A method for sampling and collecting microorganisms in a saline aquifer using a piston container

Abstract

The application discloses a piston container and a method for sampling and collecting microorganisms in a saline aquifer, and the piston container comprises a first cavity, a side wall of the first cavity is provided with a first valve, and the first valve is used for connecting a liquid outlet end of a saline aquifer well mouth; a second cavity below the first cavity, a side wall of the second cavity is provided with a second valve and a third valve below the second valve, the second valve is used for connecting a suction filter pump, and the third valve is used for connecting a sterile vacuum liquid collection bag; and a filter funnel between the first cavity and the second cavity, the filter funnel is used for filtering liquid in the first cavity to the second cavity. The piston container can be used to simply and quickly sample and collect microorganisms in the saline aquifer on site, and can guarantee the integrity of underground microorganism community information.

Description

Technical Field

[0001] This invention relates to the fields of microorganisms, carbon dioxide storage (CCS), utilization and storage (CCUS), and particularly to a method for sampling and collecting microorganisms in a piston container and a saline layer. Background Technology

[0002] Carbon dioxide capture and storage (CCS) is a technology for capturing and storing carbon dioxide (CO2). CCS is an option in a portfolio of mitigation actions to stabilize atmospheric greenhouse gas concentrations, with the potential to reduce overall mitigation costs and increase flexibility in achieving greenhouse gas emission reductions. The Earth's deep saline aquifers possess enormous CCS storage potential. The carbon dioxide sequestration mechanisms in deep saline aquifers can be categorized into physical sequestration, chemical sequestration, and biomineralization. Biomineralization is mediated by microorganisms; therefore, sampling and collecting microorganisms from saline aquifers and studying their species and carbon sequestration functions is of great significance.

[0003] Existing technologies primarily utilize conventional groundwater or oilfield produced fluid sampling devices, which are mostly used for sampling and transportation at atmospheric pressure, making them unsuitable for microbial sampling and collection. Furthermore, existing technologies are limited by objective factors such as the complexity and high cost of high-fidelity sampling procedures, and the limited availability of well site operations, making it impossible to easily and quickly collect microbial samples at the wellhead and ensuring the integrity of underground microbial community information.

[0004] There is currently no effective solution to the above problems. Summary of the Invention

[0005] This specification provides an embodiment of a piston container and a method for sampling and collecting microorganisms in a saline aquifer. The piston container can be used to simply and quickly sample and collect microorganisms in a saline aquifer on-site, ensuring the integrity of underground microbial community information.

[0006] Firstly, embodiments of this specification provide a piston container, comprising:

[0007] A first cavity, wherein a first valve is provided on the side wall of the first cavity, and the first valve is used to connect to the liquid outlet end of the saline wellhead;

[0008] A second cavity is located below the first cavity. A second valve and a third valve located below the second valve are provided on the side wall of the second cavity. The second valve is used to connect to a vacuum pump, and the third valve is used to connect to a sterile vacuum liquid collection bag.

[0009] A filter funnel located between the first chamber and the second chamber is used to filter liquid from the first chamber to the second chamber.

[0010] In some embodiments, a filter screen plate is movably disposed on the filter funnel, and filter paper is placed above the screen holes of the filter screen plate. The filter paper is used to filter the liquid flowing out of the outlet end of the saline layer well.

[0011] In some embodiments, the filter paper includes at least one of the following: qualitative filter paper with a pore size of 0.45 μm, microbial cell collection filter paper with a pore size of 0.22 μm, and microbial cell collection filter paper with a pore size of 0.1 μm.

[0012] In some embodiments, the first valve and the second valve are located on the same side, and the third valve is located on the other side of the first valve or the second valve.

[0013] In some embodiments, the piston container further includes an upper end cap and a lower end cap, wherein when the upper end cap and the lower end cap are closed, the first cavity and the second cavity form a sealed cavity.

[0014] In some embodiments, the piston container further includes: a fourth valve and a piston. The fourth valve is disposed outside the lower end cover and is connected to a manual pump. When the fourth valve is opened, liquid is injected into the lower part of the piston through the manual pump to increase the pressure of the first chamber and the second chamber until it reaches the underground pressure of the saline aquifer.

