Integrated sediment methanogenesis anaerobic culture bottle

Abstract

The invention discloses an integrated sediment methanogenesis anaerobic culture bottle, and relates to the field of microorganism culture and detection.The integrated sediment methanogenesis anaerobic culture bottle comprises a bottle body and an integrated bottle cap assembly, the integrated bottle cap assembly comprises a main cap body, a barometer, an integrated guide pipe and a sampling one-way valve, the main cap body is in sealed connection with a bottle opening of the bottle body, and the integrated guide pipe is connected with the sampling one-way valve; the barometer is installed on the main cover body, a pressure sensing probe of the barometer is communicated with a headspace in a bottle, one end of the integrated guide pipe is communicated with the headspace in the bottle, and the other end of the integrated guide pipe penetrates through the main cover body to be connected with the sampling one-way valve. The integrated sediment methanogenesis anaerobic culture bottle provided by the invention has the functions of air pressure monitoring, lossless gas taking and convenient sampling, and the accuracy, convenience and data output quality of a sediment methanogenesis long-term culture experiment are remarkably improved.

Description

An integrated sediment-based anaerobic culture flask for methanogenesis Technical Field

[0001] This invention relates to the field of microbial culture and detection, and in particular to an integrated anaerobic culture flask for methanogenic sediment production. Background Technology

[0002] Methanogenesis in sediments is a crucial link in the carbon cycle of ecosystems such as wetlands, lakes, and oceans, and is also an important topic for studying the mechanisms and regulatory factors of methane production, a greenhouse gas. In laboratory studies, anaerobic culture flasks are typically used to simulate the in-situ environment. Sediments are cultured in a closed system, and gas, liquid, and solid samples are collected periodically from the flasks to analyze changes in methane production, related intermediate products, and microbial community structure.

[0003] Existing anaerobic culture devices for methanogenesis from sediments are mostly modified from traditional serum bottles or similar glass containers. The standard procedure involves placing the sediment and overlying water into the bottle, sealing it with a butyl rubber stopper and aluminum cap to create an anaerobic environment. During cultivation, researchers typically use a glass syringe or gas chromatograph needle to directly puncture the rubber gasket of the stopper to extract air from the top of the bottle for gas composition (such as CH4 and CO2) analysis. Furthermore, if monitoring the physicochemical properties and microbiological indicators of the sediment or overlying water is required, destructive sampling is often necessary. This involves opening the bottle cap or inserting a pipette or sampler from the bottle opening to obtain samples, or dismantling the entire culture system at the end of the experiment for sample separation.

[0004] While such existing technologies are widely used, they have significant limitations in long-term, dynamic monitoring studies: First, the gas pressure inside the culture flask cannot be known in real time, and the methanogenesis process itself causes pressure changes. Gas sampling also instantly alters the pressure inside the flask. This pressure fluctuation may affect microbial activity (especially methanogenic archaea) and gas dissolution balance, leading to experimental errors. However, existing devices lack simple, in-situ gas pressure monitoring methods. Second, repeatedly puncturing the rubber gasket with a needle to extract gas leaves pinholes. As the number of gas extractions increases, the sealing performance of the gasket will inevitably decrease, potentially leading to external oxygen infiltration, disrupting the anaerobic environment, or minor internal gas leakage, affecting the accuracy of gas quantification. Finally, obtaining sediment or overlying water samples from the flask opening is extremely inconvenient, easily disturbing the culture system, and making it difficult to achieve precise, undisturbed sampling of the overlying water-sediment interface, thus restricting multi-dimensional, high-temporal-resolution monitoring.

[0005] Therefore, those skilled in the art are dedicated to developing an integrated sediment methanogenic anaerobic culture flask with functions such as pressure monitoring, non-destructive gas extraction, and convenient sampling, to improve research efficiency and data quality, more reliably assess environmental methanogenic potential, and provide a more solid data foundation for greenhouse gas emission reduction policy formulation. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to enable anaerobic culture bottles to have functions of gas pressure monitoring, non-destructive gas extraction and convenient sampling.

