Portable anaerobic culture device and application thereof

Abstract

A portable anaerobic culture device and its application, including a box, composed of a gas mixing and purifying block, a gas filling and pumping block and a gas injection block, the device continuously injects oxygen-free gas into the environment medium through the gas injection block to perform oxygen removal pretreatment on the environment medium in an open system, replaces the gas in the culture bottle through the gas filling and pumping block in a closed system, and improves the oxygen removal efficiency in the environment sample through the two-step treatment. The device is provided with a ball valve and a one-way valve to provide double protection for the gas path pipeline and the purification column, solve the problem of easy aging that may occur in the non-connected state of the system, improve the experimental stability, and prolong the service life of the device. When in use, the device only needs to be connected with a conventional gas cylinder and a vacuum pump in the laboratory to start working. The box of the device is currently made, and the improvement of the internal pipeline layout of the box can further reduce the volume. The device occupies less space and is easy to move, and can be conveniently used in laboratories with different research backgrounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of anaerobic culture device, and particularly relates to a device for preparing anaerobic culture system for environmental samples and application thereof. BACKGROUND

[0002] In the field of environmental science, it is a very important content to study biological transformation processes under different oxygen conditions. For example, in the study of site soil, pollutants are discharged into the soil and then seep into groundwater. The surface soil pollutants mainly undergo aerobic degradation, while the pollutants in deep soil and groundwater undergo anaerobic transformation. For example, in the study of farmland soil, aerobic degradation is studied for dry soil remediation, while anaerobic degradation under flooded conditions is mainly studied in paddy fields. Moreover, in the actual environment, aerobic-anaerobic conditions exist alternately in space and time, and there is no obvious boundary. It is of practical significance to analyze the aerobic and anaerobic biological transformation processes and contributions in a polluted environment for pollution remediation. Generally, aerobic research can be carried out in an open environment, and no special equipment is needed. In contrast, anaerobic research requires high equipment, not only a large anaerobic incubator, but also a complex gas pipeline on the wall, a special oxygen removal device and other supporting facilities to support a strict anaerobic system, which leads to high prices of imported incubators. In fact, some laboratories are not specialized in anaerobic research, and the demand for configuration of anaerobic devices in terms of land occupation and use cost limits these laboratories to carry out anaerobic culture work.

[0003] For the creation of anaerobic culture system, some simple solutions are proposed in China to replace the anaerobic incubator, the basic principle is through the way of alternating gas / vacuum to replace the gas in the culture bottle, such as patent application publication number CN210237634U, the name is anaerobic culture medium preparation system; patent application publication number CN107175242A, the name is anaerobic culture bottle gas filling and vacuum cleaning system; patent application publication number CN104450508A, the name is anaerobic microbial inoculation device, anaerobic microbial culture system and anaerobic microbial inoculation and culture method, these systems need to set up the gas circuit in the wall or metal support, which has certain demand for laboratory floor area. Patent application publication number CN203794901A, the name is anaerobic culture device and anaerobic culture system, provides a portable gas filling / vacuum equipment. However, at present, these systems are mainly used for preparing anaerobic microbial culture medium or anaerobic gas bottle, and lack of anaerobic system preparation for environmental samples. First of all, environmental samples such as soil, sediment and straw are solid objects, and the internal porous structure itself adsorbs a lot of oxygen, so it is not easy to remove the gas / vacuum of the headspace gas. Secondly, the traditional equipment adopts fine needle to insert into the culture bottle, and the fine needle directly inserted into the environmental samples such as soil will cause the needle to be blocked. Although there are organic matter and microorganism in the environmental sample, the oxygen will also be consumed after the system is closed for a period of time, but the biological factors need to be excluded in the research, and it is important to improve the data reliability and stability to unify the system conditions before culture. SUMMARY

[0004] The technical problem solved by the present application is to provide a portable anaerobic culture device and its application to improve the oxygen removal efficiency of environmental samples and to be suitable for popularization and application in different laboratories.

