Method for preserving embedded electrochemically active microbial film and application thereof

Abstract

The invention specifically discloses a method for preserving an embedded type electrochemical activity microbial film and a method for preparing an electrochemical activity microbial sensor. The method for preserving the embedded type electrochemical activity microbial film comprises the following steps: preparing the embedded type electrochemical activity microbial film; screening a high-activity embedded electrochemical active microbial film; preparing a protective agent coupled embedded electrochemical active microbial film; carrying out gradient cooling on the protective agent coupled embedded electrochemical active microbial film; carrying out gradient heating drying on the frozen embedded electrochemical active microbial film; and storing the dried embedded electrochemical active microbial film. According to the invention, the preservation conditions of the embedded electrochemical activity microbial film are broadened, the preservation time of the EAB biological film is prolonged, and the problem that the freeze-dried pure EAB is not prone to being frozen due to low-temperature freezing stress is solved through the embedded electrochemical activity microbial film; most of thalli die, the electricity generation capacity is reduced, and the adhesive force to a carrier material is reduced.

Description

Technical Field

[0001] The present application belongs to the technical field of biosensors, and specifically relates to a method for preserving an embedded electrochemically active microbial membrane and a method for preparing an electrochemically active microbial sensor. Background Art

[0002] Microbial electrochemical sensors (MES) with electrochemically active bacteria (EAB) as the core can directly transduce the substances to be tested in water into bioelectric signals. They have the advantages of rapid detection, high sensitivity, low detection cost, and strong anti-interference ability. They have good application prospects in the fields of biomedicine and environmental monitoring. Fresh EAB is very easy to deteriorate and difficult to transport over long distances and store for a long time. Fresh EAB can be prepared into EAB lyophilized powder to solve this problem. However, after fresh EAB is freeze-dried and prepared into lyophilized powder preparations, most of the pure bacteria EAB that have been freeze-dried will die and their electricity production capacity will decrease due to low-temperature freezing stress. The electrical signal will be greatly reduced when tested after rehydration, which is difficult to meet the needs of on-site testing. The reduction in the electrical signal of EAB after rehydration is mainly due to the reduction in its reverse electron transfer ability and the reduction in adhesion with the conductive medium. The detection signal can be increased by adding electron acceptors such as trimethylamine oxide to the reaction system. However, the actual on-site water conditions are complex and changeable, and some complex water samples will bind to electron acceptors, causing interference in the test.

[0003] In addition, the natural mixed EAB biofilm has strong tolerance to toxic substances, low sensitivity to toxic substances, and cannot detect low-concentration toxic substances. In order to improve the sensitivity to toxic substances, or to improve the sensitivity to a certain type / category of toxic substances, a pure EAB biofilm is needed. However, this biofilm formed by the specific pure EAB artificially screened and domesticated in a sterile clean space, although more sensitive to toxic substances than the natural mixed EAB biofilm, is often simple in structure and extremely fragile. There are fewer EABs attached to the carrier and they are easy to fall off, which greatly interferes with the test results. Summary of the invention

[0004] The present application aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the purpose of the present application is to propose a method for preserving an embedded electrochemically active microbial membrane and a method for preparing an electrochemically active microbial sensor. The present application broadens the storage conditions of the embedded electrochemically active microbial membrane. The dried embedded electrochemically active microbial membrane obtained by the method is no longer restricted to strict storage requirements. It can be stored in an ultra-low temperature environment, at a conventional refrigerator refrigeration temperature, or at room temperature, which is convenient for storage and transportation. At the same time, the method prolongs the storage time of the EAB biofilm, and the field test can quickly and simply obtain an EAB biofilm with guaranteed performance at any time. In addition, the present application significantly improves the bioelectric signal of the embedded EAB biofilm after cryopreservation, and significantly improves the sensitivity to heavy metal toxic substances. At the same time, the present application solves the problem that most of the pure bacteria EAB that have been freeze-dried will die, the power generation capacity will be reduced, and the adhesion to the carrier material will be reduced due to the low-temperature freezing stress.

[0005] In one aspect of the present application, the present application proposes a method for preserving an embedded electrochemically active microbial membrane. According to an embodiment of the present application, the method comprises:

[0006] (1) preparing an embedded electrochemically active microbial membrane, wherein the electrochemically active microorganisms in the embedded electrochemically active microbial membrane are a single species;

[0007] (2) Screening of highly active embedded electrochemically active microbial membranes;

[0008] (3) immersing the embedded electrochemically active microbial membrane in a protective agent solution to form a protective agent-coupled embedded electrochemically active microbial membrane;

[0009] (4) subjecting the protective agent-coupled embedded electrochemically active microbial membrane to a gradient cooling to form a frozen embedded electrochemically active microbial membrane;

[0010] (5) drying the frozen embedded electrochemically active microbial membrane by gradient temperature increase to form a dry embedded electrochemically active microbial membrane wrapped by a protective agent;

[0011] (6) preserving the dry embedded electrochemically active microbial membrane;

[0012] Step (1) comprises the following steps:

[0013] (1-1) Cultivate fresh bacterial culture;

[0014] (1-2) trapping the electrochemically active microorganisms in the fresh bacterial liquid in a porous conductive material;

[0015] (1-3) trapping activated carbon powder in the porous conductive material;

[0016] (1-4) Dense conductive material wrapping layers are respectively provided on both sides of the porous conductive material on which the electrochemically active microorganisms and the activated carbon powder are trapped, so as to form an embedded electrochemically active microorganism membrane.

