-time monitoring of methanogenic bacteria activity as described in any one of claims 1 to 7; comprising the following steps: Culture medium, methanogenic bacteria sample and simulated stratum sample were added to the anaerobic culture container body (1) under anaerobic conditions, and the liquid volume and effective gas volume were recorded. The airtight sealing cover (2) is airtightly connected to the anaerobic culture container body (1) to form a pressure-resistant and airtight anaerobic culture chamber (4). Inert gas is introduced into the pressure-resistant sealed anaerobic culture chamber (4) through the gas injection branch (12) to replace the residual oxygen, and then the target gas containing hydrogen and carbon dioxide is introduced, and the pressure in the pressure-resistant sealed anaerobic culture chamber (4) is adjusted to the preset value. The culture temperature is maintained within a preset range by the constant temperature control component (8), and the culture pressure is continuously monitored by the pressure monitoring component (10). The methane concentration in the gas phase space of the pressure-resistant sealed anaerobic culture chamber (4) is continuously detected by a micro methane sensor (5), and the methane concentration, culture temperature and culture pressure data are collected synchronously by the data acquisition and processing component. A predetermined volume of liquid phase sample is collected at a preset incubation time point using the liquid phase sampling component (21), and the changes in incubation pressure caused by liquid phase sampling are compensated. Analyze liquid samples to obtain data on methanogen concentration, metabolic activity, or proliferation. The amount and rate of methane production are calculated based on methane concentration, culture pressure, culture temperature, and effective gas phase volume. The amount or rate of methane production is then time-matched with the detection results of liquid phase samples to obtain the evaluation results of methanogenic bacteria activity.

9. A method for in-situ real-time monitoring of methanogenic bacteria activity according to claim 8, characterized in that, The collection of liquid phase samples via the liquid phase sampling assembly (21) includes: Keep the second airtight valve (25) closed and open the first airtight valve (23) so that the liquid sample enters the quantitative buffer chamber (24) under the pressure inside the pressure-resistant sealed anaerobic culture chamber (4); When the liquid sample entering the quantitative buffer chamber (24) reaches the predetermined volume, the first airtight valve (23) is closed to isolate the quantitative buffer chamber (24) from the pressure-resistant sealed anaerobic culture chamber (4); The pressure in the quantitative buffer chamber (24) is slowly reduced by the pressure regulating branch (27); Connect the sterile sample container to the sterile sampling interface (26), open the second airtight valve (25) and output the liquid sample; The target gas is supplied to the pressure-resistant sealed anaerobic culture chamber (4) through the pressure balance branch (29) to restore the culture pressure to the preset range.

10. A method for in-situ real-time monitoring of methanogenic bacteria activity according to claim 8, characterized in that, The data acquisition and processing component calculates the amount of gaseous methane using the following formula: in, This represents the amount of gaseous methane. This represents the volume fraction of methane. The absolute pressure of the culture chamber; This represents the effective volume of the gas phase. It is the gas compressibility factor; It is the gas constant; This refers to absolute temperature.

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