[0015] In some embodiments, the bottom inner diameter of the piston container ranges from 10cm to 15cm, the height ranges from 40cm to 60cm, and the volume ranges from 3.1L to 10.6L.

[0016] Secondly, embodiments of this specification provide a method for sampling and collecting microorganisms in a saline aquifer, based on the aforementioned piston container, the method comprising:

[0017] The second valve connected to the filtration pump, the first valve connected to the outlet of the saline well, and the third valve connected to the sterile vacuum liquid collection bag are opened in sequence so that the liquid flowing out from the outlet of the saline well flows into the second chamber after being filtered by the qualitative filter paper with the first aperture, and is collected into the sterile vacuum liquid collection bag.

[0018] When the liquid collection volume exceeds the capacity of the sterile vacuum liquid collection bag, the qualitative filter paper is collected and replaced with microbial cell collection filter paper with a second pore size. The third valve is connected to a new first sterile vacuum liquid collection bag, and the liquid collected by the sterile vacuum liquid collection bag is added to the first chamber so that the liquid flowing out of the first chamber flows into the second chamber after being filtered by the microbial cell collection filter paper with a second pore size, and is collected into the new first sterile vacuum liquid collection bag.

[0019] When the liquid collection volume exceeds the capacity of the new first sterile vacuum liquid collection bag, collect the microbial cell collection filter paper with the second pore size and replace it with the microbial cell collection filter paper with the third pore size. Repeat the filtration and collection process until the number of collected filter papers reaches the preset number.

[0020] The collected microbial cells were collected from filter paper into sterile centrifuge tubes, which were placed in a dry ice bucket containing dry ice.

[0021] In some embodiments, the duplicate filtering collection process includes:

[0022] Connect the third valve to a new second sterile vacuum liquid collection bag, and add the liquid collected in the new first sterile vacuum liquid collection bag to the first chamber, so that the liquid flowing out of the first chamber flows into the second chamber after being filtered by the third microbial collection filter paper with a third pore size, and is collected into the new second sterile vacuum liquid collection bag. The first pore size is 0.45 μm, the second pore size is 0.22 μm, and the third pore size is 0.1 μm.

[0023] In some embodiments, the method further includes:

[0024] Open the top cover of the piston container, disconnect the vacuum pump connected to the second valve and the sterile vacuum liquid collection bag connected to the third valve, and close the first valve, second valve and third valve. Remove the filter screen plate on the filter funnel.

[0025] The liquid flowing out from the wellhead of the saline layer flows into the first chamber and the second chamber through the upper end cover. When the amount of liquid flowing in exceeds the capacity of the chamber, the upper end cover is closed.

[0026] Open the fourth valve on the lower end cover, and inject liquid into the lower part of the piston through the manual pump connected to the fourth valve to increase the pressure of the first and second chambers until it reaches the underground pressure of the saline aquifer. Then, put the entire chamber into an insulated box for transportation.

[0027] This specification provides a piston container, comprising: a first cavity with a first valve on its side wall for connecting to the liquid outlet of a saline well; a second cavity below the first cavity, with a second valve on its side wall and a third valve below the second valve, the second valve for connecting to a filtration pump and the third valve for connecting to a sterile vacuum liquid collection bag; and a filter funnel located between the first and second cavities for filtering liquid from the first cavity into the second cavity. The piston container has a simple structure and can quickly filter suspended solids and collect microorganisms from samples taken from the saline aquifer. Simultaneously, after microbial collection, the piston container collects the original produced fluid and pressurizes it to the saline aquifer pressure before transportation, obtaining relatively complete original produced fluid.