[0017] According to the method for preserving the embedded electrochemically active microbial film according to the embodiment of the present application, first, the method broadens the storage conditions of the embedded electrochemically active microbial film, and the dry embedded electrochemically active microbial film obtained by the method is no longer bound by strict storage requirements, and can be stored in an ultra-low temperature environment, at a conventional refrigerator refrigeration temperature, or at room temperature, which is convenient for storage and transportation. Second, the method prolongs the shelf life of the embedded EAB biofilm, which can be stored at -60°C for more than two years and at room temperature for more than 3 months. Third, the dry embedded electrochemically active microbial film preserved by the method can be used after activation and recovery, and the embedded EAB biofilm with guaranteed performance can be quickly and simply obtained at any time for on-site testing. A series of processes for obtaining embedded EAB biofilms, preparing MES, and testing on the machine are simplified. Fourth, the present application prepares the electrochemically active microorganisms into a three-layer embedded electrochemically active microbial membrane before freeze-drying the electrochemically active microorganisms, so as to trap the pure bacteria EAB in the porous conductive material. The trapped pure bacteria EAB can continue to proliferate in the porous conductive material, and finally form a high-density pure bacteria EAB biofilm inside the material. Even after the freeze-drying operation, the electrical signal of the embedded electrochemically active microbial membrane can still meet the test needs, and no additional electron acceptors are required, which solves the problem that most of the pure bacteria EAB that have been freeze-dried will die, the electricity production capacity will be reduced, and the adhesion to the carrier material will be reduced due to the stress of low-temperature freezing. Therefore, the present application significantly improves the bioelectric signal of the embedded EAB biofilm after cryopreservation, and significantly improves the sensitivity to heavy metal toxic substances.

[0018] At the same time, the present application prepares the electrochemically active microorganisms into a three-layer embedded electrochemically active microbial membrane before freeze-drying the electrochemically active microorganisms, so as to trap the pure bacteria EAB in the porous conductive material. The trapped pure bacteria EAB can continue to proliferate in the porous conductive material, and finally form a high-density pure bacteria EAB biofilm (i.e., embedded electrochemically active microbial membrane) inside the material. Even after the freeze-drying operation, the electrical signal of the embedded electrochemically active microbial membrane can still meet the test needs, and no additional electron acceptor is required, which solves the problem that most of the pure bacteria EAB treated by freeze-drying will die due to low-temperature freezing stress, the power generation capacity is reduced, and the adhesion to the carrier material is reduced.

[0019] In addition, the method according to the above embodiment of the present application may also have the following additional technical features:

[0020] In some embodiments of the present application, the electrochemically active microorganisms in the embedded electrochemically active microbial film include one of Acinetobacter bayleyii BD4, Shewanella PV-4 and Shewanella MR-1.

[0021] In some embodiments of the present application, the porous conductive material includes at least one of carbon cloth and carbon felt; and / or the dense conductive material wrapping layer includes at least one of carbon paper and carbon cloth.

[0022] In some embodiments of the present application, in step (2), the screening of highly active embedded electrochemically active microbial membranes comprises:

[0023] (2-1) assembling the original embedded electrochemically active microbial membrane into a microbial electrochemical sensor, and connecting the 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;

[0024] (2-2) When the current of the microbial electrochemical sensor is lower than 10 -8 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 -8 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;

[0025] 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 judgment criteria of the highly active embedded electrochemically active microbial membrane meet the following requirements at the same time: (I2-I1) / (I3-I1)≥90%; (I4-I5) / (I3-I5)≥80%; I2, I3 and I4 are all greater than or equal to 5.0*10 -5 A.

[0026] In some embodiments of the present application, 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 lactate, sodium acetate and glucose; and / or, the organic substrate in the second culture solution includes at least one of sodium lactate, sodium acetate and glucose; and / or, in step (2-2), the second culture solution is pumped into the culture system at a flow rate of 10 mL / min to 50 mL / min.

[0027] In some embodiments of the present application, in step (3), the protective agent solution includes skimmed milk powder, trehalose and sodium chloride, the content of the skimmed milk powder in the protective agent solution is 3wt% to 10wt%, the content of the trehalose in the protective agent solution is 2wt% to 10wt%, and the content of the sodium chloride in the protective agent solution is 0.8wt% to 3wt%.

[0028] In some embodiments of the present application, in step (4), the protective agent-coupled embedded electrochemically active microbial film is cooled from T1 to T2, T3, and T4 in sequence, and the protective agent-coupled embedded electrochemically active microbial film 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; and / or, in step (5), the frozen embedded electrochemically active microbial film is sequentially cooled to T2, T3, and T4. The biofilm is heated to T'1, T'2, and T'3; the freeze-embedded electrochemically active microbial film is maintained at T'1 for 20 min to 40 min, with a vacuum degree of ≤5 Pa; the freeze-embedded electrochemically active microbial film is maintained at T'2 for 1 h to 2 h, with a vacuum degree of ≤5 Pa; the freeze-embedded electrochemically active microbial film 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 to 5°C) of the protective agent solution, the T'2 is 0°C to 5°C, and the T'3 is 20°C to 30°C.

[0029] In some embodiments of the present application, the temperature of the frozen-embedded electrochemically active microbial film is increased from T4 to T'1 at a heating rate of 5°C / min to 10°C / min, the temperature of the frozen-embedded electrochemically active microbial film is increased from T'1 to T'2 at a heating rate of 1°C / min to 5°C / min, and the temperature of the frozen-embedded electrochemically active microbial film is increased from T'2 to T'3 at a heating rate of 5°C / min to 10°C / min.

