Method for preserving electrochemically active microbial film and use thereof

Abstract

The invention belongs to the technical field of biosensors, and particularly discloses a method for preserving an electrochemically active microbial film and a method for preparing an electrochemically active microbial sensor, and the method for preserving the electrochemically active microbial film comprises the following steps: providing the electrochemically active microbial film; soaking the electrochemical active microbial film in a protective agent solution to form a protective agent coupled electrochemical active microbial film; carrying out gradient cooling on the protective agent coupled electrochemical active microbial film to form a frozen electrochemical active microbial film; carrying out gradient heating drying on the frozen electrochemical active microbial film to form a dry electrochemical active microbial film wrapped by a protective agent; and storing the dried electrochemically active microbial film. According to the method, the storage conditions of the electrochemical active microbial film are widened and are not limited by strict storage requirements any more. Meanwhile, the preservation time of the EAB biological membrane is prolonged, and the EAB biological membrane with guaranteed performance can be quickly and simply obtained at any time in field test.

Description

Technical Field

[0001] This application belongs to the field of biosensor technology, specifically relating to a method for preserving electrochemically active microbial membranes and a method for preparing electrochemically active microbial sensors. Background Art

[0002] Microbial electrochemical sensors (MES) based on electrochemically active microorganisms (EAB) can directly convert analytes in water into bioelectrical signals. They offer advantages such as rapid detection, high sensitivity, low cost, and strong anti-interference capabilities, showing promising application prospects in biomedicine and environmental monitoring. However, EABs are difficult to preserve and transport, requiring temporary activation and cultivation before being prepared into a biofilm and installed in the MES, which significantly limits the widespread application of this technology.

[0003] In existing technologies, biofilms are usually acclimatized in the laboratory in advance and then mailed to the site when needed. However, the acclimatized biofilms must be sent to the site equipment for use within one week and cannot be stored alone for a long time. The storage temperature must be between 0℃ and 25℃. Too low or too high a temperature or too long a storage time will cause changes in the activity of the biofilm or even inactivation, affecting the test performance or even making it unusable.

[0004] There are also existing technologies that involve directly inoculating electrochemically active bacteria at the testing site to acclimate the biofilm, and then conducting tests after the biofilm has matured and stabilized and the signal meets the requirements. However, the on-site acclimatization time is long and the operation is complicated, making it difficult to guarantee the performance of the biofilm test. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems in related technologies. Therefore, the purpose of this application is to propose a method for preserving electrochemically active microbial membranes and a method for preparing electrochemically active microbial sensors. This application broadens the preservation conditions for electrochemically active microbial membranes. The dried electrochemically active microbial membranes obtained by this method are no longer subject to strict preservation requirements; they can be preserved in ultra-low temperature environments (e.g., -60°C), at conventional refrigerator temperatures, or at room temperature, facilitating storage and transportation. Simultaneously, this method extends the preservation time of EAB biofilms, allowing for rapid and simple on-site testing to obtain EAB biofilms with guaranteed performance.

[0006] In one aspect of this application, a method for preserving electrochemically active microbial membranes is provided. According to an embodiment of this application, the method includes: (1) Provide an electrochemically active microbial membrane, wherein the electrochemically active microorganisms in the electrochemically active microbial membrane include at least two of the following: Geobacterium, Laryngobacterium, Pseudomonas, Shewanella, Azotobacter and Acinetobacter; (2) The electrochemically active microbial membrane is immersed in a protective agent solution to form a protective agent coupled electrochemically active microbial membrane; (3) The protective agent coupled with the electrochemically active microbial membrane is subjected to gradient cooling to form a cryo-electrochemically active microbial membrane; (4) The frozen electrochemically active microbial membrane is subjected to gradient temperature increase drying to form a dry electrochemically active microbial membrane coated with a protective agent; (5) Preserve the dried electrochemically active microbial membrane.

[0007] The method for preserving electrochemically active microbial membranes according to embodiments of this application has the following advantages: First, this method broadens the preservation conditions for electrochemically active microbial membranes (i.e., EAB biofilms). The dried electrochemically active microbial membranes obtained by this method are no longer subject to strict preservation requirements; they can be preserved in ultra-low temperature environments (e.g., -60°C), at conventional refrigerator temperatures, or at room temperature, facilitating storage and transportation. Second, this method extends the preservation time of EAB biofilms, allowing them to be preserved for more than two years at -60°C and for more than three months at room temperature. Third, the dried electrochemically active microbial membranes (i.e., EAB biofilms) preserved by this method can be used immediately after activation and revival, enabling rapid and simple on-site testing to obtain EAB biofilms with guaranteed performance. This simplifies the entire process of EAB biofilm acquisition, MES preparation, and on-machine testing.

