Closed system culture device and culture method for cable bacteria

Abstract

The invention discloses a closed system culture device and culture method.The culture device comprises a culture test tube, a rubber plug for sealing the culture test tube, an air inlet needle and an air outlet needle, and the culture test tube comprises a tube body and a pressure gauge communicated with the tube body; a sediment layer, an ultrapure water layer and a headspace gas layer are sequentially arranged in the pipe body from bottom to top, the air inlet needle and the air outlet needle penetrate through the rubber plug in parallel and are inserted into the pipe body, the tip end of the air inlet needle is lower than that of the air outlet needle, an air inlet is formed in the top end of the air inlet needle, and the air inlet is connected with a pure oxygen bottle through an air pipe to conduct aeration in the pipe body. And the pressure gauge is communicated with the headspace gas layer. According to the closed system culture device for the cable bacteria, a new method is provided for quickly, simply and conveniently culturing the cable bacteria, the pollution risk can be remarkably reduced, the culture purity and reliability of the cable bacteria are improved, and the consistency and repeatability of experimental results are improved by accurately controlling culture conditions and enhancing the repeatability of culture experiments.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial cultivation, and more specifically, to a closed system cultivation device and a cultivation method for cable bacteria. Background Art

[0002] Cable bacteria are a special type of filamentous bacteria in the Desulfobulbaceae family. They form a long chain of cells up to several centimeters long by connecting multiple cells end to end. Cable bacteria play an important role in the natural environment, especially in promoting the degradation of organic pollutants, regulating the migration and circulation of metal elements, alleviating the toxicity of sulfides, and reducing greenhouse gas emissions. However, the main culture method for cable bacteria is continuous aeration culture in an open system. Although this culture method fits the in situ environment, the limitations brought by the open system also appear. For example, the open system lacks strict isolation measures and is easily disturbed by external microorganisms, thus affecting the growth of cable bacteria and experimental results; in the open system, we cannot provide the most favorable growth conditions according to the growth characteristics of cable bacteria, resulting in the growth rate and abundance of cable bacteria failing to meet expectations; the conditions in the open system are unstable and difficult to control, and the reproducibility of the experimental results is poor, which increases the uncertainty of the experimental results of cable bacteria. Therefore, it is urgent to provide a method for culturing cable bacteria under closed system conditions to solve the problems of contamination risk, difficulty in condition control, resource consumption, complexity of experimental operation, and data reproducibility faced by microbial culture in open systems. Summary of the invention

[0003] The object of the present invention is to provide a device and method for culturing cable bacteria in a closed system.

[0004] The first aspect of the present invention provides a closed system culture device for cable bacteria, comprising a culture test tube, a rubber plug for sealing the culture test tube, an air inlet needle and an air outlet needle, the culture test tube comprising a tube body and a pressure gauge connected to the tube body, a sediment layer, an ultrapure water layer and a headspace gas layer are arranged in the tube body from bottom to top, the air inlet needle and the air outlet needle pass through the rubber plug in parallel and are inserted into the tube body, the tip of the air inlet needle is lower than the tip of the air outlet needle, an air inlet is arranged at the top of the air inlet needle, the air inlet is connected to a pure oxygen cylinder through an air pipe to aerate the tube body, and the pressure gauge is connected to the headspace gas layer.

[0005] In some embodiments, the thickness of the ultrapure water layer is 2 mm, the tip of the air inlet needle is located 5 mm above the ultrapure water layer, and the tip of the air outlet needle is located 5 mm below the rubber plug.

[0006] In some embodiments, the volume ratio of the sediment layer to the headspace gas layer is 1:2.

[0007] In some embodiments, the culture tube further comprises a one-way valve, one end of which is connected to the headspace gas layer of the tube body, and the other end of which is connected to the pressure gauge.

[0008] In some embodiments, a filter membrane is provided in the air inlet, and the filter membrane is a microporous filter membrane with a pore size of 0.22 μm.

[0009] In some embodiments, the tube body is a test tube with a specification of 18*18 mm, and the rubber plug is a butyl rubber plug, which matches the tube opening of the tube body.

[0010] The second aspect of the present invention provides a closed system culture method for cable bacteria, which uses a closed system culture device for cable bacteria to culture cable bacteria, and comprises the following steps:

[0011] S1. Pre-treating sediments collected from typical black and smelly rivers to obtain pre-treated sediments;

[0012] S2. The pretreated sediment obtained in step S1 is loaded into the tube body itself to form a sediment layer, ultrapure water is added above the sediment layer to form a 2 mm thick ultrapure water layer, the tube mouth is sealed with a butyl rubber plug, and the capping device is sealed;

[0013] S3. Insert an air inlet needle and an air outlet needle in parallel on the outside of the butyl rubber plug, wherein the tip of the air inlet needle is located 5 mm above the ultrapure water layer, and the tip of the air outlet needle is located 5 mm below the butyl rubber plug. Aerate the culture tube with pure oxygen through the air inlet needle for 5 minutes to make the oxygen concentration in the headspace gas layer reach 0.2 MPa, thus completing the establishment of a closed system culture device for cable bacteria;

[0014] S4. The closed system culture device for cable bacteria is placed at 25° C. in the dark to culture the cable bacteria contained in the sediment layer.