[0030] In a second aspect of the present application, the present application proposes a method for preparing an electrochemically active microbial sensor. According to an embodiment of the present application, the method comprises:

[0031] (a) activating and resuscitating the dry embedded electrochemically active microbial membrane preserved by the method described in the above embodiment;

[0032] (b) assembling an electrochemically active microorganism sensor using the activated and revived electrochemically active microorganisms.

[0033] According to the method for preparing an electrochemically active microbial sensor according to the embodiment of the present application, a dry embedded electrochemically active microbial membrane is used. The dry embedded electrochemically active microbial membrane can be used after activation and recovery. The embedded EAB biofilm with guaranteed performance can be obtained quickly and simply at any time for field testing. The process of obtaining the embedded EAB biofilm, preparing MES, and testing on the machine is simplified. In addition, the bioelectric signal of the embedded EAB biofilm after cryopreservation is significantly improved, and the sensitivity to heavy metal toxic substances is significantly improved.

[0034] In addition, the method according to the above embodiment of the present application may also have the following additional technical features:

[0035] In some embodiments of the present application, in step (a), the dry embedded 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 lactate, sodium acetate and glucose.

[0036] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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:

[0038] Figure 1 Schematic diagram of the process of preserving an embedded electrochemically active microbial membrane according to an embodiment of the present application. DETAILED DESCRIPTION

[0039] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0040] In one aspect of the present application, the present application provides a method for preserving an embedded electrochemically active microbial membrane. Figure 1 , the method includes: S100: preparing an embedded electrochemically active microbial membrane; S200: screening an embedded electrochemically active microbial membrane with high activity; S300: immersing the embedded electrochemically active microbial membrane in a protective agent solution to form a protective agent-coupled embedded electrochemically active microbial membrane; S400: gradient cooling the protective agent-coupled embedded electrochemically active microbial membrane to form a frozen embedded electrochemically active microbial membrane; S500: gradient heating and drying the frozen embedded electrochemically active microbial membrane to form a dry embedded electrochemically active microbial membrane wrapped by a protective agent; S600: preserving the dry embedded electrochemically active microbial membrane. Therefore, the present application broadens the preservation conditions of the embedded electrochemically active microbial membrane, and the dry embedded electrochemically active microbial membrane obtained by the method is no longer constrained by strict preservation requirements, and can be stored in an ultra-low temperature environment, at a conventional refrigerator refrigeration temperature, or at room temperature, which is convenient for storage and transportation. At the same time, the method prolongs the storage time of EAB biofilm, and the field test can quickly and easily obtain EAB biofilm with guaranteed performance at any time. In addition, the present application significantly improves the bioelectrical signal of the embedded EAB biofilm after cryopreservation, and significantly improves the sensitivity to heavy metal toxic substances.

[0041] The method for preserving the embedded electrochemically active microbial membrane proposed in this application is described in detail below:

[0042] Specifically, refer to the attached Figure 1 The method for preserving the embedded electrochemically active microbial membrane comprises the following steps:

[0043] S100: Preparation of embedded electrochemically active microbial membranes;

[0044] In the embodiment of the present application, the electrochemically active microorganisms are prepared into a three-layer embedded electrochemically active microbial membrane before freeze-drying, so that pure bacteria EAB can be trapped in the porous conductive material. The trapped pure bacteria EAB can continue to proliferate in the porous conductive material, and finally form a high-density pure bacteria EAB biofilm (i.e., embedded electrochemically active microbial membrane) inside the material. Even after the freeze-drying operation, the electrical signal of the embedded electrochemically active microbial membrane can still meet the test requirements, and no additional electron acceptor is required, which solves the problem that most of the pure bacteria EAB treated by freeze-drying will die due to low-temperature freezing stress, the power generation capacity is reduced, and the adhesion to the carrier material is reduced.

[0045] In an embodiment of the present application, the electrochemically active microorganisms in the embedded electrochemically active microbial membrane include one of Acinetobacter bayleyii BD4, Shewanella PV-4 and Shewanella MR-1. The biofilm of the present application is suitable for water quality biological toxicity testing of special water bodies, such as requiring the detection of low-concentration toxic substances and requiring the detection of heavy metal pollutants (sensitive to heavy metal toxicity, but not sensitive to pesticide toxic pollutants).

[0046] According to some specific embodiments of the present application, step S100 includes the following steps:

[0047] S110: Cultivate fresh bacterial liquid;

[0048] In this step, the EAB strains frozen in a low-temperature (e.g. -80°C) refrigerator can be taken out, thawed at room temperature, and then streaked on LB plate medium, and cultured at a constant temperature (e.g. 30°C) for activation. Select a single colony and inoculate it into LB liquid test tube medium, and culture it on a shaking table for 10h to 12h. Take a small amount of test tube bacterial solution and expand it in LB liquid triangular flask medium, and obtain fresh bacterial solution after culturing for 10h to 12h.

[0049] Furthermore, the concentration of the organic substrate in the above-mentioned culture medium is 1mmol / L to 5mmol / L (for example, it can be 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L, etc.), and the organic substrate in the above-mentioned culture medium includes at least one of sodium lactate, sodium acetate and glucose.