[0005] Technical solution: A portable anaerobic culture device, comprising a box body, composed of a gas mixing and purification block, an air charging and pumping block and a gas injection block, the gas mixing and purification block comprises a group of parallel float flow meters and gas purification columns, the gas inlet end of the float flow meter is connected with the gas cylinder interface through the two-way ball valve A pipeline, the gas outlet end of the float flow meter is connected with the gas inlet end of the gas purification column through the one-way valve A, and the gas outlet end of the gas purification column is connected with the three-way ball valve A; the air charging and pumping block comprises a three-way ball valve B, a vacuum pump interface, a stable pressure gauge and a group of parallel exhaust port small holes, one end of the three-way ball valve B is connected with the three-way ball valve A through the pipeline, the vacuum pump interface is connected with one end of the three-way ball valve B through the one-way valve B, one end of the stable pressure gauge is connected with the three-way ball valve B, and the other end is connected with a group of parallel gas outlet small holes through the one-way valve C, and a pressure relief valve is arranged on the parallel pipeline, and a two-way ball valve B is arranged on each pipeline connected with the gas outlet small hole; the gas injection block comprises a group of parallel gas injection small holes and micro-adjusting valves, the gas injection small holes are connected with the micro-adjusting valves through the one-way valve D respectively, and the micro-adjusting valves are connected with the three-way ball valve A through the two-way ball valve C.

[0006] The gas cylinder interface is a 1 / 8 in cartridge joint, and the vacuum pump interface is a 1 / 8 in cartridge joint.

[0007] The exhaust port small hole is connected with a sharp needle, and the gas injection small hole is connected with a flat needle.

[0008] The float flow meter is connected with N2, CO2 and H2 gas cylinders through the gas cylinder interface respectively.

[0009] The gas injection small hole is connected with N2, CO2 and H2 gas cylinders through the gas cylinder interface respectively.

[0010] The exhaust port small hole is connected with N2, CO2 and H2 gas cylinders through the gas cylinder interface respectively.

[0011] The internal pipeline of the box body is connected with copper pipes, and the external pipeline of the box body is connected with PTFE pipes.

[0012] The application of the portable anaerobic culture device includes the following steps: (1) connecting a gas cylinder through a gas cylinder interface and connecting a vacuum pump through a vacuum pump interface; (2) opening the gas cylinder, opening two-way ball valve A, opening two-way ball valve C, rotating three-way ball valve A into the gas injection block, adjusting the fine adjustment valve to reduce the overall gas flow rate, and adjusting the gas ratio by adjusting the knob of the float flowmeter; (3) adjusting the fine adjustment valve knob to increase the gas flow rate, inserting the connected flat needle into the soil, slurry or culture medium, injecting mixed gas, and performing medium oxygen removal pretreatment; after a period of aeration, the medium is divided into culture bottles that have been aerated, and a butyl rubber plug is covered and sealed with an aluminum cap; (4) rotating three-way ball valve A into the aeration and gas extraction block, and then rotating three-way ball valve B into the aeration gas path; (5) inserting the sharp needle connected to the aeration and gas extraction block into the sealed culture bottle to inject gas, then inserting a separate sharp needle into the culture bottle cap, and maintaining the gas replacement for a period of time; (6) pulling out the separate sharp needle, observing the pressure rise in the bottle through the pressure stabilizing pressure gauge, and protecting the bottle when the pressure rises to 25 psi and no longer rises; (7) opening the vacuum pump, rotating three-way ball valve B into the gas extraction path, and observing the pressure drop in the bottle through the pressure gauge, and when the pressure drops to below -10 psi, rotating three-way ball valve B, and the gas cylinder enters the aeration state again; (8) rotating three-way ball valve B to realize aeration / gas extraction circulation, and after 3-5 reciprocations, the gas replacement in the bottle is completed, and finally the gas in the bottle is slightly higher than atmospheric pressure; all two-way valves are closed, the gas cylinder and the vacuum pump are closed, and the connection with the device is disconnected.

