A constant-volume culture tank with sensors and a method thereof

Abstract

This invention discloses a constant-volume culture vessel with integrated sensors and its method, belonging to the field of microbiology technology. The constant-volume culture vessel includes an upper cover, a culture vessel cylinder, and a lower cover, as well as an upper guide tube and a multi-parameter probe fixed to the upper cover, and a lower guide tube and a pressure sensor fixed to the lower cover. An isolating sliding cover in the middle of the culture vessel cylinder divides the cylinder into independent upper and lower compartments. The upper compartment is used to contain samples containing target microorganisms and culture media, while the lower compartment is used to fill with liquid media to regulate the internal pressure of the culture vessel cylinder. During culture, the multi-parameter probe and pressure sensor can automatically and in real-time detect changes in chemical composition and pressure. This culture vessel has advantages such as simple structure, convenient manufacturing, and low usage and maintenance costs. It can dissolve a specified volume of gas into the culture system through pressurization, making it suitable for in-situ real-time monitoring and research of metabolic intermediates and metabolic mechanisms of target microorganisms on gas components.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a constant-volume culture vessel with built-in sensors and its method. Background Technology

[0002] Many chemical reactions involving microorganisms in nature involve gaseous components. For example, in cold seep areas and hydrate-producing areas on the seabed, hydrocarbon gases provide essential nutrients for microbial communities. Methanophiles and sulfate-reducing bacteria can work together to trigger the following chemical reactions in sediments:

[0003] CH4+SO4 2- = CO3 2- +H2S+H2O (1)

[0004] CO3 2- +(Mg, Ca) 2+ = (Mg, Ca)CO3 (2)

[0005] H₂S + Fe(OH)₂ = FeS + 2H₂O (3)

[0006] Methane in equation (1) participates in microbial metabolism and provides energy for the microbial community. However, due to the low solubility of methane in seawater, it is difficult to establish a sufficient concentration in the culture system. Directly pressurizing and introducing methane gas into the culture system poses a safety risk due to excessive compressed gas. Similar problems exist in other microbial metabolic reactions involving gaseous components.

[0007] Therefore, there is an urgent need for a culture vessel that can dissolve a specified volume of gas into the culture system by pressurization, so as to provide research methods and observation tools for studying the metabolic processes of microorganisms on gaseous components. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a constant-volume culture vessel with built-in sensors and its method. This microbial culture device, based on the principle that gas solubility increases with pressure, dissolves a specified volume of gas into the culture system through pressurization. Furthermore, it can observe and record changes in chemical parameters and pressure over time within the culture vessel using built-in sensors, providing a research method and observation tool for studying the metabolic processes of microorganisms on gaseous components.

[0009] The specific technical solution adopted in this invention is as follows:

[0010] In a first aspect, the present invention provides a volumetric culture vessel with a built-in sensor, comprising an upper end cover, a culture vessel cylinder, and a lower end cover. A sealing ring is provided between the upper port of the top of the culture vessel cylinder and the upper end cover to form a sealed connection. A sealing ring is provided between the lower port of the bottom of the culture vessel cylinder and the lower end cover to form a sealed connection. An isolation sliding cover is provided in the inner cavity of the culture vessel cylinder, and a sealing ring is provided between the inner cavity of the culture vessel cylinder and the isolation sliding cover, thereby dividing the inner cavity of the culture vessel cylinder into an independent upper compartment and a lower compartment. The upper compartment is used to contain a sample containing the target microorganism and a culture medium, and the lower compartment is used to fill a liquid medium to regulate the pressure inside the inner cavity of the culture vessel cylinder.

[0011] The upper end cover is equipped with an upper guide pipe for gas phase inlet and outlet and a multi-parameter probe for real-time monitoring of chemical components in the upper chamber. One end of the multi-parameter probe is located in the upper chamber of the culture vessel, and the other end is connected to the host computer via a watertight connector outside the upper end cover. A first valve for controlling the gas phase inlet and outlet is installed on the upper guide pipe.

[0012] The lower end cover is equipped with a lower guide pipe for liquid phase inflow and outflow and a pressure sensor for real-time monitoring of pressure changes within the lower compartment. A second valve for controlling the inflow and outflow of liquid phase is installed on the lower guide pipe. The pressure sensor is connected to the host computer via a connecting cable.