[0050] S120: trapping the electrochemically active microorganisms in the fresh bacterial liquid in a porous conductive material;

[0051] In this step, a loose porous conductive material (such as carbon cloth, carbon felt, etc.) can be spread flat on the bottom of the filtration cup, and the fresh bacterial liquid cultured in step S110 is poured in. The filtration bottle and the vacuum pump are connected for filtration. EAB is trapped in the loose porous conductive material, and the liquid is pumped into the filtration bottle. The filtration can be repeated multiple times to allow more EAB to adhere to the conductive material.

[0052] The reason why this application uses loose porous conductive materials (such as carbon cloth, carbon felt, etc.) is that under natural conditions, it is difficult for pure EAB to automatically attach to conductive materials, and the amount of attachment is limited, resulting in a low biofilm signal that is not conducive to detection. Loose porous conductive materials such as carbon cloth and carbon felt are used, and a pumping device is used to force the bacterial suspension to pass through the loose porous conductive material. While ensuring the flow of liquid, pure bacteria EAB can be trapped in the conductive material, and the trapped pure bacteria EAB can continue to proliferate in the conductive material, and finally a high-density pure bacteria EAB biofilm is formed inside the conductive material.

[0053] S130: trapping activated carbon powder in the porous conductive material;

[0054] In this step, activated carbon powder can be added to a 0.8wt% to 3wt% NaCl solution (5g to 10g of carbon powder is added to every 100mL of NaCl solution), and filtered while stirring, so that the carbon powder is also retained in the porous conductive material to fill the gaps in the material. Finally, 0.8wt% to 3wt% NaCl solution is poured in for filtering to wash the culture medium remaining in the porous material and the floating carbon powder.

[0055] S140: providing dense conductive material wrapping layers on both sides of the porous conductive material having the electrochemically active microorganisms and the activated carbon powder trapped therein, respectively, to form an embedded electrochemically active microorganism membrane;

[0056] In this step, high-density carbon paper and / or carbon cloth (which is conductive and water-permeable, through which test reagents can pass but bacteria cannot) can be fully soaked with 0.8wt% to 3wt% NaCl solution and then laid flat on the front and back sides of the conductive material with EAB and carbon powder trapped as a dense conductive material wrapping layer, forming a three-layer embedded structure of, for example, "carbon paper-material with EAB and carbon powder trapped-carbon paper". Finally, the three-layer embedded structure is cut into small pieces of uniform size (circular or square pieces, which can be selected according to the MES configuration) to obtain an embedded electrochemically active microbial membrane. In the subsequent test process, when water flows through the embedded electrochemically active microbial membrane, the carbon paper in the outermost layer can effectively prevent part of the bacteria / carbon powder from being washed away by water.

[0057] S200: Screening of highly active embedded electrochemically active microbial membranes;

[0058] According to some specific embodiments of the present application, the above screening method comprises:

[0059] S210: Assembling the original embedded electrochemically active microbial membrane into a microbial electrochemical sensor, and connecting the 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;

[0060] In this step, the original embedded electrochemically active microbial membrane (a small piece of loose porous conductive material with EAB attached) prepared in step S100 can be taken out and assembled into microbial electrochemical sensors (MES) one by one. The culture medium is replaced with the first culture medium with low concentration of organic substrate, and the multi-channel electrochemical workstation is connected to continuously monitor the output current of each channel MES. At this time, because the concentration of organic matter in the system is extremely low, it will be exhausted quickly. Within 30 minutes after the electrochemical workstation is connected, the output current of a single MES will be less than 10 -8 A.

[0061] According to some specific embodiments of the present application, the concentration of the organic substrate in the first culture solution is 0.01 mmol / L to 0.05 mmol / L (for example, it can be 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 solution includes at least one of sodium lactate, sodium acetate and glucose. The other components and concentrations in the first culture solution are the same as those in the original culture solution.

[0062] S220: When the current of the microbial electrochemical sensor is lower than 10 -8 After A, the second culture solution was pumped into the culture system for 20min to 60min, and the current of the microbial electrochemical sensor was lower than 10 for the first time. -8 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.

[0063] In this step, wait until all single MES output currents are lower than 10 -8 After A, the second culture solution 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, the MES output current of each channel continues to be monitored for 10 min.

[0064] According to some further specific embodiments of the present 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 lactate, sodium acetate and glucose.

[0065] When the second culture solution was pumped into the culture system for 3 minutes, the output current of the microbial electrochemical sensor was I2; during the process of pumping the second culture solution into the culture system, the maximum current of the microbial electrochemical sensor was I3; when the pumping of the second culture solution into the culture system ended, the output current of the microbial electrochemical sensor was I4; when the pumping of the second culture solution into the culture system stopped for 3 minutes, the output current of the microbial electrochemical sensor was I5.

[0066] The criteria for determining an embedded electrochemically active microbial membrane with high activity and a large number of attached EABs need to meet the following requirements at the same time: (I2-I1) / (I3-I1) ≥ 90%; (I4-I5) / (I3-I5) ≥ 80%; I2, I3 and I4 are all greater than or equal to 5.0-10 -5 A, thus the embedded electrochemically active microbial membrane with high activity and more attached EAB can be screened out.