[0013] Beneficial effects: 1. Functional integration: the device continuously injects anaerobic gas into the environment medium for oxygen removal pretreatment through the gas injection block in the open system, and replaces the gas in the culture bottle in the closed system through the aeration and gas extraction block, thereby improving the oxygen removal efficiency in the environmental sample. 2. Portability: the device is provided with ball valves and check valves to provide double protection for the gas path pipeline and the purification column, solve the problem of easy aging of the system in the non-connected state, improve the experimental stability, and prolong the service life of the device. When used, the device only needs to be connected to the laboratory conventional gas cylinder and vacuum pump to start working. The size of the device box is 50cm(L)*10cm(w)*40cm(H), and the internal pipeline layout of the improved box can further reduce the volume. The device occupies less space and is easy to move, and can be conveniently used in laboratories with different research backgrounds. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The appearance schematic diagram of the device of the application;

[0015] Figure 2 The internal mechanism schematic diagram of the device of the application;

[0016] Figure 3 The actual picture of the device of the present application;

[0017] In the above figure: 1 box, 2 gas cylinder interface, 3 vacuum pump interface, 4 two-way ball valve A, 5 float flowmeter, 6 three-way ball valve A, 7 three-way ball valve B, 8 stable pressure gauge, 9 two-way ball valve B, 10 exhaust port small hole, 11 two-way ball valve C, 12 fine adjustment valve, 13 gas injection port small hole, 14 one-way valve A, 15 gas purification column, 16 one-way valve B, 17 one-way valve C, 18 one-way valve D, 19 pressure relief valve;

[0018] Figure 4 The device is used for studying the application of anaerobic bioremediation of polychlorinated biphenyl contaminated soil.

[0019] In the figure: FCK: anaerobic non-flooded; F1: anaerobic non-flooded + zero-valent iron; GCK: anaerobic complete flooding; G1: anaerobic complete flooding + zero-valent iron; HCK: aerobic non-flooded; H1: aerobic non-flooded + zero-valent iron;

[0020] Figure 5 The device is used for studying the application of anaerobic bioremediation of polycyclic aromatic hydrocarbon contaminated site soil.

[0021] In the figure: CK: control of contaminated soil; T1: aerobic; T2: anaerobic. DETAILED DESCRIPTION

[0022] The present application is further described below in combination with specific examples.

[0023] Example 1:

[0024] The portable anaerobic culture device comprises a box body 1, a gas mixing and purifying block, a gas filling and exhausting block and a gas injection block are arranged in the box body, the gas mixing and purifying block comprises a group of parallel float flow meters 5 and a gas purifying column 15, the gas inlet end of the float flow meter 5 is connected with the gas cylinder interface 2 through a two-way ball valve A 4 pipeline, the gas outlet end of the float flow meter 5 is connected with the gas inlet end of the gas purifying column 15 through a one-way valve A 14 pipeline, and the gas outlet end of the gas purifying column 15 is connected with a three-way ball valve A 6; the gas filling and exhausting block comprises a three-way ball valve B 7, a vacuum pump interface 3, a constant pressure pressure gauge 8 and a group of parallel exhaust port small holes 10, one end of the three-way ball valve B 7 is connected with the three-way ball valve A 6, the vacuum pump interface 3 is connected with one end of the three-way ball valve B 7 through a one-way valve B 16, one end of the constant pressure pressure gauge 8 is connected with the three-way ball valve B 7, the other end is connected with the group of parallel exhaust port small holes 10 through a one-way valve C 17, a pressure relief valve 19 is arranged on the parallel pipeline, and a two-way ball valve B 9 is arranged on each pipeline connected with the exhaust port small hole; the gas injection block comprises a group of parallel gas injection port small holes 13 and a fine adjustment valve 12, the gas injection port small holes 13 are respectively connected with the fine adjustment valve 12 through a one-way valve D 18, and the fine adjustment valve 12 is connected with the three-way ball valve A 6 through a two-way ball valve C 11. The gas cylinder interface 2 is a 1 / 8 in card sleeve joint, the vacuum pump interface 3 is a 1 / 8 in card sleeve joint. The exhaust port small hole 10 is connected with a sharp needle, and the gas injection port small hole 13 is connected with a flat needle. The float flow meter 5 is connected with N2, CO2 and H2 gas cylinders through a gas cylinder interface respectively. The gas injection port small hole 13 is connected with three parallel holes. The exhaust port small hole 10 is connected with five parallel holes. The internal pipeline of the box body is connected with copper pipes, and the external pipeline of the box body is connected with PTFE pipes.