[0015] In some embodiments, the sediment collected from a typical black and smelly river is pretreated in step S1 by removing large foreign matter from the sediment using a 0.50 mm pore size sieve, mixing and standing for 24 hours, and then removing the supernatant to obtain pretreated sediment.

[0016] In some embodiments, the physical and chemical parameters of the sediments collected from the typical black and smelly river in step S1 are: AVS content is not less than 1.50 mg / g, and TOC content is not less than 40 mg / g.

[0017] In some embodiments, the volume of the pretreated sediment loaded in step S2 is 11 mL, and the aeration volume during the aeration process in step S3 is 1 cm 3 / min, and during the aeration process, the culture tube was placed in a constant temperature water bath at 25℃ for continuous aeration.

[0018] Beneficial effects of the present invention: The closed system culture device for cable bacteria constructed by the present invention provides a new method for quickly and easily culturing cable bacteria, which can significantly reduce the risk of contamination, improve the culture purity and reliability of cable bacteria, and by precisely controlling the culture conditions, enhance the repeatability of the culture experiment, improve the consistency and repeatability of the experimental results, make long-term culture experiments feasible, maintain a stable growth environment for cable bacteria, and have important value for studying the adaptability and evolution of cable bacteria. At the same time, it reduces human errors, improves accuracy, and provides suitable growth conditions for microorganisms that are difficult to grow in an open environment or are sensitive to environmental changes, thereby maximizing the utilization of resources and saving culture costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic structural diagram of a closed system culture device for cable bacteria according to Example 1 of the present invention.

[0020] Figure 2 This is a photo of the cable bacteria cultured in Example 2 of the present invention under an optical microscope.

[0021] Figure 3 This is a photograph of the cable bacteria cultured in Example 2 of the present invention under an electron microscope. DETAILED DESCRIPTION

[0022] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0023] Example 1

[0024] A closed system culture device for cable bacteria comprises a culture test tube 1, a rubber plug 2 for sealing the culture test tube, an air inlet needle 3 and an air outlet needle 4. The culture test tube 1 comprises a tube body 11, a one-way valve 12 connected to the tube body, and a pressure gauge 13 connected to the one-way valve 12. One end of the one-way valve 12 is connected to the headspace air layer 16 of the tube body 11, and the other end is connected to the pressure gauge 13. The tube body 11 is a test tube with a specification of 18*18mm, the rubber plug 2 is a butyl rubber plug, and the rubber plug 2 matches the pipe mouth of the tube body 11. A sediment layer 14, an ultrapure water layer 15 and a headspace air layer 16 are arranged in sequence in the tube body 11 from bottom to top. The volume ratio of the sediment layer 14 to the volume of the headspace air layer 16 is 1:2, and the thickness of the ultrapure water layer 15 is 2mm.

[0025] The air inlet needle 3 and the air outlet needle 4 pass through the rubber plug 2 in parallel and are inserted into the tube body 11, and the tip of the air inlet needle 3 is lower than the tip of the air outlet needle 4. The tip of the air inlet needle 3 is located 5 mm above the ultrapure water layer 15, and the tip of the air outlet needle 4 is located 5 mm below the rubber plug 2, and is located in the headspace air layer 16. An air inlet 31 is provided at the top of the air inlet needle 3, and a microporous filter membrane with a pore size of 0.22 μm is provided in the air inlet 31. The air inlet 31 is connected to a pure oxygen cylinder through an air pipe to aerate the inside of the tube body 11.

[0026] Example 2

[0027] A closed system culture method for cable bacteria, wherein the cable bacteria are cultured by constructing a closed system culture device for cable bacteria of Example 1, comprising the following steps:

[0028] S1. Pre-treat the sediment collected from typical black and smelly rivers, remove large foreign matter in the sediment with a 0.50 mm pore size sieve, mix and let stand for 24 hours, then remove the supernatant to obtain pre-treated sediment, wherein the physical and chemical parameters of the collected sediment are: AVS content not less than 1.50 mg / g, TOC content not less than 40 mg / g;

[0029] S2. 11 mL of the pretreated sediment obtained in step S1 was loaded into the tube body 11 to form a sediment layer 14. Ultrapure water was added above the sediment layer 14 to form a 2 mm thick ultrapure water layer 15. The tube mouth was sealed with a butyl rubber plug and sealed with a capping device;

[0030] S3. Insert an air inlet needle 3 and an air outlet needle 4 in parallel on the outside of the butyl rubber plug, wherein the tip of the air inlet needle 3 is located 5 mm above the ultrapure water layer 15, and the tip of the air outlet needle 4 is located 5 mm below the butyl rubber plug. 3 / min aeration volume for pure oxygen aeration for 5 minutes, during the aeration process, the culture tube was placed in a 25°C constant temperature water bath for continuous aeration, so that the oxygen concentration in the headspace gas layer 16 reached 0.2MPa, and a closed system culture device for cable bacteria was established;

[0031] S4. The cable bacteria closed system culture device is placed at 25° C. in the dark to culture the cable bacteria contained in the sediment layer 14 .