[0028] This specification provides a method for sampling and collecting microorganisms in a saline aquifer. The method includes: sequentially opening a second valve connected to a filtration pump, a first valve connected to the outlet of the saline aquifer well, and a third valve connected to a sterile vacuum liquid collection bag. This allows liquid flowing from the outlet of the saline aquifer well to be filtered through qualitative filter paper of a first pore size before flowing into a second chamber and being collected into the sterile vacuum liquid collection bag. When the collected liquid volume exceeds the capacity of the sterile vacuum liquid collection bag, the qualitative filter paper is collected and replaced with microbial cell collection filter paper of a second pore size. The third valve is then connected to a new first sterile vacuum liquid collection bag. Liquid collected in a sterile vacuum liquid collection bag is added to the first chamber, allowing the liquid flowing out of the first chamber to be filtered through a second-pore microbial cell collection filter paper before flowing into the second chamber and being collected into a new first sterile vacuum liquid collection bag. When the collected liquid volume exceeds the capacity of the new first sterile vacuum liquid collection bag, the second-pore microbial cell collection filter paper is collected and replaced with a third-pore microbial cell collection filter paper, and the filtration and collection process is repeated until the number of collected filter papers reaches a preset number. The collected microbial cell collection filter paper is then collected into a sterile centrifuge tube, which is placed in a dry ice bucket containing dry ice. This invention allows for simple and rapid on-site sampling and collection of microorganisms in saline aquifers using this piston container, ensuring the integrity of underground microbial community information. It also enables pressure- and temperature-controlled transportation, significantly improving the fidelity of liquid samples. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0030] Figure 1 This is a front view of the piston container provided in the embodiments of this specification;

[0031] Figure 2 This is a top view of the filter funnel provided in the embodiments of this specification;

[0032] Figure 3 This is a schematic flowchart illustrating a method for sampling and collecting microorganisms in a saline aquifer, as provided in the embodiments of this specification.

[0033] [Explanation of Labels in the Attached Image]

[0034] A. Upper end cap; B. Lower end cap; C. First cavity; D. Second cavity; E. Piston; 1. First valve; 2. Second valve; 3. Third valve; 4. Filter funnel; 5. Internal protrusion of the cavity; 6. Fourth valve; 7. Sieve hole; 8. Weight. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this specification, and not all embodiments. Based on the embodiments in this specification, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this specification.

[0036] The Earth's deep saline aquifers possess enormous CCS (carbon dioxide sequestration) potential. The sequestration mechanisms in deep saline aquifers can be categorized into physical sequestration, chemical sequestration, and biomineralization. Physical sequestration mainly includes geological structural sequestration (hydraulic traps) and residual gas sequestration; chemical sequestration mainly includes dissolution sequestration and mineralization sequestration; and biomineralization is primarily microbial-mediated carbon dioxide mineralization and fixation. Specifically, hydraulic traps refer to the upward migration of carbon dioxide to dense strata where it is blocked and accumulates within the geological body, forming a gaseous phase of carbon dioxide sequestration; residual gas sequestration occurs due to the lag in displacement and adsorption phases, where some carbon dioxide is trapped as residual gas; dissolution sequestration refers to carbon dioxide dissolving in water to form a salt solution, achieving carbon dioxide capture; and both chemical and biomineralization involve the plasma reaction of carbon dioxide with calcium, magnesium, and iron in the water to form carbonate minerals, achieving safe carbon dioxide sequestration. Biomineralization is mediated by microorganisms; therefore, sampling and collecting microorganisms from saline aquifers to study the types and carbon sequestration functions of these microorganisms is of great significance.

[0037] Currently, research on biomineralization and biocarbon fixation in deep saline aquifers is limited. Existing technologies mainly utilize conventional groundwater or oilfield produced fluid sampling equipment, which in most cases involves sampling and transporting at atmospheric pressure. This process loses much information about deep-seated microorganisms and is unsuitable for microbial sampling and collection. For example, the survival rate of barophilic microorganisms decreases rapidly over time under atmospheric pressure; and it is difficult to maintain the temperature during transportation for extended periods, making it difficult to preserve information about mesophilic microorganisms.

[0038] Furthermore, due to limitations such as the complexity and high cost of high-fidelity sampling procedures and the scarcity of well site operations, surface wellhead sampling is currently the most commonly used method in scientific research. However, it has been reported that subsurface microbial communities are significantly affected by temperature and pressure. After wellhead sampling, it is necessary to quickly collect the microbial cells within a short period of time using simple operations to ensure a more complete understanding of the subsurface microbial community. Current technologies cannot easily and quickly collect microbial samples at the wellhead, thus failing to guarantee the integrity of subsurface microbial community information.