[0008] In addition, the method according to the above embodiments of this application may also have the following additional technical features: In some embodiments of this application, step (1) further includes: screening highly active and mature electrochemically active microbial membranes, wherein the screening method includes: (1-1) The incubated original electrochemically active microbial membrane is assembled into a microbial electrochemical sensor and connected to an electrochemical workstation to monitor the output current of the microbial electrochemical sensor, wherein the culture system of the microbial electrochemical sensor includes a first culture medium; (1-2) When the current of the microbial electrochemical sensor is below 10 -7 After step A, a second culture medium is pumped into the culture system for 20-60 minutes, at which point the current of the microbial electrochemical sensor first drops below 10. -7 The current at point A is I1, and the concentration of the organic substrate in the second culture medium is greater than the concentration of the organic substrate in the first culture medium; When the second culture medium is pumped into the culture system for 3 minutes, the output current of the microbial electrochemical sensor is I2; during the process of pumping the second culture medium into the culture system, the maximum current of the microbial electrochemical sensor is I3; when the pumping of the second culture medium into the culture system ends, the output current of the microbial electrochemical sensor is I4; when the pumping of the second culture medium into the culture system stops for 3 minutes, the output current of the microbial electrochemical sensor is I5. The criteria for determining highly active and mature electrochemically active microbial membranes are as follows: (I2-I1) / (I3-I1)≥90%; (I4-I5) / (I3-I5)≥80%; and I2, I3, and I4 are all greater than 0.2mA.

[0009] In some embodiments of this application, the concentration of the organic substrate in the first culture medium is 0.01 mmol / L to 0.05 mmol / L; and / or, the concentration of the organic substrate in the second culture medium is 1 mmol / L to 5 mmol / L; and / or, the organic substrate in the first culture medium includes at least one of sodium acetate, sodium lactate, and glucose; and / or, the organic substrate in the second culture medium includes at least one of sodium acetate, sodium lactate, and glucose; and / or, in step (1-2), the second culture medium is pumped into the culture system at a flow rate of 10 mL / min to 50 mL / min.

[0010] In some embodiments of this application, in step (2), the protective agent solution includes skim milk powder, trehalose and sodium chloride, wherein the content of skim milk powder in the protective agent solution is 3wt%~10wt%, the content of trehalose in the protective agent solution is 2wt%~10wt%, and the content of sodium chloride in the protective agent solution is 0.8wt%~3wt%.

[0011] In some embodiments of this application, the protective agent solution further includes at least one of glycerol, Tween, and chitosan.

[0012] In some embodiments of this application, in step (3), the protective agent coupled electrochemically active microbial membrane is cooled sequentially from T1 to T2, T3, and T4. The protective agent coupled electrochemically active microbial membrane is maintained at temperatures of T1, T2, and T3 for 20 min to 40 min, and at temperature of T4 for 4 h to 12 h. T1 is 20 °C to 30 °C, T2 is 8 °C to 15 °C, T3 is 0 °C to 5 °C, and T4 is -50 °C to -80 °C.

[0013] In some embodiments of this application, in step (4), the cryo-electrochemically active microbial membrane is sequentially heated to T'1, T'2, and T'3; the cryo-electrochemically active microbial membrane is maintained at temperature T'1 for 20 min to 40 min with a vacuum degree ≤ 5 Pa; the cryo-electrochemically active microbial membrane is maintained at temperature T'2 for 1 h to 2 h with a vacuum degree ≤ 5 Pa; the cryo-electrochemically active microbial membrane is maintained at temperature T'3 for 2 h to 3 h with a vacuum degree ≤ 5 Pa; T'1 is equal to the eutectic point temperature T0-(0℃ to 10℃) of the protective agent solution, T'2 is 0℃ to 5℃, and T'3 is 20℃ to 30℃.

[0014] In some embodiments of this application, the cryo-electrochemically active microbial membrane is heated from T4 to T'1 at a heating rate of 5°C / min to 10°C / min, from T'1 to T'2 at a heating rate of 1°C / min to 5°C / min, and from T'2 to T'3 at a heating rate of 5°C / min to 10°C / min.

[0015] In a second aspect, this application provides a method for preparing an electrochemically active microbial sensor. According to embodiments of this application, the method includes: (a) Activation and revival of the dried electrochemically active microbial membrane preserved using the method described in the above embodiments; (b) An electrochemically active microbial sensor is assembled using the activated and revived electrochemically active microorganisms.

[0016] The method for preparing an electrochemically active microbial sensor according to the embodiments of this application uses a dried electrochemically active microbial membrane (i.e., EAB biomembrane). The dried electrochemically active microbial membrane can be used immediately after activation and revival, allowing for rapid and simple on-site testing to obtain EAB biomembranes with guaranteed performance. This simplifies the entire process of EAB biomembrane acquisition, MES preparation, and on-machine testing.

[0017] In addition, the method according to the above embodiments of this application may also have the following additional technical features: In some embodiments of this application, in step (a), the dried electrochemically active microbial membrane is activated and revived using a third culture medium, wherein the concentration of the organic substrate in the third culture medium is 1 mmol / L to 5 mmol / L, and the organic substrate in the third culture medium includes at least one of sodium acetate, sodium lactate, and glucose.