[0013] Preferably, the materials of the above-mentioned culture vessel body, upper end cover, lower end cover and isolation sliding cover are titanium, aluminum, titanium alloy or aluminum alloy.

[0014] Preferably, both the upper and lower guide pipes are high-strength seamless stainless steel pipes, and the stainless steel pipes are one of 304, 304L, 316L, 304H, 347H, 304LN, 321H, 316Ti, 316H, 317, 317L, 321, 310S, S31803, and 904L.

[0015] Preferably, the pressure sensor is a sapphire pressure sensor or a diffused silicon pressure sensor. The first valve and the second valve are general-purpose airtight shut-off valves.

[0016] Preferably, the pressure sensor described above has a range of 0–60 MPa.

[0017] Preferably, the aforementioned multi-parameter probe is a set of solid ion-selective electrodes, including a common reference electrode, a hydrogen sulfide monitoring electrode, a pH electrode, an Eh electrode, and a carbonate ion-selective electrode.

[0018] Secondly, the present invention provides a method for using a constant-volume culture vessel with a built-in sensor as described in the first aspect, the specific steps of which are as follows:

[0019] S1: Invert the culture vessel so that the lower end faces upward. Push the isolation sliding cover into the inner cavity of the culture vessel. The volume of the lower chamber should be set according to the volume of gas components required by the target microorganisms in the cultured material contained in the upper chamber. Secure the isolation sliding cover with a temporary support. Then fill the lower chamber with liquid medium, install the lower end cover and sealing ring, and press down the lower end cover to discharge excess liquid medium in the lower chamber through the lower guide pipe. Close the second valve on the lower guide pipe and flip the culture vessel so that the upper end faces upward.

[0020] S2: Place the culture medium containing the target microorganism into the upper chamber of the culture vessel, fill the upper chamber with the prepared culture medium, install the upper end cover and sealing ring, press down the upper end cover until the culture medium overflows from the upper guide tube, and then close the first valve on the upper guide tube.

[0021] S3: Open the first valve on the upper guide pipe to inject gas components into the upper compartment through the upper guide pipe. At the same time, open the second valve on the lower guide pipe so that the isolation slider moves downward during inflation, and the water in the lower compartment is discharged through the lower guide pipe. When the water discharge from the lower guide pipe stops, stop injecting gas components and close the second valve and the first valve.

[0022] S4: Open the second valve and inject high-pressure water into the lower chamber through the lower guide pipe until the pressure sensor reading on the lower end cover reaches the rated value. Then close the second valve. The gas components in the upper chamber dissolve into the culture medium and the cultured material containing the target microorganism under high pressure for culturing.

[0023] S5: During cultivation, a multi-parameter probe monitors changes in chemical composition in the upper chamber in real time, while a pressure sensor monitors changes in pressure in the lower chamber in real time. Signals detected by the multi-parameter probe are transmitted to the host computer for recording and storage via a watertight connector. Pressure values ​​recorded by the pressure sensor are transmitted to the host computer for recording and storage via a connecting cable.

[0024] S6: After the culture is completed, first slowly open the second valve to slowly discharge the high-pressure water in the lower chamber. After the lower chamber is drained, slowly open the first valve to slowly discharge the gas in the upper chamber and depressurize it.

[0025] Preferably, the filling degree of the culture medium containing the target microorganism in the upper compartment is 1 / 5 to 1 / 3.

[0026] Preferably, the target microorganisms are anaerobic methanogens or sulfate-reducing bacteria, requiring operation under anaerobic aseptic conditions. The culture medium containing the target microorganisms is seabed sediment.

[0027] Furthermore, the culture medium is water, artificial seawater, or an artificially prepared nutrient solution. Artificial seawater is a 3.5% sodium chloride solution. The gaseous components are the gaseous nutrients required for the metabolism of the target microorganisms, namely methane, hydrogen sulfide, or carbon dioxide.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] (1) The fixed-volume culture vessel with built-in sensor provided by the present invention can determine the volume ratio of gas phase to liquid phase (including solid phase) in the culture vessel by setting the position of the isolation sliding cover before filling the culture medium containing the target microorganism. After filling, by controlling the water injection pressure of the lower chamber, the gas in the upper chamber of the culture vessel is dissolved into the culture medium, thereby dissolving the specified volume of gas into the culture system by pressurization, promoting the metabolic process of the target microorganism.

