A directional anaerobic enrichment culture device

Abstract

The utility model provides a kind of directional anaerobic enrichment culture device, belong to anaerobic bacteria culture technical field, comprising: base, reactor and communication conduit, multiple mounting reactor's jack is provided on base, multiple are provided in series connection to reactor, anaerobic environment is provided in the accommodating cavity of reactor, and sealing cover is provided at the opening of reactor;Communication conduit is inserted in reactor, including sample inlet tube and sample outlet tube;Between two adjacent reactors, the sample inlet tube of first reactor is communicated with the sample outlet tube of second reactor by conveying device.The bacteria solution in first reactor is conveyed to second reactor by conveying device after reacting for a period of time, after a period of time, continue to extract bacteria solution to add to next reactor, pass on multiple times in turn, complete enrichment domestication, reduce the steps of manual operation.The directional anaerobic enrichment culture device provided by the utility model solves the problem of frequent manual operation of nitrogen-fixing campylobacter culture in the laboratory in the prior art.

Description

Technical Field

[0001] This utility model relates to the field of anaerobic bacteria culture technology, specifically to a directional anaerobic enrichment culture device. Background Technology

[0002] *Campylobacter*, belonging to the phylum Pseudomonas, class Betaproteobacteria, order Rhodocyales, family Zoyraceae, is a genus of nitrogen-fixing bacteria. Related strains are generally divided into two categories: plant strains, usually nitrogen-fixing bacteria; and soil strains, isolated from contaminated soil, which typically live in anaerobic environments and use nitrate as a terminal electron acceptor to achieve dissimilatory nitrate reduction. According to literature, some directed-cultured *Campylobacter* strains also possess the ability to degrade aromatic compounds, exhibiting significant ecological value.

[0003] Nitrogen-fixing Campylobacter can be detected in high abundance in activated sludge used for wastewater treatment in wastewater treatment plants through metagenomic detection. When the nitrogen-fixing Campylobacter in the sludge community is subjected to toxic inhibition / organic load shock, mass mortality may occur, affecting the treatment of wastewater by activated sludge. Therefore, culturing nitrogen-fixing Campylobacter in a good environment to supplement the species richness of the sludge community is also a means to improve the wastewater treatment effect.

[0004] Currently, most laboratories use serum bottles to hold culture medium and then culture it in an anaerobic incubator to cultivate nitrogen-fixing Campylobacter.

[0005] However, for bacterial solutions that need to be enriched and domesticated, frequent manual operations are required, which may introduce contaminating bacteria or disrupt the anaerobic environment. Utility Model Content

[0006] Therefore, the technical problem to be solved by this invention is to overcome the problem of frequent manual operation in the cultivation of nitrogen-fixing Campylobacter in existing laboratories, thereby providing a directional anaerobic enrichment culture device.

[0007] To address the aforementioned technical problems, this utility model provides a nitrogen-fixing, uniformly oriented anaerobic enrichment culture device, comprising: a base, a reactor, and a connecting conduit. The base is provided with multiple insertion holes, and the reactor is inserted into the insertion holes. Multiple reactors are arranged in series, and each reactor has an internal receiving cavity configured as an anaerobic environment. A sealing cap is provided at the opening of each reactor. The connecting conduit is inserted into the reactor and includes an inlet pipe and an outlet pipe. Between adjacent reactors, the inlet pipe of the preceding reactor and the outlet pipe of the following reactor are connected via a conveying device.

[0008] In use, the sealing cap seals the reactor opening, maintaining an anaerobic environment within the reactor's internal chamber. The bacterial solution is added to the chamber of the first reactor through the inlet tube. After a reaction period, the bacterial solution is extracted through the outlet tube of the first reactor and added to the second reactor through the inlet tube. This process is repeated for a period, and then the bacterial solution is extracted and added to the next reactor, repeating this process multiple times to complete the multiple subculturing and enrichment of *Campylobacter nigra*. This cultivation device reduces manual operation steps, saves manpower, ensures personnel safety during the reaction, and avoids the possibility of misoperation. The directional anaerobic enrichment cultivation device provided by this invention solves the problem of frequent manual operations in the laboratory cultivation of *Campylobacter nigra* in existing technologies.