[0067] S300: immersing the embedded electrochemically active microbial membrane in a protective agent solution to form a protective agent-coupled embedded electrochemically active microbial membrane;

[0068] In this step, firstly prepare the protective agent solution, dissolve skimmed milk powder, trehalose, glycerol, Tween, chitosan and other protective agents in a certain proportion in a NaCl aqueous solution, completely dissolve and shake well to obtain a protective agent solution. Then the protective agent solution is dispensed into vials, with a liquid layer thickness of 2cm to 5cm; the highly active EAB porous material screened out in step S200 is also added to the vials, 1 piece per bottle, and the bottle is gently shaken to expel bubbles, so that the porous material attached with EAB is completely immersed in the protective agent solution; stand at room temperature (20°C to 30°C) for 30 minutes, so that the porous material attached with EAB is fully infiltrated with the protective agent solution, forming a protective agent-coupled embedded electrochemically active microbial membrane, and the protective agent coupled to the embedded electrochemically active microbial membrane can effectively prevent the subsequent embedded electrochemically active microbial membrane from being frostbitten by ice crystals in a low temperature environment, and effectively protect the embedded electrochemically active microbial membrane.

[0069] According to some specific embodiments of the present application, the protective agent solution includes skimmed milk powder, trehalose and sodium chloride, the content of skimmed milk powder in the protective agent solution is 3wt% to 10wt% (for example, it can be 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, etc.), the content of trehalose in the protective agent solution is 2wt% to 10wt% (for example, it can be 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% (for example, it can be 0.8wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, etc.).

[0070] According to some further specific embodiments of the present application, the protective agent solution also 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%.

[0071] In the embodiments of the present application, a eutectic point tester may be used to measure the eutectic point temperature T0 of the protective agent solution.

[0072] S400: gradient cooling the protective agent-coupled embedded electrochemically active microbial membrane to form a frozen embedded electrochemically active microbial membrane;

[0073] In this step, the protective agent-coupled embedded EAB biofilm in step S300 can be placed in various temperature environments in the order of T1, T2, T3, and T4, and each temperature environment is left to stand for a period of time to form a frozen embedded EAB biofilm.

[0074] According to some other specific embodiments of the present application, the protective agent-coupled embedded electrochemically active microbial film is cooled down from T1 to T2, T3, and T4 in sequence, and the protective agent-coupled embedded electrochemically active microbial film 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; 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.

[0075] S500: performing gradient temperature drying on the frozen embedded electrochemically active microbial membrane to form a dry embedded electrochemically active microbial membrane wrapped by a protective agent;

[0076] In this step, the frozen embedded EAB biofilm in step S400 is placed in a vacuum freeze dryer that has been pre-frozen to T4. After placing the material temperature probe, the hatch is closed and the gradient temperature vacuum freeze drying program is started. The water in the protective agent solution and the embedded EAB biofilm is directly sublimated in a vacuum and frozen environment, and finally a dry embedded EAB biofilm wrapped only by the protective agent is formed. At this time, there is almost no water in the dry embedded EAB biofilm, and the microorganisms in the dry embedded EAB biofilm are in a dormant state.

[0077] According to some other specific embodiments of the present application, the above-mentioned gradient temperature increase vacuum freeze-drying procedure is: 1) heating the frozen-embedded electrochemically active microbial film from T4 to T'1 at a heating rate of 5°C / min to 10°C / min, maintaining it at T'1 for 20min to 40min, and the vacuum degree is ≤5Pa; 2) heating the frozen-embedded electrochemically active microbial film from T'1 to T'2 at a heating rate of 1°C / min to 5°C / min, maintaining it at T'2 for 1h to 2h, and the vacuum degree is ≤5Pa; 3) heating the frozen-embedded electrochemically active microbial film from T'2 to T'3 at a heating rate of 5°C / min to 10°C / min, maintaining it at T'3 for 2h to 3h, and the vacuum degree is ≤5Pa, sublimating all the water to form a dry embedded EAB biofilm wrapped only by a protective agent. T'1 is equal to the eutectic temperature T0-(0℃~5℃) of the protective agent solution, T'2 is 0℃~5℃, and T'3 is 20℃~30℃. By controlling the heating rate and maintenance time of each stage of gradient heating, the injury or death of microorganisms in the embedded electrochemically active microbial film can be effectively avoided, and at the same time, the purpose of effectively drying the embedded EAB biofilm can be ensured.

[0078] S600: Preservation of dry embedded electrochemically active microbial membranes.

[0079] In this step, there is almost no water in the dry embedded electrochemically active microbial membrane, and the microorganisms in the dry embedded electrochemically active microbial membrane are in a dormant state. Therefore, the temperature for storing the dry embedded electrochemically active microbial membrane is not particularly limited. It can be stored in an ultra-low temperature environment (e.g., -60°C), at a conventional refrigerator temperature, or at room temperature. This broadens the storage conditions for the biofilm and no longer adheres to strict storage requirements; it also extends the storage time of the embedded EAB biofilm, so that the embedded EAB biofilm with guaranteed performance can be quickly and easily obtained at any time during on-site testing.

[0080] As some preferred embodiments, at the end of the above step S600, the vacuum seal is applied, the air inlet valve is opened to take out the freeze-dried embedded EAB biofilm, and then the biofilm is placed at -20°C for two weeks for equilibrium, and then stored at -60°C for preservation and taken out before use.

[0081] The method for preserving an embedded electrochemically active microbial membrane according to an embodiment of the present application has at least the following advantages:

[0082] 1) This method broadens the storage conditions of embedded electrochemically active microbial films (i.e., embedded EAB biofilms). The dry embedded electrochemically active microbial films obtained by this method are no longer subject to strict storage requirements. They can be stored in ultra-low temperature environments (e.g., -60°C), at conventional refrigerator temperatures, or at room temperature, making them convenient for storage and transportation.