[0018] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. Attached Figure Description

[0019] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 This is a schematic flowchart of a method for preserving electrochemically active microbial membranes according to an embodiment of this application. Detailed Implementation

[0020] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0021] In one aspect of this application, a method for preserving electrochemically active microbial membranes is provided. According to embodiments of this application, refer to the appendix... Figure 1 The method includes: S100: providing an electrochemically active microbial membrane; S200: immersing the electrochemically active microbial membrane in a protective agent solution to form a protective agent-coupled electrochemically active microbial membrane; S300: subjecting the protective agent-coupled electrochemically active microbial membrane to gradient cooling to form a cryo-electrochemically active microbial membrane; S400: subjecting the cryo-electrochemically active microbial membrane to gradient heating and drying to form a dry electrochemically active microbial membrane coated with a protective agent; S500: preserving the dry electrochemically active microbial membrane. Therefore, firstly, this method broadens the preservation conditions for electrochemically active microbial membranes (i.e., EAB biofilms). The dry electrochemically active microbial membranes obtained by this method are no longer subject to strict preservation requirements; they can be preserved in ultra-low temperature environments (e.g., -60℃), at conventional refrigerator temperatures, or at room temperature, facilitating storage and transportation. Secondly, this method extends the preservation time of EAB biofilms, allowing them to be preserved for more than two years at -60℃ and more than three months at room temperature. Third, the dried electrochemically active microbial membranes (i.e., EAB biofilms) preserved by this method can be used immediately after activation and revival, and high-performance EAB biofilms can be obtained quickly and easily on-site for testing. This simplifies the entire process of EAB biofilm acquisition, MES preparation, and on-machine testing.

[0022] The method for preserving electrochemically active microbial membranes proposed in this application is described in detail below: Specifically, see the attached document. Figure 1 The above method for preserving electrochemically active microbial membranes includes the following steps: S100: Provides electrochemically active microbial membranes; In this step, following conventional EAB biofilm culture methods (as described in Comparative Example 1), several mature EAB biofilms are first incubated, and then highly active, fully mature electrochemically active microbial membranes are screened. The electrochemically active microorganisms in the aforementioned electrochemically active microbial membranes include at least two of the following: Geobacterium, Lornae, Pseudomonas, Shewanella, Azotobacter, and Acinetobacter.

[0023] The microbial strains used in this application are naturally mixed multi-strain strains obtained through direct domestication and cultivation of natural sludge. The EAB biofilm, domesticated using conventional EAB biofilm cultivation methods, consists of natural EAB microbial communities, with the original community structure largely unchanged. Therefore, it exhibits a higher bioelectrical signal, strong tolerance to external factors, and stable data, and is generally used to test higher concentrations of toxic substances. The biofilm of this application is suitable for general water quality biotoxicity testing, requiring responsiveness to all toxic substances and testing the comprehensive toxicity of all substances (e.g., the comprehensive toxicity response of all substances in water, such as pesticide pollutants and heavy metal pollutants).

[0024] The mixed-bacterial EAB of this application readily adheres to various materials to form a stable biofilm. Therefore, the biofilm carrier of this application, in addition to loose, porous conductive materials such as carbon cloth and carbon felt, can also be carbon paper (conductive, but with pores smaller than 50 micrometers that bacteria cannot penetrate), conductive glass, conductive silicone (conductive, but liquids cannot pass through), etc. Loose, porous conductive materials such as carbon cloth and carbon felt are preferred because their loose porosity allows the mixed-bacterial EAB to grow inside the material, forming a high-density biofilm from the inside out, making the biofilm more stable.

[0025] According to some specific embodiments of this application, the above screening method includes: S110: Assemble the incubated original electrochemically active microbial membrane into a microbial electrochemical sensor, and connect it to an electrochemical workstation to monitor the output current of the microbial electrochemical sensor. The culture system of the microbial electrochemical sensor includes a first culture medium. In this step, the original electrochemically active microbial membranes incubated using the conventional EAB biofilm culture method are removed from the original culture system and assembled one by one into microbial electrochemical sensors (MES). The culture medium is replaced with a first culture medium containing a low concentration of organic substrate, and a multi-channel electrochemical workstation is connected to continuously monitor the output current of each MES channel. At this time, because the concentration of organic matter in the system is extremely low, it will be quickly depleted, and the output current of a single MES will be less than 10 within 30 minutes after the electrochemical workstation is connected. -7 A.

[0026] According to some specific embodiments of this application, the concentration of the organic substrate in the first culture medium is 0.01 mmol / L to 0.05 mmol / L (e.g., 0.01 mmol / L, 0.02 mmol / L, 0.03 mmol / L, 0.04 mmol / L, 0.05 mmol / L, etc.), and the organic substrate in the first culture medium includes at least one of sodium acetate, sodium lactate, and glucose. Other components and concentrations in the first culture medium are the same as those in the original culture medium.

[0027] S120: When the current of the microbial electrochemical sensor is below 10 -7 After step A, the second culture medium was pumped into the culture system for 20-60 minutes, at which point the current of the microbial electrochemical sensor first dropped below 10. -7 The current at point A is I1, and the concentration of the organic substrate in the second culture medium is greater than that in the first culture medium.

[0028] In this step, wait until the output current of all individual MES is below 10. -7 After step A, the second culture medium can be pumped into the culture system at a flow rate of 10 mL / min to 50 mL / min for 20 min to 60 min. After the pumping is completed, continue to monitor the MES output current of each channel for 10 min.

[0029] According to some specific embodiments of this application, the concentration of the organic substrate in the second culture medium is 1 mmol / L to 5 mmol / L (for example, it can be 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, etc.), and the organic substrate in the second culture medium includes at least one of sodium acetate, sodium lactate and glucose.

[0030] When the second culture medium is pumped into the culture system for 3 minutes, the output current of the microbial electrochemical sensor is I2; during the process of pumping the second culture medium into the culture system, the maximum current of the microbial electrochemical sensor is I3; when the pumping of the second culture medium into the culture system ends, the output current of the microbial electrochemical sensor is I4; when the pumping of the second culture medium into the culture system stops for 3 minutes, the output current of the microbial electrochemical sensor is I5.