[0009] Optionally, the reactor is threadedly connected to the sealing cap. With the above arrangement, the reactor and the sealing cap achieve a sealed connection via threads.

[0010] Optionally, an anaerobic gas-generating bag is provided inside the sealing cover. With this arrangement, the anaerobic gas-generating bag can absorb oxygen from the reactor's containment chamber and generate carbon dioxide, thus creating an anaerobic environment within the reactor's containment chamber.

[0011] Optionally, an oxygen indicator is provided inside the reactor. With this configuration, the oxygen indicator turns blue upon contact with oxygen and pink in an anaerobic environment. The color change of the oxygen indicator can be observed to determine whether an anaerobic environment has been generated within the reactor's containment chamber.

[0012] Optionally, the base is equipped with an oscillation module for oscillating and mixing the solution within the reactor. This configuration enables automatic oscillation, reducing manual operation.

[0013] Optionally, the base is also equipped with a heating module to maintain a constant temperature. This configuration ensures the reactor remains at a constant temperature, guaranteeing the effectiveness of the microbial culture.

[0014] Optionally, a control module is provided on the base, and the control module is electrically connected to the conveying device, the oscillation module, and the heating module. With the above configuration, the control module can control the automatic start and stop of the conveying device, the oscillation module, and the heating module, regulate the temperature of the heating module, and, after the automatic control system of the control module is programmed, can complete the bacterial culture process with a single click.

[0015] Optionally, the inlet end of the sample inlet tube is provided with a first branch and a second branch. The first branch is used to connect to the bacterial solution supply component, and the second branch is used to connect to the culture medium supply component. With the above configuration, the bacterial solution supply component delivers the bacterial solution to the reactor through the first branch, and the culture medium supply component delivers the culture medium nutrient solution to the reactor through the second branch, achieving precise mixing of the bacterial solution and the culture medium nutrient solution, and ensuring the stability and efficiency of the culture process.

[0016] Optionally, the sample outlet tube of the reactor is used to connect to the sampling component. With the above configuration, multiple reactors connected in series can be passaged multiple times, and the sampling component can extract bacterial solution through the sample outlet tube for subsequent plate culture on agar medium.

[0017] Optionally, the delivery device is configured as a peristaltic pump. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of this utility model or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this utility model. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of one embodiment of the directional anaerobic enrichment culture device provided in this utility model.

[0020] Figure 2 for Figure 1 A schematic diagram of the reactor.

[0021] Figure 3 for Figure 1 A perspective view of the central sealing cap.

[0022] Explanation of reference numerals in the attached figures:

[0023] 1. Base; 2. Insertion hole; 3. Reactor; 4. Sealing cap; 5. Anaerobic gas generating bag; 6. Oxygen indicator; 7. Connecting conduit; 8. Sample inlet tube; 9. First branch tube; 10. Second branch tube; 11. Sample outlet tube; 12. Conveying device; 13. Control module. Detailed Implementation

[0024] The technical solution of this utility model will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this utility model. Based on the embodiments of this utility model, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this utility model.

[0025] In the description of this utility model, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings and are only for the convenience of describing this utility model and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this utility model. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0026] In the description of this utility model, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this utility model according to the specific circumstances.

[0027] Furthermore, the technical features involved in the different embodiments of this utility model described below can be combined with each other as long as they do not conflict with each other.

[0028] This embodiment provides a structure for a directional anaerobic enrichment culture device that can complete the bacterial culture process with one click through an automatic control system, for the enrichment and domestication of nitrogen-fixing Campylobacter.

[0029] like Figure 1 The image shows a specific implementation of a directional anaerobic enrichment culture device provided in this embodiment, comprising: a base 1, a reactor 3, and a connecting conduit 7. The base 1 is provided with multiple insertion holes 2, and the reactor 3 is inserted into the insertion holes 2. The reactor 3 has multiple reactors arranged in series. The reactor 3 has a receiving cavity inside, and the receiving cavity is set as an anaerobic environment. The opening of the reactor 3 is provided with a sealing cap 4. The connecting conduit 7 is inserted into the reactor 3, and the connecting conduit 7 includes an inlet pipe 8 and an outlet pipe 11. Among two adjacent reactors 3, the inlet pipe 8 of the previous reactor 3 and the outlet pipe 11 of the next reactor 3 are connected by a conveying device 12.