[0039] To address the aforementioned issues, this specification provides a method for sampling and collecting microorganisms in a piston container and a saline layer. This method enables simple and rapid on-site sampling and collection of microorganisms from a saline layer, and allows for high-pressure preservation and pressure- and temperature-controlled transportation of microorganisms in the water. This results in obtaining more complete bacterial cells and high-fidelity samples, which are then used for more valuable scientific research.

[0040] To better understand the inventive concept of this application, a piston container provided by an embodiment of this specification is first introduced. For example... Figure 1 As shown, the piston container may include:

[0041] A first cavity C, the side wall of which may be provided with a first valve 1, which may be used to connect to the liquid outlet end of the saline wellhead;

[0042] The second cavity D is located below the first cavity. The side wall of the second cavity D can be provided with a second valve 2 and a third valve 3 located below the second valve 2. The second valve 2 can be used to connect a vacuum pump, and the third valve 3 can be used to connect a sterile vacuum liquid collection bag.

[0043] The filter funnel 4 is located between the first cavity C and the second cavity D. The filter funnel 4 can be used to filter the liquid in the first cavity C into the second cavity D.

[0044] In some embodiments, the filter funnel 4 may include a filter screen plate, on which filter paper may be placed above the sieve holes of the filter screen plate, and the filter paper may be used to filter the liquid flowing out of the outlet end of the saline well.

[0045] In some embodiments, the internal protrusion 5 of the cavity can be used to support the filter funnel 4, which can be removed when not in use to save space and facilitate maintenance. After the filter funnel 4 is removed, the extracted liquid concentrate can be collected based on the piston container.

[0046] In some embodiments, the filter paper may include at least one of the following: qualitative filter paper with a pore size of 0.45 μm, microbial cell collection filter paper with a pore size of 0.22 μm, and microbial cell collection filter paper with a pore size of 0.1 μm.

[0047] For details, please refer to Figure 2 As shown, Figure 2 This is a top view of the filter funnel 4. Filter paper with different pore sizes can be placed above the sieve holes 7 of its filter screen plate, such as qualitative filter paper with a pore size of 0.45 μm, microbial cell collection filter paper with a pore size of 0.22 μm, or microbial cell collection filter paper with a pore size of 0.1 μm. To prevent the filter paper from moving during the filtration process, a ring of steel weights 8 can be placed in the center of the filter screen plate to compact the edges of the filter paper.

[0048] By placing filter paper with different pore sizes on the filter screen plate of the filter funnel 4, bacterial cells and ultra-small microorganisms in the saline water layer can be collected quickly, and a relatively complete microbial community sample can be obtained.

[0049] In some embodiments, the first valve 1 and the second valve 2 are located on the same side, and the third valve 3 is located on the other side of the first valve 1 or the second valve 2.

[0050] Specifically, the first valve 1 can be located on the side wall of the first chamber, near the upper end cap A. It can be connected to the outlet end of the saline well, facilitating the entry of liquid flowing from the saline well outlet into the first chamber. The second valve 2 can be located on the side wall of the second chamber, near the filter funnel 4. It can be connected to a filtration pump, which accelerates the flow of liquid through the filter paper. The third valve 3 can be located on the side wall of the second chamber, near the lower end cap B. It can be connected to a sterile vacuum liquid collection bag for collecting the filtered liquid. The sterile vacuum liquid collection bag can have a capacity threshold (maximum capacity), such as 1L. Once this value is exceeded, the third valve 3 can be connected to a new sterile vacuum liquid collection bag. The second valve 2 is located below the first valve 1, and the third valve 3 is located below the second valve 2.

[0051] In some embodiments, the piston container may further include: an upper end cover A and a lower end cover B, wherein when the upper end cover A and the lower end cover B are closed, the first cavity C and the second cavity D form a sealed cavity.

[0052] The second cavity D is located below the first cavity C.