[0007] To achieve the above objectives, the present invention provides an integrated anaerobic culture flask for methanogenesis from sediments, characterized in that it comprises a flask body and an integrated cap assembly, wherein the integrated cap assembly includes a main cap body, a barometer, an integrated conduit, and a sampling check valve. The main cap body is sealed to the mouth of the flask body, the barometer is mounted on the main cap body, and the pressure sensing probe of the barometer is in communication with the headspace inside the flask. One end of the integrated conduit is in communication with the headspace inside the flask, and the other end passes through the main cap body and is connected to the sampling check valve.

[0008] Furthermore, the bottle body is made of high borosilicate glass.

[0009] Furthermore, the main cap is connected to the bottle mouth of the bottle body via threads, and a sealing gasket is provided at the connection between the main cap and the bottle body.

[0010] Furthermore, the sampling check valve is a Luer locking connector valve with a threaded protective cap, and the inlet of the sampling check valve is sealed to the top end of the integrated conduit.

[0011] Furthermore, the integrated bottle cap assembly is also provided with a spare sealing plug, which is a spare opening pre-reserved on the main cap body and sealed by a rubber gasket.

[0012] Furthermore, the integrated conduit is a hollow stainless steel tube, and the position where the integrated conduit passes through the main cover body is sealed and fixed.

[0013] Furthermore, the bottle body also includes a mud inlet assembly, which is located in the lower half of the side wall of the bottle body. The mud inlet assembly includes a first interface tube and a first sealing plug. The first interface tube is inclined towards the bottle mouth. One end of the first interface tube is fixedly connected to the bottle body, and the other end is sealed by the first sealing plug.

[0014] Furthermore, the bottle body also includes a water inlet assembly, which is located in the upper middle part of the side wall of the bottle body. The water inlet assembly is higher than the mud inlet assembly. The water inlet assembly includes a second interface tube, one end of which is fixedly connected to the bottle body, and the second interface tube is inclined towards the bottle mouth.

[0015] Furthermore, the water intake assembly also includes a second sealing plug, through which the other end of the second interface tube is sealed.

[0016] Furthermore, the other end of the second interface tube is sealed by a rubber diaphragm.

[0017] The beneficial technical effects of this invention are as follows: 1. Existing technologies cannot obtain real-time, in-situ information on the gas pressure changes inside anaerobic culture bottles during cultivation and sampling. Gas pressure is a key physical parameter affecting the metabolic activities and gas-liquid balance of methanogenic microorganisms. Its unknown state may lead to inaccurate control of experimental conditions and biased data interpretation. This invention integrates a miniature barometer (or pressure sensor) into the top cap assembly of the culture bottle, enabling real-time, visual, or digital monitoring of the gas pressure inside the bottle. The sensing element of the miniature barometer is directly connected to the headspace inside the bottle through a dedicated channel on the cap, allowing direct sensing of the gas pressure inside the bottle. Its reading device (such as an analog dial or digital display) is located outside the cap, facilitating researchers to observe and record gas pressure data at any time without interfering with the culture system. This achieves in-situ, continuous, and non-destructive monitoring of the gas pressure environment inside the culture bottle, enabling researchers to accurately grasp the pressure dynamics during methanogenesis, assess the pressure disturbances caused to the system by gas sampling operations, and more accurately calculate gas production (combined with headspace volume) based on the gas pressure data, thus improving the accuracy and repeatability of the experiment. This invention reduces the risk of experimental failure or inaccurate data due to unknown stress factors, improves scientific research efficiency and data quality, helps to more reliably assess the potential for methane production in the environment, and provides a more solid data foundation for the formulation of greenhouse gas emission reduction policies.