[0030] In use, the sealing cap 4 seals the opening of the reactor 3, maintaining an anaerobic environment inside the containment chamber of the reactor 3. The bacterial solution is added to the containment chamber of the first reactor 3 through the sample inlet tube 8. After a period of reaction, the bacterial solution is extracted through the sample outlet tube 11 of the first reactor 3 via the delivery device 12 and added to the second reactor 3 through the sample inlet tube 8. After a period of reaction, the bacterial solution is extracted again and added to the next reactor 3, repeating this process multiple times to complete the multiple subculturing and enrichment of *Campylobacter nigra*. This cultivation device reduces manual operation steps, saves manpower, ensures personnel safety during the reaction process, and avoids the possibility of misoperation. The directional anaerobic enrichment culture device provided in this embodiment solves the problem of frequent manual operation in the laboratory cultivation of *Campylobacter nigra* in the prior art.

[0031] Specifically, in the directional anaerobic enrichment culture device provided in this embodiment, the base 1 is provided with four insertion holes 2, and the reactor 3 has four connected in series. Furthermore, as an alternative implementation, the number of insertion holes 2 and the reactor 3 can be adjusted according to the needs of the bacterial culture.

[0032] Specifically, the portion of the sample outlet tube 11 extending into the reactor 3 is longer than the portion of the sample inlet tube 8 extending into the reactor 3, so that the end of the sample outlet tube 11 can be immersed in the mixture of bacterial solution and nutrient solution.

[0033] Specifically, the volume of the reactor 3 is preferably, but not limited to, 250 ml.

[0034] like Figure 2 , Figure 3 As shown, in the directional anaerobic enrichment culture device provided in this embodiment, the reactor 3 is threadedly connected to the sealing cap 4. The reactor 3 and the sealing cap 4 are sealed together by the threads. Alternatively, as an alternative embodiment, the sealing cap 4 can also be configured as a flexible sealing plug, achieving a seal by interference fit between the sealing cap 4 and the reactor 3.

[0035] like Figure 3 As shown, in the directional anaerobic enrichment culture device provided in this embodiment, an anaerobic gas-generating bag 5 is disposed inside the sealing cover 4. The anaerobic gas-generating bag 5 can absorb oxygen in the containment cavity of the reactor 3 and generate carbon dioxide, thereby creating an anaerobic environment inside the containment cavity of the reactor 3. Specifically, the anaerobic gas-generating bag 5 is replaceably disposed inside the sealing cover 4. Alternatively, as an alternative implementation, the anaerobic gas-generating bag can be omitted, and the reactor 3 can be vacuumed using equipment.

[0036] like Figure 2 As shown in the embodiment, in the directional anaerobic enrichment culture device, an oxygen indicator 6 is provided inside the reactor 3. The oxygen indicator 6 turns blue upon contact with oxygen and pink in an anaerobic environment. The color change of the oxygen indicator 6 can be observed to determine whether an anaerobic environment has been generated within the containment chamber of the reactor 3. Specifically, the oxygen indicator 6 can be replaced and placed inside the reactor 3.

[0037] like Figure 1 As shown in the embodiment, in the directional anaerobic enrichment culture device, the base 1 is equipped with a shaking module, which is used for shaking and mixing the solution in the reactor 3. This allows for automatic shaking, reducing manual operation. Specifically, the rotation speed of the shaking module is preferably, but not limited to, 180 rpm.

[0038] like Figure 1 As shown in the embodiment, in the directional anaerobic enrichment culture device, the base 1 is also equipped with a heating module, which is used to maintain a constant temperature. This ensures that the reactor 3 is in a constant temperature state, guaranteeing the bacterial culture effect. Specifically, the temperature control range of the heater is preferably, but not limited to, 30-40℃.

[0039] like Figure 1 As shown, in the directional anaerobic enrichment culture device provided in this embodiment, a control module 13 is provided on the base 1. The control module 13 is electrically connected to the conveying device 12, the oscillation module, and the heating module. The control module 13 can control the automatic start and stop of the conveying device 12, the oscillation module, and the heating module, and regulate the temperature of the heating module. After the automatic control system of the control module 13 is programmed, it can complete the bacterial culture process with one click. Specifically, the control module 13 has a control panel for adjusting parameters. The control panel has adjustment buttons that can adjust parameters such as oscillation time, temperature, and the volume of bacterial solution extracted by the conveying device 12.