[0053] In some embodiments, the piston container may further include: a fourth valve and a piston. The fourth valve may be disposed outside the lower end cover. The fourth valve may be connected to a manual pump. When the fourth valve is opened, liquid is injected into the lower part of the piston through the manual pump to increase the pressure of the first chamber and the second chamber until the pressure of the saline aquifer is reached.

[0054] Specifically, the fourth valve 6 is located outside the lower end cover B, and the piston E is located on the upper end cover B. Opening the fourth valve 6 allows liquid to be injected into the lower part of the piston E via a connected manual pump, pushing the piston E upward to increase the liquid pressure in the first and second chambers until it reaches the underground pressure of the saline aquifer. The first and second chambers are considered as a single, integrated chamber. The manual pump connected to the fourth valve 6 is a pressure pump connected to the outside of the piston container, making it easy to bring to the site. The liquid injected into the lower part of the piston E can be deionized water, etc.

[0055] In some embodiments, the bottom inner diameter of the piston container can range from 10cm to 15cm, the height from 40cm to 60cm, and the volume from 3.1L to 10.6L. The ratio of the cylinder's inner cavity height to its diameter ranges from 2.6 to 6.0. Theoretically, the higher this ratio, the better. However, if the bottom diameter is too large, the design difficulty for high-pressure resistance will also increase.

[0056] The piston container described above can be used to easily and quickly collect microorganisms from the saline aquifer on-site, ensuring the integrity of the underground microbial community information and providing more comprehensive information for subsequent sequencing results.

[0057] This specification also provides an embodiment of a method for sampling and collecting microorganisms in a saline aquifer. For specific implementation details, please refer to [reference needed]. Figure 3 As shown, the method, based on the piston container described above, may include the following:

[0058] S301: Sequentially open the second valve connected to the filtration pump, the first valve connected to the saline wellhead outlet, and the third valve connected to the sterile vacuum liquid collection bag, so that the liquid flowing out from the saline wellhead outlet flows into the second chamber after being filtered by the first-diameter qualitative filter paper and is collected into the sterile vacuum liquid collection bag.

[0059] S302: When the liquid collection volume exceeds the capacity of the sterile vacuum liquid collection bag, collect the qualitative filter paper and replace it with microbial cell collection filter paper with a second pore size, connect the third valve to the new first sterile vacuum liquid collection bag, add the liquid collected by the sterile vacuum liquid collection bag to the first chamber, so that the liquid flowing out of the first chamber flows into the second chamber after being filtered by the microbial cell collection filter paper with a second pore size, and is collected into the new first sterile vacuum liquid collection bag;

[0060] S303: When the liquid collection volume exceeds the capacity of the new first sterile vacuum liquid collection bag, collect the microbial cell collection filter paper with the second pore size and replace it with the microbial cell collection filter paper with the third pore size, repeat the filtration and collection work until the number of collected filter papers reaches the preset number.

[0061] S304: The collected microbial cells are collected by filter paper into a sterile centrifuge tube, which is placed in a dry ice bucket containing dry ice.

[0062] In some embodiments, the above-described duplicate filtering collection process includes:

[0063] Connect the third valve to a new second sterile vacuum liquid collection bag, and add the liquid collected in the new first sterile vacuum liquid collection bag to the first chamber, so that the liquid flowing out of the first chamber flows into the second chamber after being filtered by the third microbial collection filter paper with a third pore size, and is collected into the new second sterile vacuum liquid collection bag. The first pore size is 0.45 μm, the second pore size is 0.22 μm, and the third pore size is 0.1 μm.

[0064] Specifically, the second valve 2 connected to the filtration pump can be opened, and the filtration pump can be used to filter for a certain period of time, such as 1 minute. Then, the first valve 1 connected to the outlet of the saline well and the third valve 3 connected to the sterile vacuum liquid collection bag can be opened, so that the liquid flowing out from the outlet of the saline well is filtered through a qualitative filter paper with a first pore size (such as 0.45μm qualitative filter paper, which is placed above the sieve holes 7 of the filter screen plate) and flows into the second chamber D. Then, it is collected into the connected sterile vacuum liquid collection bag through the third valve 3.