[0018] 2. Existing technologies involve repeatedly puncturing the bottle cap gasket with a syringe or injection needle to extract gas. This leads to an irreversible decrease in the gasket's sealing performance with repeated use, making it difficult to maintain the stable and strictly controlled anaerobic environment required for long-term cultivation (e.g., weeks to months). This invention replaces the traditional gas extraction method by incorporating a dedicated one-way sampling valve (such as a Luer connector valve with a sealing cap, a diaphragm valve, or a similar structure) on the bottle cap. This one-way valve is normally closed and sealed, with its internal channel connected to the headspace inside the bottle. When gas extraction using a syringe is required, the syringe interface is connected to the dedicated interface on the outside of the valve and tightened or locked. At this time, the internal channel of the valve opens, allowing gas to be extracted. After sampling, the syringe is disconnected, and the valve automatically or manually returns to a closed and sealed state. The entire process does not require puncturing any elastic seals. This invention permanently solves the problem of sealing failure caused by gas extraction. It ensures a high degree of sealing and stability of the anaerobic environment inside the bottle throughout the entire cultivation cycle, preventing oxygen infiltration and gas leakage, and guaranteeing the authenticity of gas sample concentration and the accuracy of quantitative gas extraction. At the same time, it also reduces the risk of sample contamination or damage due to the replacement of damaged gaskets, extends the service life of the core sealing components of the culture flask, and reduces the cost of consumable (gasket) replacement and sample loss due to seal failure.

[0019] 3. Existing technologies make it very difficult to obtain sediment or overlying water samples from the top cap of the culture flask, and this process easily and severely disturbs the culture system, making convenient, targeted, and minimally disturbed sample collection impossible. This invention provides dedicated sampling ports (such as threaded interfaces with stopcocks or ports with sealing diaphragms) with sealed caps on the sidewall of the culture flask, in the lower half (sediment layer) and the upper middle part (overlying water layer), respectively. The mud and water sampling ports are pre-embedded at specific heights within the flask, with their inner openings communicating with the flask's interior and their outer openings sealed by reusable caps. When sampling is needed, the corresponding sampling port can be operated on (e.g., piercing the diaphragm with a syringe, or unscrewing the stopper and using a micro-sampler) to obtain directional sediment or overlying water samples without opening the main cap or reaching deep into the flask opening for sampling. This invention enables convenient, targeted, and low-disturbance sampling of solid (sediment) and liquid (overlying water) samples in a culture system. This greatly facilitates the dynamic monitoring of sediment physicochemical properties (such as pH, Eh, and organic carbon concentration), microbial biomass / community, and overlying water chemical indicators (such as dissolved CH4 and CO2) during the culture process, without terminating the culture or severely disrupting the microenvironment within the flask. This invention supports more complex and refined experimental designs, enabling high-throughput, multi-indicator time-series monitoring within the same culture flask, thus advancing in-depth research into sediment microbial ecology and biogeochemical cycling.

[0020] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0021] Figure 1 is a structural schematic diagram of an integrated sediment methanogenic anaerobic culture bottle according to a preferred embodiment of the present invention; Figure 2 is a schematic diagram of a commonly used sediment methanogenic anaerobic culture device in the prior art; wherein, 1-bottle body, 2-integrated bottle cap assembly, 21-main cap body, 22-barometer, 23-integrated conduit, 24-sampling one-way valve, 25-spare sealing plug, 3-sludge inlet assembly, 31-first interface pipe, 32-first sealing stopcock, 4-water inlet assembly, 41-second interface pipe, 42-second sealing stopcock. Detailed Implementation

[0022] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0023] In the accompanying drawings, components with the same structure are indicated by the same numerical designation, and components with similar structures or functions are indicated by similar numerical designations. The dimensions and thicknesses of each component shown in the drawings are arbitrary, and the present invention does not limit the dimensions and thicknesses of each component. To make the illustrations clearer, the thickness of some components has been appropriately exaggerated in the drawings.

[0024] The structure of a commonly used anaerobic sediment methanogenesis culture device in the prior art is shown in Figure 2. This invention provides an integrated anaerobic sediment methanogenesis culture bottle with functions of pressure monitoring, non-destructive gas extraction, and convenient sampling, as shown in Figure 1. The device mainly includes: a bottle body 1, an integrated bottle cap assembly 2, a mud inlet assembly 3, and a water inlet assembly 4.