[0030] (2) The fixed-volume culture vessel with built-in sensor provided by the present invention can automatically detect changes in chemical composition and pressure during culture in real time. It is suitable for in-situ real-time monitoring of the metabolic intermediates and metabolic mechanisms of target microorganisms on gas components. It has the advantages of simple structure, easy manufacturing, and low cost of use and maintenance. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the fixed-volume culture vessel with built-in sensors provided in this embodiment;

[0032] In the diagram: 1. Upper guide pipe; 2. First valve; 3. Watertight connector; 4. Multi-parameter probe; 5. Upper end cover; 6. Sealing ring; 7. Culture vessel body; 8. Isolation sliding cover; 9. Upper chamber; 10. Lower chamber; 11. Lower end cover; 12. Pressure sensor; 13. Lower guide pipe; 14. Second valve. Detailed Implementation

[0033] The present invention will be further described and illustrated below with reference to specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0034] Example 1

[0035] This embodiment provides a constant-volume culture vessel with a built-in sensor, such as... Figure 1 As shown, it includes an upper end cover 5, a culture vessel cylinder 7, and a lower end cover 11. The culture vessel cylinder 7 is a hollow cylindrical structure, and its wall thickness design must comply with the design specifications for pressure vessels, such as GB 150-1998. The inner lining material is polytetrafluoroethylene.

[0036] The culture vessel cylinder has an upper end cover 5 at its top upper port and a lower end cover 11 at its bottom lower port. Both the upper end cover 5 and the lower end cover 11 are flange-type structures, which are fixed by external screws and nuts to maintain pressure inside the cylinder and achieve a seal. A sealing ring 6 is also provided between the upper end port of the culture vessel cylinder 7 and the upper end cover 5 to form a sealed connection. A sealing ring 6 is also provided between the lower end port of the culture vessel cylinder 7 and the lower end cover 11 to form a sealed connection.

[0037] An isolation sliding cover 8 is provided inside the culture vessel body 7, and a sealing ring 6 is provided between the inner cavity of the culture vessel body 7 and the isolation sliding cover 8. The isolation sliding cover 8 and the sealing ring 6 divide the inner cavity of the culture vessel body 7 into two independent upper chambers 9 and lower chambers 10. The upper chamber 9 is used to contain the sample containing the target microorganism and the culture medium, while the lower chamber 10 is used to fill the liquid medium and regulate the pressure inside the culture vessel body 7.

[0038] One side of the upper end cover 5 is equipped with an upper guide pipe 1 for gas phase inlet and outlet, and the other side is equipped with a multi-parameter probe 4 for real-time monitoring of chemical components in the upper chamber 9. One end of the multi-parameter probe 4 is located inside the upper chamber 9 of the culture vessel body 7, and the other end is connected to the data acquisition circuit board and the host computer outside the upper end cover 5 via a watertight connector 3. The watertight connector 3 can be a five-pin water surface connector, and a general commercial product can be used. Note that the model of the watertight connector must match the upper pressure limit of the culture vessel.

[0039] The multi-parameter probe 4 on the upper cover 5 is a set of solid ion-selective electrodes, which can be commercial products or homemade. For the cultivation of methanophiles and sulfate-reducing bacteria in the cold seep area of ​​the seabed, the recommended probe electrode combination is as follows: using an Ag / AgCl electrode as a common reference electrode, using an Ag / Ag2S electrode to detect dissolved hydrogen sulfide, an Ir / Ir(OH)x electrode to detect pH value, a Pt wire electrode to detect Eh value, and a carbon-plated nickel wire coated with a carbonate ion-sensitive membrane to detect carbonate concentration.

[0040] The lower end cap 11 has a lower guide pipe 13 for liquid phase inlet and outlet on one side, and a pressure sensor 12 for real-time monitoring of pressure changes in the lower chamber 10 on the other side. The pressure sensor 12 is connected to the host computer via a connecting cable. The upper limit of the pressure of the pressure sensor 12 needs to match the compressive strength of the lining material of the culture vessel cylinder 7. In this embodiment, the pressure sensor 12 is selected with a range of 0-60 MPa. Sapphire or diffused silicon pressure sensors are recommended, as both of these sensors can transmit pressure changes in the culture vessel in real time via an output interface.