[0065] When the liquid collection volume exceeds the capacity of the sterile vacuum liquid collection bag (e.g., exceeding 1L), the second valve 2, the first valve 1, and the third valve 3 can be closed sequentially. Open the upper cap A and collect the 0.45μm qualitative filter paper into the container. Then place the second-pore-size microbial cell collection filter paper (e.g., 0.22μm pore-size microbial cell collection filter paper) above the sieve holes 7 of the filter sieve plate, replacing the 0.45μm qualitative filter paper with 0.22μm pore-size microbial cell collection filter paper. Connect the second valve 2 to the vacuum pump and the third valve 3 to the new first sterile vacuum plastic collection bag. Add the liquid collected in the sterile vacuum liquid collection bag to the first chamber C, so that the liquid flowing out of the first chamber C, after being filtered by the second-pore-size microbial cell collection filter paper, flows into the second chamber D and is collected into the new first sterile vacuum liquid collection bag.

[0066] When the liquid collection volume exceeds the capacity of the new first sterile vacuum liquid collection bag (e.g., exceeding 1L), collect the microbial cell collection filter paper with the second pore size into a sterile 2mL centrifuge tube. After collecting enough microorganisms under the 0.22μm filter membrane for subsequent experiments, place the microbial cell collection filter paper with the third pore size (e.g., 0.1μm pore size) above the sieve holes 7 of the filter plate and repeat the filtration and collection process. When the number of collected filter papers reaches the preset number (the preset number can be set according to actual test needs; this instruction manual does not specify a specific limit), stop the filtration and collection process. Collect the microbial cell collection filter paper with the third pore size into a sterile 2mL centrifuge tube and place the centrifuge tube in a dry ice bucket containing dry ice. The ice bucket should be transported to the laboratory promptly for bacterial DNA extraction and high-throughput sequencing.

[0067] In some embodiments, after collecting the collected microbial cells into a sterile centrifuge tube using the aforementioned collection filter paper, the process may further include:

[0068] Open the top cover of the piston container, disconnect the vacuum pump connected to the second valve and the sterile vacuum liquid collection bag connected to the third valve, and close the first valve, second valve and third valve. Remove the filter screen plate on the filter funnel.

[0069] Liquid flowing from the wellhead outlet of the saline layer flows into the first and second chambers through the upper end cover. When the amount of liquid flowing in exceeds the capacity of the chambers, the upper end cover is closed.

[0070] Open the fourth valve on the lower end cover, and inject liquid into the lower part of the piston through the manual pump connected to the fourth valve to increase the pressure of the first and second chambers until it reaches the underground pressure of the saline aquifer. Then, put the entire chamber into an insulated box for transportation.

[0071] Specifically, once the number of collected filter papers reaches the preset quantity—that is, after collecting enough bacterial filter paper for testing and analysis—the upper cover A of the piston container can be opened. The suction pump connected to the second valve 2 and the sterile vacuum liquid collection bag connected to the third valve 3 can be disassembled. The first valve 1, second valve 2, and third valve 3 can be closed, and the filter funnel 4 can be removed. Liquid flowing from the wellhead outlet of the saline aquifer flows from the upper cover A into the first chamber C + second chamber D. When the amount of liquid flowing in exceeds the chamber capacity (i.e., the entire chamber is full of liquid), the upper cover A can be closed or covered. Then, the fourth valve 6 on the lower cover B can be opened, and liquid can be injected into the lower part of the piston E through the manual pump connected to the fourth valve 6 to increase the overall pressure of the chamber until it reaches the underground pressure of the saline aquifer. The entire chamber can then be placed in an insulated box for transportation.

[0072] The above method can rapidly collect microbial cells from saline aquifers at the wellhead, obtaining relatively complete microbial community samples. Simultaneously, after collecting the microbial cells, the produced fluid is collected in its original form using a piston container and pressurized on-site to saline aquifer pressure before transportation, thus obtaining relatively complete original produced fluid samples.