[0025] Bottle 1 is made of transparent or semi-transparent borosilicate glass, cylindrical in shape, and can be in common sizes such as 250mL, 500mL or 1000mL, used to hold sediment samples and overlying water; the side walls of the bottle are precision machined and have two openings that are slightly inclined towards the bottle mouth; the bottom of the bottle can be designed to be flat or slightly concave to facilitate the stability of the sample placement.

[0026] The integrated bottle cap assembly 2 consists of the following parts: main cap body 21, made of corrosion-resistant metal (such as stainless steel) or high-strength engineering plastic, which is connected to the bottle body 1 mouth by threads and a polytetrafluoroethylene sealing gasket (or butyl rubber septum) can be placed at the connection to ensure airtightness.

[0027] The barometer 22 is embedded in one side of the main cap 21. Its pressure sensing probe extends downwards and communicates directly with the headspace inside the bottle through a tiny channel inside the cap. The barometer dial or digital display is exposed upwards or to the side for easy observation. It can be either a mechanical pointer type or a digital display type, with a typical range of -100 kPa to +200 kPa (or selected according to specific requirements).

[0028] The integrated conduit 23 is a hollow, thin stainless steel tube that passes through the main sealing gasket and extends downwards into the headspace inside the bottle. Its top end is fixed to the through hole at the top of the cap by an interference fit or sealant, and is connected to the inlet of the sampling check valve 24 above. The wall thickness and inner diameter of the conduit must ensure smooth gas flow and have sufficient mechanical strength.

[0029] The sampling check valve 24 is fixedly installed on the other side of the main cover 21, off-center. This embodiment uses a Luer locking connector valve with a threaded protective cap. The valve inlet is sealed to the top of the integrated conduit 23, and the outlet is a standard external Luer connector. The internal structure of this check valve (such as an umbrella-shaped valve core or a spring valve core) gives it unidirectional conduction characteristics: under normal conditions, the valve core is closed by spring force or its own structure, isolating the inside and outside; during sampling, the protective cap is unscrewed, and a glass syringe or gas chromatograph injection needle with a corresponding Luer locking tip is screwed in and locked. When the Luer connector of the syringe is connected to it and the plunger is withdrawn, the valve core opens under the pressure difference between the inside and outside of the vial, allowing headspace gas from the vial to enter the syringe through the integrated conduit 23 and the sampling check valve 24; after evacuation, the syringe is disconnected, and the valve core automatically resets and closes, then the protective cap is screwed back on for secondary protection. This design ensures that the sampling channel is only temporarily opened when needed.

[0030] The spare sealing plug 25 and the main cover 21 can also have a spare port sealed with a butyl rubber gasket for initial vacuuming / nitrogen filling to create an anaerobic environment, or for adding reagents with a syringe. This port is not the main gas intake channel and is used infrequently.

[0031] The mud inlet assembly 3 is located on the lower half of the side wall of the bottle body 1. Its height can be set according to the typical sediment cultivation thickness. It includes a first interface tube 31 and a first sealing plug 32.

[0032] The first interface tube 31 is a short glass tube that is sintered and connected to the bottle body as one piece, and has external threads machined on its outer end.

[0033] The first sealing plug 32 is made of polytetrafluoroethylene and is hollow inside. One end has an internal thread that matches the external thread of the first interface tube 31. When tightened, it achieves a seal. During sampling, the first sealing plug 32 is unscrewed, and a long micro sampler (such as a syringe with the needle tip cut off and connected to a thin plastic tube) can be used to draw sediment slurry near the interface or at a specific depth through the first interface tube 31.

[0034] The water intake assembly 4 is located in the upper middle part of the side wall of the bottle body 1, higher than the mud intake port, and within the preset overlying water layer. Its basic structure is similar to that of the mud intake assembly 3, including a second interface tube 41 and a second sealing plug 42. The water intake 4 can also adopt a pre-opened diaphragm sealing port. That is, the outer end of the interface tube is sealed with a thickened silicone / butyl rubber diaphragm. During sampling, the overlying water is extracted directly by piercing the diaphragm with a syringe needle, which is faster, but the diaphragm has a limited puncture life.