[0041] Both the upper guide tube 1 and the lower guide tube are made of high-strength seamless stainless steel pipe, which can be one of 304, 304L, 316L, 304H, 347H, 304LN, 321H, 316Ti, 316H, 317, 317L, 321, 310S, S31803, and 904L.

[0042] The upper guide pipe 1 and the lower guide pipe 13 are respectively equipped with a first valve 2 for controlling the inflow and outflow of the gas phase and a second valve 14 for controlling the inflow and outflow of the liquid phase, which are used as pressure relief channels after the experiment. The first valve 2 and the second valve 14 are both general-purpose airtight shut-off valves, and their pressure resistance index must be consistent with the upper limit of the operating pressure.

[0043] Example 2

[0044] This embodiment provides a method for culturing seabed sediments containing methanogens using a constant-volume culture vessel with a built-in sensor as described in Embodiment 1. The specific steps are as follows:

[0045] S1: Install the upper conduit 1, the first valve 2, the watertight connector 3, the multi-parameter probe 4, and the sealing ring 6 on the upper end cover 5; install the sealing ring 6 on the isolation sliding cover 8; install the lower conduit 13, the second valve 14, the pressure sensor 12, and the sealing ring 6 on the lower end cover 11.

[0046] Invert the culture vessel 7 so that the lower end faces upward, push the isolation sliding cover 8 into the inner cavity of the culture vessel 7, and set the volume of the lower chamber 10 according to the volume of gas components required by the target microorganism in the cultured material contained in the upper chamber 9.

[0047] Under normal pressure, the volume solubility of methane gas in seawater is approximately 1 / 300, meaning that only 1 liter of methane can dissolve in 300 liters of seawater. The extremely low solubility of methane in water at normal pressure makes it difficult to provide the same support conditions as the natural seabed environment for the survival and metabolism of methanogens and sulfate-reducing bacteria in the culture system. If methane gas is pressurized to seabed pressure and then injected into the culture vessel, the compressibility of the gas and the large amount of flammable gas injected pose a risk of combustion and explosion, jeopardizing the safety of the laboratory and personnel. The core of this invention is to introduce a defined volume of gaseous nutrients into the culture vessel, satisfying the survival conditions of methanogens without violating safety regulations.

[0048] When the current port is facing upwards, it is recommended to install a temporary simple support at its lower part to fix the position of the isolation sliding cover 8, ensuring that the position of the isolation sliding cover will not move during operation. It is recommended that the isolation sliding cover be positioned in the middle of the culture vessel. This facilitates operation, and the 1:1 volume ratio ensures that the concentration of methane in the culture medium is higher than its solubility during the culture process, maintaining a supersaturated state to meet the metabolic needs of methanophilic microorganisms.

[0049] S2: Fill the lower chamber 10 with pure water, install the lower end cap 11 and sealing ring 6, and press down the lower end cap 11 to discharge excess liquid medium in the lower chamber 10 through the lower guide pipe 13; overflow of water at the outlet of the lower guide pipe 13 is an indication that the lower chamber is full of water. After the lower end cap 11 is pressed down, the second valve 14 on the lower guide pipe should be closed, and the culture vessel should be turned upside down so that the upper port faces upward in order to proceed with the next filling step.

[0050] S3: In a glove box filled with high-purity nitrogen, place the seabed sediment containing methanogens into the upper chamber 9 of the culture vessel 7, filling it to 1 / 4 full. Fill the upper chamber 9 with prepared 3.5% sodium chloride solution as artificial seawater, install the upper end cap 5 and sealing ring 6, press down the upper end cap until culture medium overflows from the upper guide pipe 1, then close the first valve 2 on the upper guide pipe 1. After completing the operation, remove the culture vessel from the glove box.

[0051] This step must adhere to standard rules and regulations for microbial culture, such as aseptic techniques, and the use of pre-sterilized containers and utensils to prevent contamination. For experiments culturing methanophiles and sulfate-reducing bacteria, since the culture subjects are anaerobic microorganisms, anaerobic operating procedures must be followed during sample transfer and culture vessel filling to prevent sample contact with air, which could kill the anaerobic bacteria.