[0031] The criteria for determining highly active and fully mature electrochemically active microbial membranes are as follows: (I2-I1) / (I3-I1)≥90%; (I4-I5) / (I3-I5)≥80%; and I2, I3, and I4 are all greater than 0.2mA. Only in this way can highly active and fully mature electrochemically active microbial membranes be screened.

[0032] S200: The electrochemically active microbial membrane is immersed in a protective agent solution to form a protective agent coupled electrochemically active microbial membrane; In this step, a protective agent solution is first prepared by dissolving skim milk powder, trehalose, glycerol, Tween, chitosan, and other protective agents in a certain proportion in an aqueous NaCl solution. The solution is then completely dissolved and shaken to obtain the protective agent solution. Next, the protective agent solution is dispensed into vials, with a liquid layer thickness of 2-5 cm. The fresh EAB biofilms screened in step S100 are also added to the vials, one vial per vial. The vials are gently shaken to remove air bubbles, ensuring the fresh EAB biofilm is completely immersed in the protective agent solution. The vials are then allowed to stand at room temperature (20℃-30℃) for 30 minutes to allow the EAB biofilm carrier to fully saturate with the protective agent solution, forming a protective agent-coupled electrochemically active microbial membrane. The protective agent coupled to the electrochemically active microbial membrane effectively prevents subsequent freezing damage by ice crystals at low temperatures, effectively protecting the electrochemically active microbial membrane.

[0033] According to some specific embodiments of this application, the protective agent solution includes skim milk powder, trehalose, and sodium chloride. The content of skim milk powder in the protective agent solution is 3wt% to 10wt% (e.g., 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, etc.), the content of trehalose in the protective agent solution is 2wt% to 10wt% (e.g., 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, etc.), and the content of sodium chloride in the protective agent solution is 0.8wt% to 3wt% (e.g., 0.8wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, etc.).

[0034] According to some specific embodiments of this application, the protective agent solution further includes at least one of glycerol, Tween, and chitosan, wherein the content of glycerol in the protective agent solution can be 1wt% to 20wt%, the content of Tween in the protective agent solution can be 1wt% to 20wt%, and the content of chitosan in the protective agent solution can be 1wt% to 20wt%.

[0035] In the embodiments of this application, the eutectic point temperature T0 of the protective agent solution can be measured using a eutectic point tester.

[0036] S300: Gradual cooling is applied to the protective agent coupled with the electrochemically active microbial membrane to form a cryo-electrochemically active microbial membrane; In this step, the protective agent coupled with the EAB biofilm in step S200 can be placed in various temperature environments in the order of T1, T2, T3, and T4, and left to stand for a period of time in each temperature environment to form a frozen EAB biofilm.

[0037] According to some specific embodiments of this application, the protective agent coupled electrochemically active microbial membrane is sequentially cooled from T1 to T2, T3, and T4. The protective agent coupled electrochemically active microbial membrane is maintained at temperatures of T1, T2, and T3 for 20 min to 40 min, and at temperature of T4 for 4 h to 12 h. T1 is 20℃ to 30℃, T2 is 8℃ to 15℃, T3 is 0℃ to 5℃, and T4 is -50℃ to -80℃.

[0038] S400: Gradient temperature drying of cryo-electrochemically active microbial membranes to form a dry electrochemically active microbial membrane coated with a protective agent; In this step, the frozen EAB biofilm from step S300 is placed in a vacuum freeze dryer pre-frozen to T4. After the material temperature probe is placed, the door is closed, and the gradient temperature vacuum freeze drying program is started. The preservative solution and the water in the EAB biofilm sublimate directly under vacuum and freezing conditions, finally forming a dried EAB biofilm coated only with the preservative. At this point, there is almost no water in the dried EAB biofilm, and the microorganisms in the dried EAB biofilm are in a dormant state.

[0039] According to some specific embodiments of this application, the above-mentioned gradient temperature vacuum freeze-drying procedure is as follows: 1) The frozen electrochemically active microbial membrane is heated from T4 to T'1 at a heating rate of 5℃ / min to 10℃ / min, and maintained at T'1 temperature for 20min to 40min, with a vacuum degree ≤5Pa; 2) The frozen electrochemically active microbial membrane is heated from T'1 to T'2 at a heating rate of 1℃ / min to 5℃ / min, and maintained at T'2 temperature for 1h to 2h, with a vacuum degree ≤5Pa; 3) The frozen electrochemically active microbial membrane is heated from T'2 to T'3 at a heating rate of 5℃ / min to 10℃ / min, and maintained at T'3 temperature for 2h to 3h, with a vacuum degree ≤5Pa, sublimating all the water to form a dried EAB biofilm coated only with the protective agent. T'1 is equal to the eutectic point temperature of the protective agent solution T0 - (0℃ to 10℃), T'2 is 0℃ to 5℃, and T'3 is 20℃ to 30℃. By controlling the heating rate and holding time of each gradient heating stage, damage or death of microorganisms in the electrochemically active microbial membrane can be effectively avoided, while ensuring the effective drying of the EAB biofilm.

[0040] S500: Preservation of dried electrochemically active microbial membranes.