[0025] Example 2

[0026] Preparation of oxygen-free water

[0027] Preparation of oxygen-free water for adjusting the proportion of water in an oxygen-free culture system.

[0028] Using the device of Example 1, connect the N2 gas cylinder to cylinder interface 2, and seal the other two cylinder interfaces with a sleeve cap. Connect the vacuum pump to vacuum pump interface 3. Open the N2 gas cylinder, open two-way ball valve A 4, open two-way ball valve C 11, and screw three-way ball valve A 6 into the gas injection block. Adjust the gas flow rate by adjusting the float flow meter 5 and the fine adjustment valve 12. Insert one of the flat needle heads connected to the gas injection block into a round-bottom flask pre-filled with distilled water. Heat the bottom of the flask, and when the distilled water boils, place the round-bottom flask on ice to cool. Keep the air on during this period. Add 0.2 mM L-cysteine and 0.2 mM sodium sulfide as reducing agents, and add 0.25 mg / L resazurin as an indicator. Dissolve and mix evenly by magnetic stirring. Use a liquid dispenser to divide the cooled distilled water into anaerobic culture bottles (previously aerated with another bottle needle). Cover with a butyl rubber plug and seal with an aluminum cap.

[0029] Screw three-way ball valve A 6 into the gas injection / exhaust block, and then screw three-way ball valve B 7 on the gas injection / exhaust line into the gas injection line. Insert the sharp needle connected to the gas injection / exhaust block into the headspace of the sealed culture bottle to inject gas, and then insert a separate sharp needle into the bottle cap. Inject and exhaust gas alternately for about 3 minutes. Remove the separate sharp needle, and observe the pressure rise in the bottle through the pressure stabilizer 8. When the pressure rises to 25 psi and no longer rises, open the vacuum pump and screw three-way ball valve B 7 on the gas injection / exhaust line into the exhaust line. Observe the pressure drop in the bottle through the pressure stabilizer 8. When the pressure drops to -10 psi or below, rotate three-way ball valve B 7 on the gas injection / exhaust line, and the gas cylinder enters the gas injection state again. Rotate three-way ball valve B 7 on the gas injection / exhaust line to achieve a cycle of gas injection / exhaust, and repeat for 3 times to complete the replacement of the gas in the bottle. Close all valves, turn off the gas cylinder and vacuum pump, and disconnect the device.

[0030] The prepared solution is subjected to high-temperature sterilization treatment, and the color will change from the initial pink to a lighter or colorless state, indicating that the water body is in a deoxygenated state.

[0031] Example 2: Study on anaerobic bioremediation of polychlorinated biphenyl contaminated soil

[0032] An artificial polychlorinated biphenyl contaminated farmland soil (initial concentration of 1.01 ± 0.96 mg·kg -1 ) is used for anaerobic bioremediation research. The anaerobic non-flooded state is set by adjusting the maximum field water capacity to 60% with anaerobic water, and the contribution of rhizosphere anaerobic microenvironment to biodegradation in upland soil is evaluated. The anaerobic complete flooding state is set by adjusting the water-soil ratio to 2:1 with anaerobic water, and the biodegradation effect in the repair scene of paddy field is simulated.

[0033] Using the device of Example 1, the N2 gas cylinder and the CO2 gas cylinder are connected with the gas cylinder interface 2 respectively, and the other interface is sealed with a sleeve plug. The vacuum pump is connected with the vacuum pump interface 3; the gas cylinder is opened, the two-way ball valve A 4 of N2 and CO2 is opened, the two-way ball valve C 11 is opened, the three-way ball valve A 6 is screwed into the gas injection block, the overall gas flow rate is adjusted by adjusting the micro-adjusting valve 12, the gas ratio is adjusted to 8:2 by adjusting the float flow meter 5 corresponding to N2 and CO2, the outflow rate is increased by adjusting the micro-adjusting valve 12 knob. Insert one of the flat needle heads connected to the gas injection block into the anaerobic culture bottle preloaded with the organic contaminated soil sample, and operate multiple culture bottles at the same time. Keep aeration for 15-20 min, cover with a butyl rubber plug and seal with an aluminum cap, add anaerobic water to adjust the soil moisture content to 60% of the maximum field water holding capacity.