[0083] 2) This method prolongs the storage time of embedded EAB biofilms, which can be stored for more than two years at -60°C and more than three months at room temperature.

[0084] 3) The dried embedded electrochemically active microbial film (i.e., embedded EAB biofilm) preserved by this method can be used after activation and recovery, and embedded EAB biofilm with guaranteed performance can be obtained quickly and easily at any time for field testing. This simplifies the process of obtaining embedded EAB biofilm, preparing MES, and testing on the machine.

[0085] 4) The present application prepares the electrochemically active microorganisms into a three-layer embedded electrochemically active microbial membrane before freeze-drying the electrochemically active microorganisms, so as to trap the pure bacteria EAB in the porous conductive material. The trapped pure bacteria EAB can continue to proliferate in the porous conductive material, and finally form a high-density pure bacteria EAB biofilm (i.e., embedded electrochemically active microbial membrane) inside the material. Even after the freeze-drying operation, the electrical signal of the embedded electrochemically active microbial membrane can still meet the test needs, and no additional electron acceptor is required, which solves the problem that most of the pure bacteria EAB that have been freeze-dried will die, the electricity generation capacity will be reduced, and the adhesion to the carrier material will be reduced due to the stress of low-temperature freezing. Therefore, the present application significantly improves the bioelectric signal of the embedded EAB biofilm after cryopreservation, and significantly improves the sensitivity to heavy metal toxic substances.

[0086] In a second aspect of the present application, the present application proposes a method for preparing an electrochemically active microbial sensor. According to an embodiment of the present application, the method comprises:

[0087] (a) activating and resuscitating the dry embedded electrochemically active microbial membrane preserved by the method of the above embodiment;

[0088] In this step, the dried embedded electrochemically active microbial membrane can be 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 lactate, sodium acetate and glucose.

[0089] As a specific example, the dried embedded EAB biofilm is taken out from a storage environment at -60°C, the bottle stopper is opened and immediately an LB culture medium of the same volume as that used for packaging is added to the vial, the vial is quickly shaken to allow the protective agent in the dried embedded electrochemically active microbial film to fully dissolve and the biofilm to fully absorb water, and then the vial is allowed to stand at room temperature for 20 to 60 minutes to obtain an activated and revived embedded EAB biofilm.

[0090] (b) Electrochemically active microorganisms are assembled and revived to form electrochemically active microbial sensors.

[0091] In this step, the activated and revived embedded EAB biofilm was assembled according to the conventional MES assembly method for on-machine testing.

[0092] According to the method for preparing an electrochemically active microbial sensor according to the embodiment of the present application, a dry embedded electrochemically active microbial membrane (i.e., embedded EAB biofilm) is used. The dry embedded electrochemically active microbial membrane (i.e., embedded EAB biofilm) can be used after activation and recovery. On-site testing can quickly and easily obtain an embedded EAB biofilm with guaranteed performance at any time. The process of obtaining the embedded EAB biofilm, preparing MES, and testing on the machine is simplified. In addition, the bioelectrical signal of the embedded EAB biofilm after cryopreservation is significantly improved, and the sensitivity to heavy metal toxic substances is significantly improved.

[0093] The embodiments of the present application are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. In addition, unless otherwise explicitly stated, all reagents used in the following embodiments are commercially available or can be synthesized according to this article or known methods, and the reaction conditions not listed are also easily available to those skilled in the art.

[0094] Comparative Example 1

[0095] Step 1: Take out the EAB strain (Shewanella PV-4) frozen in a -80℃ low-temperature refrigerator, thaw at room temperature, and then streak the strain on the LB plate medium and incubate at 30℃ for 24 hours for activation. Select a single colony and inoculate it into a 5mL LB liquid test tube culture medium and incubate it in a shaking table at 30℃ and 200rpm for 12 hours. Take 1mL of the test tube bacterial solution and expand it in a 100mL LB liquid triangular flask medium. Incubate it for 12 hours to obtain a fresh bacterial solution. Medium ingredients: The organic substrate is sodium lactate, and its concentration is 5mmol / L.

[0096] Centrifuge 300mL of fresh bacterial liquid at 10,000rpm for 3min in a 4℃ centrifuge to collect the bacteria; resuspend with 0.9% NaCl aqueous solution and centrifuge again to wash the residual culture medium to finally obtain the washed bacteria. Dissolve 8% skim milk powder, 4% trehalose, 3% glycerol and other protective agents in 0.9% NaCl aqueous solution, completely dissolve and shake well. Resuspend the washed bacteria with 100mL of protective agent solution; then dispense into vials with a liquid layer thickness of 2-5cm and cover; use a eutectic point tester to measure the eutectic point temperature of the protective agent solution T0 = -25.56℃; finally, place the vials filled with materials in a -60℃ environment for pre-freezing for 8h.

[0097] Step 2: Gradient temperature increase vacuum freeze drying. The pre-frozen material is placed in a vacuum freeze dryer that has been pre-frozen to -55°C. After placing the material temperature probe, the hatch is closed and the gradient temperature increase vacuum freeze drying program is started. The water in the protective agent solution is directly sublimated in a vacuum and frozen environment to form the Shewanella PV-4 freeze-dried powder preparation. The vacuum freeze dryer program design: (1) The material is heated from -55°C to -25°C at a heating rate of 10°C per hour. After reaching the target temperature, it is maintained for 30 minutes, and the vacuum degree is ≤5Pa; (2) The material is heated from -25°C to 5°C at a heating rate of 5°C per hour. After reaching the target temperature, it is maintained for 60 minutes, and the vacuum degree is ≤5Pa; (3) The material is heated from 5°C to 25°C at a heating rate of 10°C per hour. After reaching the target temperature, it is maintained for 120 minutes, and the vacuum degree is ≤5Pa. All the water is sublimated to form the Shewanella PV-4 freeze-dried powder preparation. At the end of freeze-drying, vacuum the lid, open the air inlet valve and take out the Shewanella PV-4 freeze-dried powder preparation, then place it at -20℃ for two weeks to equilibrate, and then store it at -60℃ and take it out before use.