[0041] In this step, the dried electrochemically active microbial membrane contains almost no moisture, and the microorganisms within it are in a dormant state. Therefore, the temperature for preserving the dried electrochemically active microbial membrane is not particularly limited; it can be stored in ultra-low temperature environments (e.g., -60°C), at conventional refrigerator temperatures, or at room temperature. This broadens the preservation conditions for biofilms, removing the constraints of strict preservation requirements; and it also extends the preservation time of EAB biofilms, enabling rapid and simple on-site testing to obtain EAB biofilms with guaranteed performance.

[0042] As some preferred embodiments, at the end of step S400 above, the vacuum cap is pressed, the air inlet valve is opened to remove the freeze-dried EAB biofilm, and then it is placed at -20°C for two weeks to equilibrate before being stored at -60°C. It can be taken out before use.

[0043] The method for preserving electrochemically active microbial membranes according to the embodiments of this application has at least the following advantages: 1) This method broadens the storage conditions for electrochemically active microbial membranes (i.e., EAB biofilms). The dried electrochemically active microbial membranes obtained by this method are no longer subject to strict storage requirements. They can be stored in ultra-low temperature environments (e.g., -60℃), at conventional refrigerator temperatures, or at room temperature, making them convenient for storage and transportation.

[0044] 2) This method extends the shelf life of EAB biofilms, allowing them to be stored at -60℃ for more than two years and at room temperature for more than three months.

[0045] 3) The dried electrochemically active microbial membrane (EAB biofilm) preserved by this method can be used immediately after activation and revival. High-performance EAB biofilms can be obtained quickly and easily on-site for testing. This simplifies the entire process of EAB biofilm acquisition, MES preparation, and on-machine testing.

[0046] In a second aspect, this application proposes a method for preparing an electrochemically active microbial sensor. According to embodiments of this application, the method includes: (a) Activation and revival of the dried electrochemically active microbial membrane preserved using the method described in the above embodiments; In this step, a third culture medium can be used to activate and revitalize the dried electrochemically active microbial membrane. The concentration of the organic substrate in the third culture medium is 1 mmol / L to 5 mmol / L, and the organic substrate in the third culture medium includes at least one of sodium acetate, sodium lactate, and glucose.

[0047] As a specific example, the dried EAB biofilm was taken out from the storage environment at -60°C, and the stopper was opened. Immediately, an aqueous solution of 1 mmol / L to 5 mmol / L sodium acetate, the same volume as that dispensed, was added to the vial. The vial was shaken rapidly to fully dissolve the protective agent in the dried electrochemically active microbial membrane and to allow the biofilm to fully absorb water. Then, it was allowed to stand at room temperature for 20 to 60 minutes to obtain the activated and revived EAB biofilm.

[0048] (b) An electrochemically active microbial sensor is formed by assembling activated and revived electrochemically active microorganisms.

[0049] In this step, the activated and revived EAB biomembrane is assembled according to the conventional MES assembly method for instrument testing.

[0050] The method for preparing an electrochemically active microbial sensor according to the embodiments of this application uses a dried electrochemically active microbial membrane (i.e., EAB biomembrane). The dried electrochemically active microbial membrane (i.e., EAB biomembrane) can be used immediately after activation and revival. On-site testing allows for the rapid and simple acquisition of EAB biomembranes with guaranteed performance. This simplifies the entire process of EAB biomembrane acquisition, MES preparation, and on-machine testing.

[0051] The embodiments of this application are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. In addition, unless otherwise specified, all reagents used in the following embodiments are commercially available or can be synthesized according to the methods described herein or known methods. For reaction conditions not listed, they are also readily available to those skilled in the art.

[0052] Comparative Example 1 In this comparative example, mature biofilms were incubated using conventional electrochemically active microbial membrane acclimation methods. After acclimation, the biofilms exhibited stable signals and were directly assembled into a microbial electrochemical sensor for instrumental testing. The process consists of six steps, detailed below: Step 1: Assemble the electrochemically active microbial membrane incubation sensor. Each sensor consists of two top covers and one tank body; it includes one bioanode (WE), one reference electrode (RE), and one counter electrode (CE); the reference electrode (RE) is assembled in the middle of the first top cover, and the bioanode (WE) is assembled in the middle of the second top cover; the counter electrode (CE) is directly assembled onto the tank body; there is a hole at the front and back of the bottom of the tank body for installing the inlet and outlet pipes; control valves that can control the flow path opening / closing are installed on the inlet and outlet pipes. Assemble all components of the sensor.

[0053] Step 2: Prepare fresh culture medium. The culture medium consists of four components: salts, trace elements, vitamins, and carbon source. First, prepare stock solutions for each of the four components separately. Then, take 200 mL of the salt stock solution, 2 mL of the trace element stock solution, 0.2 mL of the vitamin stock solution, and 5 mL of the carbon source stock solution, and dilute them with deionized water to a total volume of 3.2 L. Place the solutions in a constant temperature incubator and purge with nitrogen gas (purity >99.5%) at a rate of 500 mL / min while maintaining the constant temperature.

[0054] Step 3: Inoculate with the target bacterial strain. Mix the fresh culture medium prepared in Step 2 with the seed source effluent at a 1:1 ratio to obtain an inoculated bacterial suspension, which is then placed in a storage bottle. The bacterial strain includes 50% Geobacterium and 50% Shewanella.