[0040] like Figure 1 , Figure 3As shown, in the directional anaerobic enrichment culture device provided in this embodiment, the inlet end of the sample inlet tube 8 is provided with a first branch tube 9 and a second branch tube 10. The first branch tube 9 is used to connect to the bacterial solution supply component, and the second branch tube 10 is used to connect to the culture medium supply component. The bacterial solution supply component delivers the bacterial solution to the reactor 3 through the first branch tube 9, and the culture medium supply component delivers the culture medium nutrient solution to the reactor 3 through the second branch tube 10, realizing precise mixing of the bacterial solution and the culture medium nutrient solution, and ensuring the stability and efficiency of the culture process. In addition, as an alternative embodiment, the sample inlet tube 8 may not have a branch tube structure, and two sample inlet tubes 8 may be arranged side by side, one sample inlet tube 8 connected to the bacterial solution supply component and the other sample inlet tube 8 connected to the culture medium supply component.

[0041] like Figure 1 As shown in the embodiment, in the directional anaerobic enrichment culture device, the sample outlet tube 11 of the reactor 3 is used to connect to the sampling component. After multiple passages, the sampling component extracts bacterial solution through the sample outlet tube 11 from the multiple reactors 3 connected in series for subsequent plate culture on agar medium.

[0042] like Figure 1 As shown, in the directional anaerobic enrichment culture device provided in this embodiment, the conveying device 12 is configured as a peristaltic pump.

[0043] How to use:

[0044] like Figure 1 As shown, in the directional anaerobic enrichment culture device provided in this embodiment, the sealing cap 4 seals the opening of the reactor 3 to maintain an anaerobic environment in the containment cavity inside the reactor 3. The bacterial solution is added to the containment cavity of the first reactor 3 through the sample inlet tube 8. After reacting for a period of time, the bacterial solution is extracted through the sample outlet tube 11 of the first reactor 3 by the delivery device 12 and added to the second reactor 3 through the sample inlet tube 8 of the second reactor 3. After reacting for a period of time, the bacterial solution is extracted and added to the next reactor 3. This process is repeated multiple times to complete the multiple subculture of nitrogen-fixing Campylobacter and the enrichment and domestication are completed.

[0045] The directional anaerobic enrichment culture device provided in this embodiment operates as follows:

[0046] 1) Prepare the nutrient solution for the directional culture of nitrogen-fixing Campylobacter. The preparation method of the nutrient solution is as follows: Adjust the pH of nutrient solution 1 (main components are KH2PO4 and K2HPO4) and nutrient solution 2 (main components are NH4Cl, MgSO4, KNO3 and CaCl2) to 7.8, autoclave, cool and mix. Then add 10 mL of sterile SL-1 solution (main components are hydrochloric acid, FeCl2, ZnCl2, MnCl2, H3BO3, CoCl2, CuCl2, NiCl2 and Na2MoO4), 5 mL of vitamin solution (main components are vitamin B12, pantothenic acid, riboflavin, pyridoxine, biotin, folic acid, niacin, nicotinamide, α-lipoic acid, para-aminobenzoic acid and thiamine), 4 mM ascorbic acid and a low concentration of potentially toxic carbon source (main components are toluene, phenol and benzoate) to the mixed solution. Finally, store the prepared nutrient solution in a sterile environment.

[0047] 2) Place the base 1 of the device in the fume hood, connect the power supply, and after powering on, the heating module will automatically turn on the constant temperature mode, and the temperature will be controlled within the range of 30 to 40℃.

[0048] 3) Sterilize reactor 3, sealing cap 4, and connecting conduit 7 by autoclaving.

[0049] 4) Use pure water to centrifuge and wash the activated sludge obtained from the wastewater treatment plant twice, and prepare the washed activated sludge into a sludge suspension of 100 mg / L.

[0050] 5) Replace the oxygen indicator 6 in reactor 3 and the anaerobic gas-producing bag 5 in the sealing cap 4.