[0034] The N2 gas cylinder and the CO2 gas cylinder are connected with the gas cylinder interface 2 respectively, and the other interface is sealed with a sleeve plug. The vacuum pump is connected with the vacuum pump interface 3; the gas cylinder is opened, the two-way ball valve A 4 of N2 and CO2 is opened, the two-way ball valve C 11 is opened, the three-way ball valve A 6 is screwed into the gas injection block, the overall gas flow rate is adjusted by adjusting the micro-adjusting valve 12, the gas ratio is adjusted to 8:2 by adjusting the float flow meter 5 corresponding to N2 and CO2, the outflow rate is increased by adjusting the micro-adjusting valve 12 knob. Organic contaminated soil and anaerobic water are mixed into mud in a ratio of 1:2 and added to the anaerobic culture bottle, and one of the flat needle heads connected to the gas injection block is inserted into the anaerobic culture bottle containing the sample, and multiple culture bottles can be operated at the same time. Keep aeration for 15-20 min, cover with a butyl rubber plug and seal with an aluminum cap.

[0035] The three-way ball valve A 6 is screwed into the gas injection / exhaust block, and the three-way ball valve B 7 on the gas injection / exhaust pipeline is screwed into the gas injection pipeline; the sharp needle head connected to the gas injection / exhaust block is inserted into the headspace of the sealed culture bottle to inject gas, and then a separate sharp needle head is inserted into the culture bottle cap, the gas is injected and exhausted simultaneously, and maintained for about 3 minutes; the separate sharp needle head is pulled out, the pressure rise in the bottle is observed through the pressure stabilizing pressure gauge 8, and when the pressure rises to 25 psi, it stops rising; the vacuum pump is opened, the three-way ball valve B 7 on the gas injection / exhaust pipeline is screwed into the exhaust pipeline, and the pressure drop in the bottle is observed through the pressure stabilizing pressure gauge 8, and when the pressure drops to-10 psi or below, the three-way ball valve B 7 on the gas injection / exhaust pipeline is rotated, and the gas cylinder enters the gas injection state again; by rotating the three-way ball valve B 7 on the gas injection / exhaust pipeline, the gas injection / exhaust cycle is realized, and the gas in the bottle is replaced for 3 times; all valves are closed, the gas cylinder and the vacuum pump are closed, and the connection with the device is disconnected.

[0036] The anaerobic bottle is incubated at 28 ℃, and after 2 months, the total amount and composition of polychlorinated biphenyls in the contaminated soil are detected. The results show that Figure 4), anaerobic treatment (FCK, GCK) and aerobic treatment (HCK) did not significantly reduce the total amount of pollutants in the soil, but significantly promoted the transformation of high-chlorine components to low-chlorine components in the soil. Compared with aerobic treatment (HCK), anaerobic non-flooded treatment (FCK) reduced 6-chlorine components by 13.5%, 5-chlorine components by 18.4%, 4-chlorine components by 28.0%, and 2-chlorine components by 20.2%; anaerobic complete flooding treatment (GCK) reduced 6-chlorine components by 30.7%, 5-chlorine components by 14.8%, 4-chlorine components by 22.0%, and 2-chlorine components by 58.7%; between the two anaerobic states, the efficiency of high-chlorine to low-chlorine pollutant transformation was more obvious under anaerobic complete flooding treatment (GCK). At the same time, the addition of zero-valent iron in the treatment did not significantly change the total amount and composition of pollutants in the soil under aerobic conditions (H1), but further promoted the transformation of high-chlorine components to low-chlorine components under anaerobic conditions (F1, G1). Compared with aerobic treatment (FCK), anaerobic non-flooded + zero-valent iron (F1) reduced 6-chlorine components by 32.7%, 5-chlorine components by 27.7%, 4-chlorine components by 6.6%, and 2-chlorine components by 92.9%; anaerobic complete flooding treatment (G1) reduced 6-chlorine components by 36.9%, 5-chlorine components by 27.0%, 4-chlorine components by 8.9%, and 2-chlorine components by 100%.