[0055] Step 4: Connect the tubing. Insert the sampling tube into the cap of the storage bottle, and connect the other end of the sampling tube to a peristaltic pump. Connect the outlet of the peristaltic pump to the inlet of the sensor. Extend the sensor outlet tube and insert it into the storage bottle. Turn on the flow path controller on the sensor inlet and outlet tubes to start the peristaltic pump. The culture medium will circulate in the sensor-storage bottle.

[0056] Step 5: Connect the electrochemical workstation and start the culture. To improve culture efficiency, a multi-channel electrochemical workstation is used, with multiple sensors connected to each channel of the workstation, one sensor per channel. Start the constant potential electrochemical control mode to begin culture. The electrochemically active microorganisms in the inoculum suspension gradually accumulate on the bioanode; simultaneously, they multiply and produce new highly active electrochemical microorganisms in the nutrient-rich system. The current in each channel sensor can be observed to gradually increase, and the current in each channel sensor is recorded in real time.

[0057] Step 6: Change the medium. In Step 5, as the culture time increases, nutrients in the culture system are consumed, and the sensor current rises to its first peak (I1'', equal to 2.43mA) and then rapidly decreases. When the current of all channels decreases to 10... -3 At the mA level, the culture medium in the storage bottle is directly replaced with the fresh culture medium from step 2. After the medium change, the second stage of culture begins. The current in each channel will increase sharply, reaching the first peak (I1'', equal to 2.43 mA) within 10 minutes, and the current will continue to gradually and slowly increase from the first peak (I1''). When the sensor current rises to the second peak (I2'', equal to 5.31 mA), it will decrease rapidly again due to nutrient consumption. When the current in all channels decreases to 10 mA... -3At the mA level, the culture medium in the storage bottle is replaced again with the fresh culture medium from step 2. After the medium change, the third stage of culture begins, and the current in each channel will rise sharply again, reaching the second peak (I2'') within 10 minutes. This process is repeated to obtain the third peak I3'' = 8.02 mA, the fourth peak I4'' = 8.55 mA, and the fifth peak I5'' = 8.58 mA. When the peak current values ​​differ by no more than ±10% after the last three consecutive medium changes, the EAB biofilm is considered to have matured.

[0058] Machine testing: 1) Randomly select 3 EAB biofilms from the matured EAB biofilms and immediately assemble them into 3 MES for machine testing to evaluate the performance of EAB biofilms; 2) After the EAB biofilms mature, store them at 0-4℃ for 1 month and then randomly select 3 more to assemble into 3 MES for machine testing.

[0059] Example 1 The steps include the following: Step 1: Incubate several mature EAB biofilms according to the comparative example steps 1-6.

[0060] Step 2: Select highly active, fully mature EAB biofilms for the following steps. (1) Take the EAB biofilms incubated in Step 1 out of the original culture system and assemble them into MES one by one. Replace the culture medium with the first culture medium (sodium acetate concentration 0.03 mmol / L, other components and concentrations are the same as the original culture medium). Connect a multi-channel electrochemical workstation to continuously monitor the output current of each MES channel; (2) At this time, because the concentration of organic matter in the system is extremely low, it will be quickly depleted. Within 30 minutes after connecting the electrochemical workstation, the output current of a single MES will be less than 10. -7 A; (3) When the output current of all individual MES (denoted as I1) is less than 10 -7 After step A, the second culture medium (sodium acetate concentration 3.0 mmol / L, other components and concentrations the same as the original culture medium) was pumped into the culture system at a flow rate of 30 mL / min for 30 min. After pumping, the output current of each MES channel was monitored for 10 min. The output current of a single MES channel 3 min after the start of pumping was recorded as I2, the maximum output current of a single MES channel during the 30 min of pumping was recorded as I3, the output current of a single MES channel at the 30 min mark of pumping was recorded as I4, and the output current of a single MES channel 3 min after the pumping stopped was recorded as I5. The specific values ​​are shown in Table 1.

[0061] The criteria for determining a highly active and fully mature EAB biofilm must simultaneously meet the following requirements: Formula (1) (I2-I1) / (I3-I1)≥90%; Formula (2) (I4-I5) / (I3-I5)≥80%; (3) I2, I3, and I4 must all be greater than 0.2mA.

[0062] Table 1

[0063] It is evident that MES-1, MES-2, and MES-3 are all highly active and fully mature EAB biomembranes.

[0064] Step 3: Preparation of the preservative solution. Dissolve the preservatives, such as skim milk powder, trehalose, and glycerin, in a 0.9% NaCl aqueous solution. After complete dissolution and shaking, the concentration of skim milk powder is 8%, the concentration of trehalose is 4%, and the concentration of glycerin is 3%.

[0065] Step 4: Dispensing and coupling to form a protective agent-coupled EAB biofilm. Dispense the protective agent solution into vials, with a liquid layer thickness of 4 cm. Add one mature, fresh EAB biofilm selected in Step 2 to each vial. Gently shake the vial to remove air bubbles, ensuring the fresh EAB biofilm is completely immersed in the protective agent solution. Let it stand at room temperature (20-30℃) for 30 minutes to allow the EAB biofilm carrier to fully wet the protective agent solution, obtaining the protective agent-coupled EAB biofilm. The eutectic point temperature of the protective agent solution was measured to be T = -24.11℃ using a eutectic point tester.

[0066] Step 5: Gradient cooling pre-cooling. The protective agent coupled EAB biofilm obtained in Step 4 is placed in the following temperature environments in sequence: room temperature (20-30℃), 10℃, 0℃, and -60℃. Each temperature environment is left to stand for 30 minutes. Finally, it is placed at -60℃ for 8 hours to form a frozen EAB biofilm.