[0032] After 14 days of cultivation, the cultured cable bacteria were characterized by optical microscopy or electron microscopy to confirm the successful cultivation of cable bacteria. Figure 2 , Figure 3 .

[0033] Depend on Figure 2 and Figure 3It can be seen that the cable bacteria were successfully cultured using the method of this embodiment, and the cable bacteria were cultured in a closed system, which significantly reduced the risk of contamination during the culture process, improved the purity and reliability of the culture, and enhanced the repeatability by precisely controlling the influence of conditions. At the same time, it makes the long-term culture experiment of cable bacteria feasible and maintains a stable growth environment, which is of great value for studying the adaptability and evolution of cable bacteria.

[0034] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A closed system culture device for cable bacteria, characterized in that: It comprises a culture test tube, a rubber plug for sealing the culture test tube, an air inlet needle and an air outlet needle. The culture test tube comprises a tube body and a pressure gauge connected to the tube body. A sediment layer, an ultrapure water layer and a headspace gas layer are arranged in the tube body from bottom to top. The air inlet needle and the air outlet needle pass through the rubber plug in parallel and are inserted into the tube body. The tip of the air inlet needle is lower than the tip of the air outlet needle. An air inlet is arranged on the top of the air inlet needle. The air inlet is connected to a pure oxygen cylinder through an air pipe to aerate the tube body. The pressure gauge is connected to the headspace gas layer.

2. A closed system culture device for cable bacteria according to claim 1, characterized in that: The thickness of the ultrapure water layer is 2 mm, the tip of the air inlet needle is located 5 mm above the ultrapure water layer, and the tip of the air outlet needle is located 5 mm below the rubber plug.

3. A closed system culture device for cable bacteria according to claim 1, characterized in that: The volume ratio of the sediment layer to the headspace gas layer is 1:

2.

4. A closed system culture device for cable bacteria according to claim 1, characterized in that: The culture test tube also includes a one-way valve, one end of which is connected to the headspace gas layer of the tube body, and the other end of which is connected to the pressure gauge.

5. The closed system culture device for cable bacteria according to claim 1, characterized in that: A filter membrane is arranged in the air inlet, and the filter membrane is a microporous filter membrane with a pore size of 0.22 μm.

6. A closed system culture device for cable bacteria according to claim 1, characterized in that: The tube body is a test tube with a specification of 18*18 mm, and the rubber plug is a butyl rubber plug, which matches the tube opening of the tube body.

7. A closed system culture method for cable bacteria, characterized in that: The cable bacteria are cultured using a closed system culture device for cable bacteria according to any one of claims 1 to 6, comprising the following steps: S1. Pre-treating sediments collected from typical black and smelly rivers to obtain pre-treated sediments; S2. The pretreated sediment obtained in step S1 is loaded into the tube body itself to form a sediment layer, ultrapure water is added above the sediment layer to form a 2 mm thick ultrapure water layer, the tube mouth is sealed with a butyl rubber plug, and sealed with a capping device; S3. Insert an air inlet needle and an air outlet needle in parallel on the outside of the butyl rubber plug, wherein the tip of the air inlet needle is located 5 mm above the ultrapure water layer, and the tip of the air outlet needle is located 5 mm below the butyl rubber plug. Aerate the culture tube with pure oxygen through the air inlet needle for 5 minutes to make the oxygen concentration in the headspace gas layer reach 0.2 MPa, thus completing the establishment of a closed system culture device for cable bacteria; S4. The closed system culture device for cable bacteria is placed at 25° C. in the dark to culture the cable bacteria contained in the sediment layer.

8. The closed system culture method for cable bacteria according to claim 7, characterized in that: In step S1, the sediment collected from the typical black and smelly river is pretreated by removing large foreign matter from the sediment using a 0.50 mm pore size sieve, mixing and standing for 24 hours, and then removing the supernatant to obtain the pretreated sediment.

9. The closed system culture method for cable bacteria according to claim 7, characterized in that: The physicochemical parameters of the sediments collected from the typical black and smelly rivers in step S1 are: AVS content is not less than 1.50 mg / g, and TOC content is not less than 40 mg / g.

10. The closed system culture method for cable bacteria according to claim 7, characterized in that: The volume of the pretreated sediment loaded in step S2 is 11 mL, and the aeration volume during the aeration process in step S3 is 1 cm 3 / min, during the aeration process, the culture tube was placed in a constant temperature water bath at 25°C for continuous aeration.

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