[0073] In a specific example, taking a piston container with a bottom diameter of 10cm, a height of 50cm, and a volume of approximately 4L as an example, the sampling and collection process of microorganisms in the saline layer can be described, which may include the following steps.

[0074] (1) Connect the second valve 2 to the filtration pump and the third valve 3 to a sterile vacuum plastic collection bag (G), approximately 1L. Cover with the top cap A, connect the first valve 1 to the outlet of the saline well to form a closed system, and close the fourth valve 6.

[0075] (2) Open the second valve 2, turn on the filtration pump and filtration for one minute, then open the first valve 1 and the third valve 3, so that the liquid flows through the filter paper and into the chamber D, and then through the third valve 3 into the sterile vacuum liquid collection bag. After the liquid has been collected to 1L, quickly replace the collection bag.

[0076] (3) When the sample volume is sufficient (more than 1L), close the second valve 2, the first valve 1 and the third valve 3 in sequence. Open the upper cover A and collect the 0.45μm filter paper into the container.

[0077] (4) Place the 0.22μm pore size microbial cell collection filter paper in the center of the filter screen plate. To prevent the filter paper from moving during the filtration process, place a ring of steel weights 8 in the center of the filter screen plate to press the edges of the filter paper firmly.

[0078] (5) Connect the second valve 2 to the filtration pump and the third valve 3 to the new first sterile vacuum plastic collection bag (H). Open the second valve 2, start the filtration pump and filtration for one minute, then open the third valve 3. Add the collected liquid from G to chamber C, allowing the liquid to flow through the filter paper into chamber D, and then through the third valve 3 into H. Once 1L of liquid has been collected, quickly replace the collection bag.

[0079] (6) When the sample volume is sufficient (more than 1L), close valves 2 and 3 in sequence. Open the top cap A and quickly collect the 0.22μm filter paper into a sterile 2mL centrifuge tube. Place the centrifuge tube in a dry ice bucket containing dry ice.

[0080] (7) After collecting enough bacterial cells under the 0.22 μm filter membrane for subsequent experiments by repeating steps (4)-(6), place the 0.1 μm pore size microbial cell collection filter paper in the center of the filter sieve plate. To prevent the filter paper from moving during the filtration process, place a ring of steel weights 8 in the center of the filter sieve plate to compact the edges of the filter paper. Repeat steps (5)-(6). The dry ice bucket is promptly transported to the laboratory for bacterial DNA extraction and high-throughput sequencing.

[0081] (8) After collecting enough microbial cells for testing and analysis using filter paper, open the top cover A, disconnect the vacuum pump and collection bag connected to the second valve 2 and the third valve 3 respectively, and close the valves. Close the first valve 1. Remove the middle filter funnel 4.

[0082] (9) Liquid from the wellhead outlet of the saline aquifer flows into cavity C+D from end A of the upper cover. After filling, the upper cover A is closed. Connect the fourth valve 6 to a simple manual pump, open the fourth valve 6, and inject liquid into the lower part of the piston to increase the pressure of the entire cavity to the underground pressure of the saline aquifer.

[0083] (10) Close the fourth valve 6. Place the entire cavity into the insulated box. Transport promptly to ensure that the sampled liquid and its microbial life characteristics are kept in a high-temperature, high-pressure, and high-fidelity state.

[0084] The present invention can achieve the following beneficial effects: First, it can quickly filter suspended solids, collect bacterial cells, and transport bacterial cells with dry ice in the saline aquifer, making the bacterial cell information of underground microorganisms more complete and the subsequent sequencing results more comprehensive; Second, it can carry out pressure and heat preservation transportation, which greatly improves the fidelity of liquid samples; Third, it is simple and convenient to operate.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A piston container, characterized in that, include: A first cavity, with a first valve provided on the side wall of the first cavity, the first valve being used to connect to the liquid outlet end of the saline wellhead; A second cavity is located below the first cavity. A second valve and a third valve located below the second valve are provided on the side wall of the second cavity. The second valve is used to connect to a vacuum pump, and the third valve is used to connect to a sterile vacuum liquid collection bag. A filter funnel located between the first chamber and the second chamber is used to filter liquid from the first chamber to the second chamber.