[0037] Example 3: Study on anaerobic bioremediation of polycyclic aromatic hydrocarbon contaminated soil

[0038] The polycyclic aromatic hydrocarbon contaminated soil (initial concentration of 161 ± 29.4 mg kg -1 ) was collected for anaerobic bioremediation research. The anaerobic flooding state was set by adjusting the water-soil ratio to 1:1 with anaerobic water, simulating the biodegradation effect of the soil under the groundwater saturation state.

[0039] Using the device of Example 1, connect the N2 gas cylinder and the CO2 gas cylinder to the gas cylinder interface 2 respectively, and seal the other interface with a sleeve plug. Connect the vacuum pump to the vacuum pump interface 3; open the gas cylinder, open the two-way ball valve A 4 of N2 and CO2, open the two-way ball valve C 11, rotate the three-way ball valve A 6 into the gas injection block, adjust the fine tuning valve 12 to reduce the overall gas flow rate, and adjust the gas ratio to 8:2 by adjusting the corresponding N2 and CO2 float flow meters 5 knobs; adjust the fine tuning valve 12 knob to increase the gas flow rate. Insert one of the flat needle heads connected to the gas injection block into the anaerobic culture bottle preloaded with the organic contaminated soil sample, and operate multiple culture bottles at the same time. Keep aeration for 15~20 min, cover with a butyl rubber plug and seal with an aluminum cap, add anaerobic water to adjust the water-soil ratio to 1:1.

[0040] The three-way ball valve A 6 is screwed into the inflation / exhaustion block, and the three-way ball valve B 7 on the inflation / exhaustion pipeline is screwed into the inflation gas path; the pointed needle connected with the inflation / exhaustion block is inserted into the headspace of the sealed culture bottle to inject gas, and then a separate pointed needle is inserted into the bottle cap; the gas is inflated and exhausted simultaneously, and maintained for about 3 minutes; the separate pointed needle is pulled out, and the pressure rise in the bottle is observed through the constant pressure gauge 8; when the pressure increases to 25 psi, it stops rising; the vacuum pump is opened, the three-way ball valve B 7 on the inflation / exhaustion pipeline is screwed into the exhaust gas path, and the pressure drop in the bottle is observed through the constant pressure gauge 8; when the pressure drops to below-10 psi, the three-way ball valve B 7 on the inflation / exhaustion pipeline is rotated, and the gas cylinder enters the inflation state again; the inflation / exhaustion cycle is realized by rotating the three-way ball valve B 7 on the inflation / exhaustion pipeline, and the gas in the bottle is replaced for 3 times; all valves are closed, the gas cylinder and the vacuum pump are closed, and the connection with the device is disconnected.

[0041] The anaerobic bottle was incubated at 28°C, and the total amount and composition of polycyclic aromatic hydrocarbons in the contaminated soil were detected after 2 months. Figure 5 ), both aerobic treatment (T1) and anaerobic treatment (T2) promoted the degradation of polycyclic aromatic hydrocarbons in the soil, and the total amount of polycyclic aromatic hydrocarbons decreased by 31.0% under anaerobic treatment. Among them, the degradation effect of high-ring components was more obvious, with 34.1% for indeno(1,2,3-cd)pyrene, 33.3% for dibenzo(a,h)anthracene, and 32.6% for benzo(g,h,i)perylene.

[0042] The above-described embodiments are only preferred embodiments given to better explain the present application, and therefore the above-described embodiments are not used to limit the present application, and modifications, improvements and replacements made within the principles of the present application should be included in the protection scope of the present application.