[0051] 6) Assemble reactor 3 and sealing cap 4, ensuring the threaded connection is sealed. Then place the four assembled reactor units on base 1 and connect them in series via hoses and conveying device 12, from left to right: first reactor 3, second reactor 3, third reactor 3, and fourth reactor 3.

[0052] 7) Configure the control program through the programming control window, select the appropriate culture program, and the program will proceed automatically in 4 stages:

[0053] In the first stage, 10 mL of sludge suspension is automatically added to the first reactor 3 through the bacterial liquid injection tube 8, and 90 mL of nutrient solution is added through the nutrient solution injection tube 8.

[0054] In the second stage, after the liquid is added, the base 1 begins to vibrate (at a speed of 180 rpm). During this process, the anaerobic gas-generating bag 5 will absorb oxygen and generate carbon dioxide. The surface of the oxygen indicator 6 can be observed to change from blue to pink, indicating that an anaerobic environment has been generated in the reactor 3.

[0055] In the third stage, the oscillation automatically stopped after 48 hours of reaction.

[0056] In the fourth stage, 10 mL of bacterial solution is automatically extracted from the first reactor 3 through the sample outlet tube 11, and the extracted bacterial solution is added to the second reactor 3 through the first branch tube 9 of the sample inlet tube 8. 90 mL of nutrient solution is added to the reactor 2 through the second branch tube 10.

[0057] In the fifth stage, after the liquid addition is completed, base 1 starts to oscillate again (at a speed of 180 rpm);

[0058] The process of stages 3-5 was repeated twice more to complete four passages of the curved nitrogen-fixing bacteria, thus completing the enrichment and domestication.

[0059] 8) Obtain the enriched culture of nitrogen-fixing Campylobacter broth for subsequent plate culture on agar medium.

[0060] 9) Finally, clean reactor 3, turn off the power to base 1, disinfect the experimental apparatus and experimental environment, and the experiment ends.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the protection scope of this utility model.

Claims

1. A directional anaerobic enrichment culture device, characterized in that, include: A base (1) having multiple insertion holes (2) on it. The reactor (3) is inserted into the socket (2). The reactor (3) has multiple reactors connected in series. The reactor (3) has a receiving cavity inside. The receiving cavity is set as an anaerobic environment. The opening of the reactor (3) is provided with a sealing cover (4). A connecting conduit (7) is inserted into the reactor (3). The connecting conduit (7) includes an inlet tube (8) and an outlet tube (11). The length of the inlet tube (8) extending into the receiving cavity is less than the length of the outlet tube (11) extending into the receiving cavity. Among them, the sample inlet tube (8) of the first reactor (3) and the sample outlet tube (11) of the second reactor (3) are connected by a conveying device (12) between two adjacent reactors (3).

2. The directional anaerobic enrichment culture device according to claim 1, characterized in that, The reactor (3) is threadedly connected to the sealing cap (4).

3. The directional anaerobic enrichment culture device according to claim 2, characterized in that, An anaerobic gas-generating bag (5) is provided inside the sealing cover (4).

4. The directional anaerobic enrichment culture device according to claim 3, characterized in that, An oxygen indicator (6) is provided inside the reactor (3).

5. The directional anaerobic enrichment culture device according to claim 1, characterized in that, The base (1) is equipped with an oscillation module, which is used for oscillating and mixing the solution in the reactor (3).

6. The directional anaerobic enrichment culture device according to claim 5, characterized in that, The base (1) is also provided with a heating module, which is used to maintain a constant temperature.

7. The directional anaerobic enrichment culture device according to claim 6, characterized in that, A control module (13) is provided on the base (1), and the control module (13) is electrically connected to the conveying device (12), the oscillation module and the heating module.

8. The directional anaerobic enrichment culture device according to claim 1, characterized in that, The inlet end of the sample inlet tube (8) is provided with a first branch tube (9) and a second branch tube (10). The first branch tube (9) is used to connect to the bacterial solution supply component, and the second branch tube (10) is used to connect to the culture medium supply component.

9. The directional anaerobic enrichment culture device according to claim 1, characterized in that, The sample outlet tube (11) of the reactor (3) is used to connect to the sampling assembly.

10. The directional anaerobic enrichment culture device according to any one of claims 1-9, characterized in that, The conveying device (12) is configured as a peristaltic pump.

Images