[0067] Step 6: Gradient temperature rise vacuum freeze drying. Place the frozen EAB biofilm obtained in Step 5 into a vacuum freeze dryer pre-frozen to -60℃. After placing the material temperature probe, close the door and start the gradient temperature rise vacuum freeze drying program. The protective agent solution and the water in the EAB biofilm sublimate directly under vacuum and freezing conditions, finally forming a dried EAB biofilm only coated with the protective agent.

[0068] Vacuum freeze dryer program design: (1) Heat the material from -60℃ to -30℃ at a heating rate of 10℃ per hour, maintain the target temperature for 30 minutes, and the vacuum degree ≤5Pa; (2) Heat the material from -30℃ to 5℃ at a heating rate of 5℃ per hour, maintain the target temperature for 60 minutes, and the vacuum degree ≤5Pa; (3) Heat the material from 5℃ to 25℃ at a heating rate of 10℃ per hour, maintain the target temperature for 120 minutes, and the vacuum degree ≤5Pa, sublimate all the moisture, and form a dry EAB biofilm with only protective agent coating.

[0069] Step 7: After completing Step 6 above, vacuum cap the membrane, open the air inlet valve to remove the freeze-dried EAB biofilm, then place it at -20℃ for two weeks to equilibrate, and then store it at -60℃. Remove it before use.

[0070] Step 8: Activation and recovery. Remove the dried EAB biofilm from -60℃, immediately open the stopper and add the same volume of 3.0 mmol / L sodium acetate aqueous solution as when it was dispensed into the vial. Shake the vial quickly to fully dissolve the preservative and allow the biofilm to fully absorb water. Then let it stand at room temperature for 30 minutes to obtain the activated and recovered EAB biofilm.

[0071] Step 9: Assemble the activated and revived EAB biomembrane using the standard MES assembly method for instrument testing.

[0072] On-machine testing of freeze-dried biofilm performance: 1) Take out the newly prepared dry EAB biofilm stored at -60℃ and activate and revive it immediately, then assemble 3 MES for instrument testing; 2) Take out the newly prepared dry EAB biofilm stored at -60℃, store it at 0-4℃ for 1 month, then activate and revive it, and assemble 3 MES for instrument testing; 3) Take out the dry EAB biofilm that has been prepared and stored at -60℃ for two years, activate and revive it immediately, and assemble 3 MES for instrument testing.

[0073] Example 2 The preparation method in this embodiment is basically the same as that in the previous embodiment, except that: Step 2: First culture medium (sodium acetate concentration 0.05 mmol / L, other components and concentrations are the same as the original culture medium), second culture medium (sodium acetate concentration 1.0 mmol / L, other components and concentrations are the same as the original culture medium). The current values ​​output by the MES are shown in Table 2.

[0074] Table 2

[0075] It is evident that MES-1', MES-2', and MES-3' are all highly active and fully mature EAB biomembranes.

[0076] Step 3: Preparation of the protective agent solution. Dissolve the skim milk powder and trehalose, the two protective agents, in a 2.5% NaCl aqueous solution. After complete dissolution and shaking, the concentration of the skim milk powder is 8%, and the concentration of the trehalose is 4%. The eutectic point temperature of the protective agent solution is T0 = -30.13℃.

[0077] Step 6: Vacuum freeze dryer program design: (1) Heat the material from -55℃ to -35℃ at a heating rate of 10℃ per hour, maintain the target temperature for 30 minutes, and the vacuum degree ≤ 5Pa; (2) Heat the material from -35℃ to 5℃ at a heating rate of 5℃ per hour, maintain the target temperature for 60 minutes, and the vacuum degree ≤ 5Pa; (3) Heat the material from 5℃ to 25℃ at a heating rate of 10℃ per hour, maintain the target temperature for 120 minutes, and the vacuum degree ≤ 5Pa, sublimate all the moisture, and form a dry EAB biofilm with only protective agent coating.

[0078] The results of the MES on-machine test in Examples 1-2 are shown in Table 3.

[0079] Table 3

[0080] As can be seen from Table 3, Comparative Example 1, stored at 0-4℃ for only one month, had a reference current that was too low to run the test. Example 1, stored at 0-4℃ for one month, had a reference current slightly lower than the fresh biofilm in the comparative example, but this did not affect the test. Example 1, stored at -60℃ for two years, had a reference current significantly lower than the fresh biofilm in the comparative example, but this did not affect the test. Example 2, stored at 0-4℃ for one month, had a reference current slightly lower than the fresh biofilm in the comparative example, but this did not affect the test. Example 2, stored at -60℃ for two years, had a reference current significantly lower than the fresh biofilm in the comparative example, but this did not affect the test. Therefore, compared to Comparative Example 1, Examples 1-2 of this application broaden the storage conditions for biofilms, no longer adhering to strict storage requirements. They can be stored in ultra-low temperature environments such as -60℃, in conventional refrigerator temperatures, or at room temperature, and the storage time of EAB biofilms is extended.