Claims

1. A portable anaerobic culture device, comprising a housing (1), characterized in that, The chamber contains a gas mixing and purification zone, a gas filling and evacuation zone, and a gas injection zone. The gas mixing and purification zone includes a set of parallel float flowmeters (5) and a gas purification column (15). The inlet end of the float flowmeter (5) is connected to the gas cylinder interface (2) via a two-way ball valve A (4). The exhaust end of the float flowmeter (5) is connected to the inlet end of the gas purification column (15) via a one-way valve A (14). The exhaust end of the gas purification column (15) is connected to a three-way ball valve A (6). The gas filling and evacuation zone includes a three-way ball valve B (7), a vacuum pump interface (3), a pressure gauge (8), and a set of parallel exhaust ports (10). One end of the three-way ball valve B (7) is connected to the three-way ball valve A (6). The vacuum pump interface (3) is connected to one end of the three-way ball valve B (7) via a one-way valve B (16). One end of the pressure gauge (8) is connected to the three-way ball valve A (6). Valve B (7) is connected, and the other end is connected to a set of outlet holes (10) in parallel via a one-way valve C (17). A pressure relief valve (19) is provided on the parallel pipeline. When the pressure inside the bottle exceeds 25 psi, it will automatically release pressure. A two-way ball valve B (9) is provided on each pipeline connected to the outlet hole. The gas injection block includes a set of parallel gas injection holes (13) and a fine-tuning valve (12). The gas injection holes (13) are connected to the fine-tuning valve (12) via a one-way valve D (18). The gas injection holes (13) are connected to a flat needle for direct insertion into soil, mud or culture medium for open system ventilation and deoxygenation. The fine-tuning valve (12) is connected to the three-way ball valve A (6) via a two-way ball valve C (11). The exhaust end of the gas purification column (15) is connected to both the gas filling and gas extraction block and the gas injection block. The gas path direction is switched via the three-way ball valve A (6).

2. The portable anaerobic culture device according to claim 1, characterized in that, The gas cylinder interface (2) is a 1 / 8-inch compression fitting, and the vacuum pump interface (3) is a 1 / 8-inch compression fitting.

3. The portable anaerobic culture device according to claim 1, characterized in that, The small hole (10) of the exhaust port is connected to a pointed needle.

4. The portable anaerobic culture device according to claim 1, characterized in that, The float flowmeter (5) consists of three parallel units, which are connected to N2, CO2, and H2 gas cylinders respectively through the gas cylinder interface.

5. The portable anaerobic culture device according to claim 1, characterized in that, The air injection port (13) consists of three small holes connected in parallel.

6. The portable anaerobic culture device according to claim 1, characterized in that, The exhaust port (10) consists of 5 holes connected in parallel.

7. The portable anaerobic culture device according to claim 1, characterized in that, The internal piping of the enclosure is connected by copper pipes, and the external piping is connected by PTFE pipes.

8. The application of the portable anaerobic culture device according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Connect the gas cylinder to the required gas cylinder through the gas cylinder interface (2), and connect the vacuum pump to the vacuum pump through the vacuum pump interface (3); (2) Open the gas cylinder, open the two-way ball valve A (4), open the two-way ball valve C (11), turn the three-way ball valve A (6) into the gas injection block, adjust the fine adjustment valve (12) to reduce the overall gas flow rate, and adjust the gas ratio by adjusting the knob of the float flowmeter (5); (3) Adjust the knob of the fine adjustment valve (12) to increase the outflow rate, insert the connecting flat-mouth needle into the soil, mud or culture medium, inject the mixed gas, and perform pretreatment for medium deoxygenation; After a period of time, the medium is dispensed into culture bottles that have been ventilated, and the bottles are capped with butyl rubber stoppers and sealed with aluminum caps; (4) Three-way ball valve A (6) is screwed into the inflation and degassing block, and then three-way ball valve B (7) is screwed into the inflation gas path; (5) The pointed needle connected to the inflation and degassing block is inserted into the sealed culture bottle to inject gas, and then another separate pointed needle is inserted into the cap of the culture bottle. The gas is vented and released at the same time, and the gas in the bottle is replaced for a period of time; (6) The separate pointed needle is removed, and the pressure inside the bottle is observed to rise through the pressure gauge (8). Due to the pressure stabilization effect, the pressure increases to 25. (7) Turn on the vacuum pump and turn the three-way ball valve B (7) into the evacuation air path. Observe the pressure drop in the bottle through the pressure gauge. When it drops to below -10 psi, turn the three-way ball valve B (7) and the gas cylinder will enter the filling state again. (8) By turning the three-way ball valve B (7), the filling / evacuation cycle is realized. Repeat the cycle 3 to 5 times to complete the gas replacement in the bottle. Finally, adjust the gas pressure in the bottle to be slightly higher than atmospheric pressure. Close all two-way valves, turn off the gas cylinder and vacuum pump, and then disconnect the connection with the device.

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