[0035] Assembly and use of integrated sediment methanogenic anaerobic culture flasks: During the experiment, first unscrew the integrated cap assembly 2, and then fill the flask body 1 with the sediment and top layer of water in sequence. Tighten the integrated cap assembly 2. Using the gasket at the spare sealing plug 25, connect the vacuum pump and nitrogen source with a double-ended needle to perform a vacuum-nitrogen-filling cycle to create an anaerobic environment. Then, place the culture flask in a constant temperature incubator.

[0036] Pressure monitoring: Throughout the culture period, the pressure inside the bottle is recorded by observing the reading of barometer 22.

[0037] Gas sampling: Headspace gas is periodically collected by connecting a syringe to the sampling check valve 24 for gas chromatography analysis. Gas pressure changes are recorded after sampling.

[0038] Solid / Liquid Sampling: According to the experimental design, at specific time points, small amounts of sediment and overlying water samples are collected through the mud sampling assembly 3 and the water sampling assembly 4, respectively, for analysis of microorganisms (such as DNA extraction) and chemicals (such as organic carbon and dissolved CH4). After sampling, each port is quickly closed.

[0039] The culture flask of this invention achieves pressure visualization through an integrated barometer; ensures long-term sealing through a one-way valve for gas extraction; and enables convenient and low-disturbance acquisition of solid and liquid samples through a side-wall sampling port. The combination of these three features significantly improves the accuracy, convenience, and data output quality of long-term methanogenic culture experiments on sediments.

[0040] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. An integrated anaerobic culture flask for methanogenesis from sediments, characterized in that, The device includes a bottle body and an integrated cap assembly. The integrated cap assembly includes a main cap body, a barometer, an integrated conduit, and a sampling check valve. The main cap body is sealed to the bottle mouth of the bottle body. The barometer is mounted on the main cap body. The pressure sensing probe of the barometer is in communication with the headspace inside the bottle. One end of the integrated conduit is in communication with the headspace inside the bottle, and the other end passes through the main cap body and is connected to the sampling check valve.

2. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 1, characterized in that, The bottle body is made of borosilicate glass.

3. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 1, characterized in that, The main cap is connected to the bottle mouth of the bottle body by threads, and a sealing gasket is provided at the connection between the main cap and the bottle body.

4. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 1, characterized in that, The sampling check valve is a Luer locking connector valve with a threaded protective cap, and the inlet of the sampling check valve is sealed to the top of the integrated conduit.

5. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 1, characterized in that, The integrated bottle cap assembly is also provided with a spare sealing plug, which is a spare opening pre-reserved on the main cap body and sealed by a rubber gasket.

6. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 1, characterized in that, The integrated conduit is a hollow stainless steel tube, and the position where the integrated conduit passes through the main cover is sealed and fixed.

7. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 1, characterized in that, The bottle body also includes a mud inlet assembly, which is located on the lower half of the side wall of the bottle body. The mud inlet assembly includes a first interface tube and a first sealing stopper. Tilt towards the bottle opening One end of the first interface tube is fixedly connected to the bottle body, and the other end is sealed by the first sealing plug.

8. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 7, characterized in that, The bottle body also includes a water inlet assembly, which is located in the upper middle part of the side wall of the bottle body. The water inlet assembly is higher than the mud inlet assembly. The water inlet assembly includes a second interface tube, one end of which is fixedly connected to the bottle body and is inclined towards the bottle mouth.

9. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 8, characterized in that, The water intake assembly also includes a second sealing plug, and the other end of the second interface pipe is sealed by the second sealing plug.

10. The integrated sediment-based methanogenic anaerobic culture flask as described in claim 8, characterized in that, The other end of the second interface tube is sealed by a rubber diaphragm.

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