[0052] S4: Open the first valve 2 on the upper guide pipe 1 to inject methane into the upper compartment 9 through the upper guide pipe 1; simultaneously open the second valve 14 on the lower guide pipe 13 so that the isolation slider 8 moves downward during inflation, and the water in the lower compartment 10 is discharged through the lower guide pipe 13. When the water discharge from the lower guide pipe 13 stops, the methane injection stops, and the second valve 14 and the first valve 2 are closed. At this time, the volume of methane gas injected into the upper compartment is equal to the volume of water discharged from the lower compartment. In this way, precise control of the amount of methane gas injected can be achieved.

[0053] For the cultivation of methanogens and sulfate-reducing bacteria, it is recommended to inject methane as an essential nutrient for the microorganisms; the other essential nutrient, sulfate, is added to the culture medium in the form of soluble salt. When injecting methane gas, open valve 14 on the lower end cover. The gas pressure will push the isolation slider 8 downward, and the pre-filled water in the lower chamber will flow out. After the drainage is completed, close valve 14 and the first valve 2.

[0054] S5: Open the second valve 14 and inject high-pressure water into the lower chamber 10 through the lower guide pipe 13 until the pressure sensor 12 on the lower end cover 11 reaches the rated value. Then close the second valve 14 and the methane in the upper chamber 9 dissolves into the artificial seawater and seabed sediments under high pressure for cultivation.

[0055] During the process of increasing the pressure inside the culture vessel by injecting water, the isolation slider 8 located between the upper and lower chambers gradually moves towards the upper chamber as high-pressure water is injected. This compresses the methane gas in the upper chamber, thus maintaining pressure balance between the two chambers. During the pressurization process, the total amount of methane in the upper chamber remains constant, but as the pressure increases, the amount of methane dissolved in the culture medium increases, while the amount of free methane on the liquid surface decreases. Only the methane dissolved in the culture medium can be utilized by methanogenic bacteria and sulfate-reducing bacteria.

[0056] S6: During the cultivation period, the multi-parameter probe 4 monitors the changes in chemical composition in the upper chamber 9 in real time, and the pressure sensor 12 monitors the changes in pressure in the lower chamber 10 in real time; the signals detected by the multi-parameter probe 4 are transmitted to the host computer for recording and storage through the watertight connector 3; the pressure values ​​recorded by the pressure sensor 12 are transmitted to the host computer for recording and storage through the connecting line.

[0057] The multi-parameter probe 4 is a set of solid ion-selective electrodes. The recommended electrode combination is: Ag / AgCl as a common reference electrode, Ag / Ag2S as the total dissolved hydrogen sulfide electrode, Ir / Ir(OH)x as the pH electrode, platinum wire as the Eh electrode, and carbon-plated nickel wire-carbonate ion carrier as the total dissolved carbonate electrode.

[0058] During the cultivation process, the temperature, pressure, and composition conditions inside the culture vessel are similar to those of the cultured material in its natural environment. Due to the metabolism of microorganisms, the following chemical reactions will occur:

[0059] CH4+SO4 2- = CO3 2- +H2S+H2O (1)

[0060] CO3 2- +(Mg, Ca) 2+ = (Mg, Ca)CO3 (2)

[0061] H₂S + Fe(OH)₂ = FeS + 2H₂O (3)

[0062] Equation (1) represents the anaerobic oxidation of methane and the reduction of sulfate, which simultaneously leads to a decrease in the Eh value of the system. The carbonate and hydrogen sulfide formed in the products, as well as the change in the Eh value of the system, can be detected in real time by an ion-selective electrode. The reactions represented by Equations (2) and (3) can be regarded as the reaction of carbonate and hydrogen sulfide, the metabolites of Equation (1), with sediment and pore water, to generate carbonates and metal sulfides. The consumption of volatile components will lead to a decrease in the pressure of the system, which can be detected and recorded by a pressure sensor installed on the lower end cap 11.

[0063] S7: After the culture is completed, first slowly open the second valve 14 to discharge the high-pressure water in the lower chamber 10 in a controllable manner. After the lower chamber 10 is drained, slowly open the first valve 2 to discharge and depressurize the gas in the upper chamber 9 in a controllable manner.