2. The piston container according to claim 1, characterized in that, A filter screen plate is movably mounted on the filter funnel, and filter paper is placed above the sieve holes of the filter screen plate. The filter paper is used to filter the liquid flowing out of the wellhead of the saline layer.

3. The piston container according to claim 2, characterized in that, The filter paper includes at least one of the following: qualitative filter paper with a pore size of 0.45 μm, microbial cell collection filter paper with a pore size of 0.22 μm, and microbial cell collection filter paper with a pore size of 0.1 μm.

4. The piston container according to claim 1, characterized in that, The first valve and the second valve are located on the same side, and the third valve is located on the other side of the first valve or the second valve.

5. The piston container according to claim 1, characterized in that, The piston container further includes an upper end cover and a lower end cover, wherein when the upper end cover and the lower end cover are closed, the first cavity and the second cavity form a sealed cavity.

6. The piston container according to claim 5, characterized in that, The piston container also includes a fourth valve and a piston. The fourth valve is located outside the lower end cover and is connected to a manual pump. When the fourth valve is opened, liquid is injected into the lower part of the piston through the manual pump to increase the pressure of the first chamber and the second chamber until it reaches the underground pressure of the saline aquifer.

7. The piston container according to claim 1, characterized in that, The piston container has a bottom inner diameter ranging from 10cm to 15cm, a height ranging from 40cm to 60cm, and a volume ranging from 3.1L to 10.6L.

8. A method for sampling and collecting microorganisms in a saline aquifer, characterized in that, Based on the piston container according to any one of claims 1-7, the method comprises: The second valve connected to the filtration pump, the first valve connected to the outlet of the saline well, and the third valve connected to the sterile vacuum liquid collection bag are opened in sequence so that the liquid flowing out from the outlet of the saline well flows into the second chamber after being filtered by the qualitative filter paper with the first aperture, and is collected into the sterile vacuum liquid collection bag. When the liquid collection volume exceeds the capacity of the sterile vacuum liquid collection bag, the qualitative filter paper is collected and replaced with microbial cell collection filter paper with a second pore size. The third valve is connected to a new first sterile vacuum liquid collection bag, and the liquid collected by the sterile vacuum liquid collection bag is added to the first chamber so that the liquid flowing out of the first chamber flows into the second chamber after being filtered by the microbial cell collection filter paper with a second pore size, and is collected into the new first sterile vacuum liquid collection bag. When the liquid collection volume exceeds the capacity of the new first sterile vacuum liquid collection bag, collect the microbial cell collection filter paper with the second pore size and replace it with the microbial cell collection filter paper with the third pore size. Repeat the filtration and collection process until the number of collected filter papers reaches the preset number. The collected microbial cells were collected from filter paper into sterile centrifuge tubes, which were placed in a dry ice bucket containing dry ice.

9. The method according to claim 8, characterized in that, The repeated filtering and collection process includes: Connect the third valve to a new second sterile vacuum liquid collection bag, and add the liquid collected in the new first sterile vacuum liquid collection bag to the first chamber, so that the liquid flowing out of the first chamber flows into the second chamber after being filtered by the third microbial collection filter paper with a third pore size, and is collected into the new second sterile vacuum liquid collection bag. The first pore size is 0.45 μm, the second pore size is 0.22 μm, and the third pore size is 0.1 μm.

10. The method according to claim 8, characterized in that, The method further includes: Open the top cover of the piston container, disconnect the suction pump connected to the second valve and the sterile vacuum liquid collection bag connected to the third valve, and close the first valve, second valve and third valve. Remove the filter screen plate on the filter funnel. The liquid flowing out from the wellhead of the saline layer flows into the first chamber and the second chamber through the upper end cover. When the amount of liquid flowing in exceeds the capacity of the chamber, the upper end cover is closed. Open the fourth valve on the lower end cover, and inject liquid into the lower part of the piston through the manual pump connected to the fourth valve to increase the pressure of the first and second chambers until it reaches the underground pressure of the saline aquifer. Then, put the entire chamber into an insulated box for transportation.

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