[0098] Step 3: Activation and resuscitation. Take out the Shewanella PV-4 lyophilized powder preparation from -60℃, open the bottle stopper and immediately add the same volume of LB medium as that used for dispensing into the vial, shake the vial quickly to fully dissolve the protective agent, and then let it stand at room temperature for 30 minutes to obtain the activated and resuscitated Shewanella PV-4 bacterial suspension.

[0099] Step 4: Assemble 3 sets of MES, consisting of working electrode, counter electrode, reference electrode and electrolytic cell. The working electrode is 4 cm 2 Square carbon cloth (WOS1009, China Carbon Energy Company) was soaked in acetone overnight before use, dried and then subjected to high-temperature ammoniation treatment. The counter electrode and reference electrode were 1 cm 2 Square platinum sheet (Pt210, Tianjin Aida Hengsheng Technology Co., Ltd.) and Ag / AgCl electrode (R0303, Tianjin Aida Hengsheng Technology Co., Ltd.; 222.4mV vs. standard hydrogen electrode). The total volume of the electrolytic cell is 50mL and is sealed with a PTFE lid. Five holes are set on the lid, three of which are inserted into the working electrode, counter electrode and reference electrode respectively, and the other two holes are used as the water inlet and outlet during self-circulation. The reference electrode is soaked in 75% medical alcohol overnight, and all other accessories are sterilized by high temperature and high pressure. Finally, the electrochemical system is assembled in a clean workbench.

[0100] 20 mL of the Shewanella PV-4 suspension activated and revived in step 3 was added to MES containing 20 mL of sterile electrolyte, each liter of electrolyte containing 1 g NaHCO3, 0.13 g KCl, 0.027 g CaCl2·2H2O, 0.2 g MgCl2·6H2O, 5.85 g NaCl and 7.2 g HEPES. After inoculation, it was placed in a constant temperature incubator (HPS500, Harbin Donglian Electronic Technology Development Co., Ltd.) for cultivation, and a peristaltic pump was used to self-circulate the bacteria at a constant temperature of 3 mL / min to resuscitate the bacteria. While resuscitating the bacteria, the bacterial respiratory metabolism was used to remove the dissolved oxygen in the solution. The working electrode potential was set to -0.5 V (vs. Ag / AgCl electrode) using a multi-channel constant potentiostat (CHI1030C, Shanghai Chenhua Instrument Co., Ltd.), and the electrical signal of MES was continuously recorded and monitored in real time. After the current stabilized, 1 mmol / L (final concentration) of trimethylamine oxide was added, and the changes in the electrical signal were recorded and observed until the electron acceptor was exhausted and the current stabilized to the baseline level again. Then, 0.5 mg / L Cd 2+ 1mmol / L (final concentration) of trimethylamine oxide was added, and the changes in the electrical signals were recorded again.

[0101] After adding trimethylamine oxide, a peak current of 30μA appeared immediately. 2+ After adding 1mmol / L (final concentration) of trimethylamine oxide, a peak current of 25μA appeared immediately. Compared with the peak current without adding toxic substances, the inhibition rate was 16.67%.

[0102] Example 1

[0103] Step 1: Take out the EAB strain (Shewanella PV-4) frozen in a -80℃ low-temperature refrigerator, thaw at room temperature, and then streak the strain on the LB plate medium and incubate at 30℃ for 24 hours for activation. Select a single colony and inoculate it into a 5mL LB liquid test tube culture medium and incubate it in a shaking table at 30℃ and 200rpm for 12 hours. Take 1mL of the test tube bacterial solution and expand it in a 100mL LB liquid triangular flask medium. Incubate it for 12 hours to obtain a fresh bacterial solution. Medium ingredients: The organic substrate is sodium lactate, and its concentration is 5mmol / L.

[0104] Step 2: comprises the following steps: (1) spreading the loose porous conductive material carbon felt on the bottom of the filtration cup, pouring the fresh bacterial solution of step 1, connecting the filtration bottle and the vacuum pump for filtration, the EAB in the fresh bacterial solution is trapped in the loose porous conductive material, the liquid is pumped into the filtration bottle, and the filtration can be repeated for multiple times to allow more EAB to adhere to the conductive material; (2) adding activated carbon powder to 0.9% NaCl solution (adding 5g of carbon powder to every 100mL of NaCl solution), stirring and filtration, so that the carbon powder is also trapped in the conductive material, further filling the gaps in the conductive material, and forming a compact EAB bacterial body inside - the carbon powder is filled in The mesh-like conductive material can improve the adhesion ability of EAB bacteria to the conductive material; (3) Pour 0.9% NaCl solution into the porous material for the last time to filter and wash the residual culture medium and the floating carbon powder in the porous material; (4) High-density carbon paper (which is conductive and permeable to water, test reagents can pass through, but bacteria cannot) is fully soaked with 0.9% NaCl solution and then laid flat on the front and back sides of the conductive material with EAB and carbon powder trapped, forming a three-layer embedded structure of "carbon paper-conductive material with EAB and carbon powder trapped-carbon paper"; (5) Finally, the three-layer embedded structure is cut into small pieces of uniform size to adapt to the MES configuration.