[0081] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0082] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for preserving an electrochemically active microbial membrane, characterized in that: include: (1) providing an electrochemically active microbial membrane, wherein the electrochemically active microorganisms in the electrochemically active microbial membrane include at least two of Geobacter, Laryngobacter, Pseudomonas, Shewanella, Azospirillum and Acinetobacter; (2) immersing the electrochemically active microbial membrane in a protective agent solution to form a protective agent-coupled electrochemically active microbial membrane; (3) Gradual cooling of the protective agent-coupled electrochemically active microbial film to form a frozen electrochemically active microbial film; (4) drying the frozen electrochemically active microbial membrane by gradient temperature increase to form a dry electrochemically active microbial membrane wrapped by a protective agent; (5) Preserving the dried electrochemically active microbial film.

2. The method according to claim 1, characterized in that Step (1) further includes: screening highly active and mature electrochemically active microbial membranes, wherein the screening method includes: (1-1) assembling the incubated original electrochemically active microbial membrane into a microbial electrochemical sensor, and connecting the microbial electrochemical sensor to an electrochemical workstation to monitor the output current of the microbial electrochemical sensor, wherein the culture system of the microbial electrochemical sensor includes a first culture solution; (1-2) When the current of the microbial electrochemical sensor is lower than 10 -7 A, the second culture solution is pumped into the culture system for 20min to 60min, and the current of the microbial electrochemical sensor is lower than 10 -7 The current at A is I1, and the concentration of the organic substrate in the second culture solution is greater than the concentration of the organic substrate in the first culture solution; When the second culture solution is pumped into the culture system for 3 minutes, the output current of the microbial electrochemical sensor is I2; during the process of pumping the second culture solution into the culture system, the maximum current of the microbial electrochemical sensor is I3; when the pumping of the second culture solution into the culture system ends, the output current of the microbial electrochemical sensor is I4; when the pumping of the second culture solution into the culture system stops for 3 minutes, the output current of the microbial electrochemical sensor is I5; The criteria for determining a highly active and mature electrochemically active microbial film simultaneously meet the following requirements: (I2-I1) / (I3-I1)≥90%; (I4-I5) / (I3-I5)≥80%; I2, I3 and I4 are all greater than 0.2 mA.

3. The method according to claim 2, characterized in that The concentration of the organic substrate in the first culture solution is 0.01 mmol / L to 0.05 mmol / L; and / or, the concentration of the organic substrate in the second culture solution is 1 mmol / L to 5 mmol / L; and / or, the organic substrate in the first culture solution includes at least one of sodium acetate, sodium lactate and glucose; and / or, the organic substrate in the second culture solution includes at least one of sodium acetate, sodium lactate and glucose; And / or, in step (1-2), the second culture solution is pumped into the culture system at a flow rate of 10 mL / min to 50 mL / min.

4. The method according to claim 1, characterized in that: In step (2), the protective agent solution includes skimmed milk powder, trehalose and sodium chloride, wherein the content of skimmed milk powder in the protective agent solution is 3wt%~10wt%, the content of trehalose in the protective agent solution is 2wt%~10wt%, and the content of sodium chloride in the protective agent solution is 0.8wt%~3wt%.

5. The method according to claim 4, characterized in that The protective agent solution also includes at least one of glycerol, Tween and chitosan.

6. The method according to any one of claims 1 to 5, characterized in that In step (3), the protective agent coupled electrochemically active microbial membrane is cooled from T1 to T2, T3, and T4 in sequence, and the protective agent coupled electrochemically active microbial membrane is maintained at the temperatures of T1, T2, and T3 for 20 min to 40 min, respectively, and at the temperature of T4 for 4 h to 12 h; The T1 is 20°C to 30°C, the T2 is 8°C to 15°C, the T3 is 0°C to 5°C, and the T4 is -50°C to -80°C.

7. The method according to any one of claims 1 to 5, characterized in that In step (4), the temperature of the frozen electrochemically active microbial membrane is raised to T'1, T'2, and T'3 in sequence; the frozen electrochemically active microbial membrane is maintained at T'1 for 20 min to 40 min, with a vacuum degree of ≤5 Pa; the frozen electrochemically active microbial membrane is maintained at T'2 for 1 h to 2 h, with a vacuum degree of ≤5 Pa; the frozen electrochemically active microbial membrane is maintained at T'3 for 2 h to 3 h, with a vacuum degree of ≤5 Pa; The T'1 is equal to the eutectic point temperature T0-(0°C~10°C) of the protective agent solution, the T'2 is 0°C~5°C, and the T'3 is 20°C~30°C.

8. The method according to claim 7, characterized in that The frozen electrochemically active microbial film is heated from T4 to T'1 at a heating rate of 5°C / min to 10°C / min, the frozen electrochemically active microbial film is heated from T'1 to T'2 at a heating rate of 1°C / min to 5°C / min, and the frozen electrochemically active microbial film is heated from T'2 to T'3 at a heating rate of 5°C / min to 10°C / min.

9. A method for preparing an electrochemically active microbial sensor, characterized in that: include: (a) activating and resuscitating the dried electrochemically active microbial membrane preserved by the method according to any one of claims 1 to 8; (b) The activated and revived electrochemically active microorganisms are assembled into an electrochemically active microbial sensor.

10. The method according to claim 9, characterized in that In step (a), the dried electrochemically active microbial membrane is activated and revived using a third culture solution, the concentration of the organic substrate in the third culture solution is 1 mmol / L to 5 mmol / L, and the organic substrate in the third culture solution includes at least one of sodium acetate, sodium lactate and glucose.

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