[0064] At the end of the culture, the culture vessel will still have high pressure. The recommended depressurization sequence is bottom to top: first drain the water from the lower chamber, and after the lower chamber is emptied, open the drain pipe on the upper cover to depressurize the upper chamber. Before depressurizing the upper chamber, consider whether it is necessary to sample and analyze the gaseous, liquid, and solid components inside the culture vessel.

[0065] S8: Read and transfer data, open the culture vessel, clean the device, and end the experiment.

[0066] The multi-parameter probe 4 mounted on the upper end cover 5 and the pressure sensor 12 mounted on the lower end cover 11 both have data interfaces and built-in data storage functions and clocks. After the experiment, the data in the built-in storage can be read to obtain the changes in chemical parameters and pressure over time during the experiment.

[0067] The application scenario designed for this invention is to simulate the deep-sea environment of cold seep areas and hydrate-producing areas in the laboratory, providing experimental means to study the microbial chemistry of methanophiles and sulfate-reducing bacteria. As the anaerobic microorganisms metabolize, an increase in hydrogen sulfide and carbonate content can be monitored, along with significant changes in pH and Eh. Simultaneously, as methane is consumed, the pressure gauge reading gradually decreases. Conversely, if no significant changes are observed in these parameters after a certain period of cultivation, it indicates that the target anaerobic microorganisms are not present in the collected seabed sediments.

[0068] The self-contained, constant-volume culture vessel with integrated sensors provided by this invention can also be applied to other similar scenarios, such as as a reaction vessel for studying the Fischer-Tropsch process. This process uses a mixture of carbon monoxide and hydrogen as raw materials to synthesize liquid hydrocarbons or hydrocarbons under appropriate conditions and with a catalyst. The dual-compartment reactor allows for precise setting and control of temperature, pressure, and composition changes during the reaction, which is beneficial for studying the relationship between condition parameters and the degree of reaction. If using this device to study the Fischer-Tropsch reaction, it is recommended to replace the multi-parameter probe mounted on the upper end cap 5 with other sensors, such as sensors for detecting the concentrations of carbon monoxide and hydrogen in the system.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A constant-volume culture vessel with integrated sensors, characterized in that, The system includes an upper end cap (5), a culture vessel cylinder (7), and a lower end cap (11). A sealing ring (6) is provided between the upper port of the culture vessel cylinder (7) and the upper end cap (5) to form a sealed connection. A sealing ring (6) is provided between the lower port of the culture vessel cylinder (7) and the lower end cap (11) to form a sealed connection. An isolation sliding cover (8) is provided in the inner cavity of the culture vessel cylinder (7), and a sealing ring (6) is provided between the inner cavity of the culture vessel cylinder (7) and the isolation sliding cover (8). The isolation sliding cover (8) and the sealing ring (6) divide the inner cavity of the culture vessel cylinder (7) into an independent upper chamber (9) and a lower chamber (10). The upper chamber (9) is used to contain samples containing target microorganisms and culture media, and the lower chamber (10) is used to fill liquid media to adjust the pressure inside the culture vessel cylinder (7). The upper end cover (5) is provided with an upper guide pipe (1) for gas phase entry and exit and a multi-parameter probe (4) for real-time monitoring of chemical components in the upper chamber (9); one end of the multi-parameter probe (4) is located in the upper chamber (9) of the culture vessel body (7), and the other end is connected to the host computer outside the upper end cover (5) through a watertight connector (3); the upper guide pipe (1) is provided with a first valve (2) for controlling the entry and exit of the gas phase. The lower end cap (11) is provided with a lower guide pipe (13) for liquid phase entry and exit and a pressure sensor (12) for real-time monitoring of pressure changes in the lower chamber (10); the lower guide pipe (13) is provided with a second valve (14) for controlling the entry and exit of liquid phase; the pressure sensor (12) is connected to the host computer via a connecting line.

2. The constant-volume culture vessel with built-in sensor as described in claim 1, characterized in that, The culture vessel body (7), upper end cover (5), lower end cover (11) and isolation sliding cover (8) are made of titanium, aluminum, titanium alloy or aluminum alloy.

3. The constant-volume culture vessel with built-in sensor as described in claim 1, characterized in that, Both the upper guide pipe (1) and the lower guide pipe (13) are high-strength seamless stainless steel pipes, and the stainless steel pipes are one of 304, 304L, 316L, 304H, 347H, 304LN, 321H, 316Ti, 316H, 317, 317L, 321, 310S, S31803, and 904L.

4. The constant-volume culture vessel with built-in sensor as described in claim 1, characterized in that, The pressure sensor (12) is a sapphire pressure sensor or a diffused silicon pressure sensor; the first valve (2) and the second valve (14) are general-purpose airtight shut-off valves.

5. The constant-volume culture vessel with integrated sensor as described in claim 1, characterized in that, The pressure sensor (12) has a range of 0~60MPa.

6. The constant-volume culture vessel with built-in sensor as described in claim 1, characterized in that, The multi-parameter probe (4) is a set of solid ion-selective electrodes, including a common reference electrode, a hydrogen sulfide monitoring electrode, a pH electrode, an Eh electrode, and a carbonate ion-selective electrode.

7. A cultivation method using a constant-volume culture vessel with a built-in sensor as described in any one of claims 1 to 6, characterized in that, The specific steps are as follows: S1: Invert the culture vessel (7) so that the lower end faces upward. Push the isolation sliding cover (8) into the inner cavity of the culture vessel (7). The volume of the lower chamber (10) is set according to the volume of gas components required by the target microorganism in the cultured material contained in the upper chamber (9). Fix the lower part of the isolation sliding cover (8) with a temporary support. Then fill the lower chamber (10) with liquid medium. Install the lower end cover (11) and sealing ring (6). Press down the lower end cover (11) so that the excess liquid medium in the lower chamber (10) is discharged through the lower guide pipe (13). Close the second valve (14) on the lower guide pipe (13). Turn the culture vessel (7) over so that the upper end faces upward. S2: Place the culture medium containing the target microorganism into the upper chamber (9) of the culture vessel (7), fill the upper chamber (9) with the prepared culture medium, install the upper end cover (5) and sealing ring (6), press down the upper end cover until the culture medium overflows from the upper guide pipe (1), and then close the first valve (2) on the upper guide pipe (1). S3: Open the first valve (2) on the upper guide pipe (1) to inject gas components into the upper compartment (9) through the upper guide pipe (1); at the same time, open the second valve (14) on the lower guide pipe (13) so that the isolation cover (8) moves downward when inflating, and the water in the lower compartment (10) is discharged through the lower guide pipe (13). When the lower guide pipe (13) stops discharging water, stop injecting gas components and close the second valve (14) and the first valve (2). S4: Open the second valve (14) and inject high-pressure water into the lower chamber (10) through the lower guide pipe (13) until the pressure sensor (12) on the lower end cover (11) reaches the rated value. Then close the second valve (14) and the gas components in the upper chamber (9) dissolve into the culture medium and the culture material containing the target microorganism under high pressure for culture. S5: During the cultivation period, the multi-parameter probe (4) monitors the changes in chemical composition in the upper chamber (9) in real time, and the pressure sensor (12) monitors the changes in pressure in the lower chamber (10) in real time; the signal detected by the multi-parameter probe (4) is transmitted to the host computer for recording and storage through the watertight connector (3); the pressure value recorded by the pressure sensor (12) is transmitted to the host computer for recording and storage through the connecting line; S6: After the culture is completed, first slowly open the second valve (14) to slowly discharge the high-pressure water in the lower chamber (10). After the lower chamber (10) is drained, slowly open the first valve (2) to slowly discharge the gas in the upper chamber (9) and depressurize it.

8. The cultivation method according to claim 7, characterized in that, The filling degree of the culture medium containing the target microorganism in the upper compartment (9) is 1 / 5 to 1 / 3.

9. The cultivation method according to claim 7, characterized in that, The target microorganism is an anaerobic microorganism, methanophilic bacteria or sulfate-reducing bacteria, and needs to be operated in an anaerobic sterile environment; the culture medium containing the target microorganism is seabed sediment.

10. The cultivation method according to claim 7, characterized in that, The culture medium is water, artificial seawater, or artificially prepared nutrient solution; the artificial seawater is a 3.5% sodium chloride solution; the gaseous component is a gaseous nutrient component required for the metabolism of the target microorganism, namely methane, hydrogen sulfide, or carbon dioxide.

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