[0105] Step 3: Select the embedded electrochemically active microbial membrane with high activity and more attached EAB. (1) Take out the carbon felt with attached EAB prepared in step 2, assemble them into MES one by one, replace the culture medium with the first culture medium (sodium lactate concentration 0.03mmol / L, other components and concentrations are the same as the original culture medium), and connect to the multi-channel electrochemical workstation to continuously monitor the output current of each channel MES; (2) At this time, because the concentration of organic matter in the system is extremely low, it will be exhausted quickly. Within 30 minutes after connecting to the electrochemical workstation, the output current of a single MES will be less than 10 -8 A; (3) When the output current of all single MES (denoted as I1) is less than 10 -8 After A, the second culture medium (sodium lactate concentration 3.0mmol / L, other components and concentrations are the same as the original culture medium) was pumped into the culture system at a flow rate of 30mL / min for 30min. After the pumping was completed, the MES output current of each channel was continuously monitored for 10min. The output current of a single MES 3min after the start of the pumping was recorded as I2, the maximum output current of a single MES during the 30min pumping process was recorded as I3, the output current of a single MES at the 30th minute of the pumping was recorded as I4, and the output current of a single MES 3min after the pumping stopped was recorded as I5.

[0106] The criteria for determining an embedded electrochemically active microbial membrane with high activity and a large number of attached EABs must meet the following requirements at the same time: (1) (I2-I1) / (I3-I1) ≥ 90%; (2) (I4-I5) / (I3-I5) ≥ 80%; (3) I2, I3, and I4 are greater than or equal to 5.0*10 -5 A.

[0107] Table 1

[0108] Current <![CDATA[I1 / μA]]> <![CDATA[I2 / μA]]> <![CDATA[I3 / μA]]> <![CDATA[I4 / μA]]> <![CDATA[I5 / μA]]> Formula (1) Formula (2) MES1 12 79 86 80 33 90.54％ 88.68％ MES2 16 70 75 75 20 91.53％ 100.00％ MES3 17 59 63 62 30 91.30％ 96.97％

[0109] It can be seen that MES-1, MES-2 and MES-3 are all embedded electrochemically active microbial membranes with high activity and a large number of attached EABs.

[0110] Step 4: Preparation of protective agent solution. Dissolve protective agents such as skimmed milk powder, trehalose, and glycerol in 0.9% NaCl aqueous solution, completely dissolve and shake well, the concentration of skimmed milk powder is 8%, the concentration of trehalose is 4%, and the concentration of glycerol is 3%.

[0111] Step 5: Packaging and coupling to form a protective agent-coupled embedded EAB biofilm. The protective agent solution is dispensed into vials, with a liquid layer thickness of 2-5 cm; the highly active embedded electrochemically active microbial membrane selected in step 3 is also added to the vials, 1 piece per bottle, and the bottle is gently shaken to expel bubbles, so that the carbon felt is completely immersed in the protective agent solution; it is allowed to stand at room temperature (20-30°C) for 30 minutes to allow the embedded EAB biofilm to fully infiltrate the protective agent solution. The eutectic point temperature T0 of the protective agent solution is measured using a eutectic point tester, which is -24.56°C.

[0112] Step 6: Gradient cooling precooling. The protective agent-coupled embedded EAB biofilm formed in step 5 is placed in various temperature environments in the order of room temperature (20-30°C), 10°C, 0°C, and -60°C. Each temperature environment is left to stand for 30 minutes, and finally placed in a -60°C freezer for 8 hours to form a frozen embedded EAB biofilm.

[0113] Step 7: Gradient temperature increase vacuum freeze drying. Place the frozen embedded EAB biofilm formed in step 6 in a vacuum freeze dryer that has been pre-frozen to -55°C. After placing the material temperature probe, close the hatch and start the gradient temperature increase vacuum freeze drying program. The water in the protective agent solution and the embedded EAB biofilm will sublime directly in a vacuum and frozen environment, and finally form a dry embedded EAB biofilm wrapped only by the protective agent.

[0114] Program design of vacuum freeze dryer: (1) Heat the material from -55°C to -25°C at a heating rate of 10°C per hour, maintain the target temperature for 30 minutes, and the vacuum degree is ≤5Pa; (2) Heat the material from -25°C to 5°C at a heating rate of 5°C per hour, maintain the target temperature for 60 minutes, and the vacuum degree is ≤5Pa; (3) Heat the material from 5°C to 25°C at a heating rate of 10°C per hour, maintain the target temperature for 120 minutes, and the vacuum degree is ≤5Pa, so as to sublimate all the water and form a dry embedded EAB biofilm wrapped only with a protective agent.

[0115] Step 8: At the end of step 7 above, vacuum seal the lid, open the air inlet valve and take out the freeze-dried embedded EAB biofilm, then place it at -20°C for two weeks to equilibrate, then store it at -60°C and take it out before use.

[0116] Step 9: Activation and resuscitation. Take out the dried embedded EAB biofilm from -60℃, open the bottle stopper and immediately add the same volume of LB medium as that used for packaging into the vial, shake the vial quickly to fully dissolve the protective agent and fully absorb water for the biofilm, and then let it stand at room temperature for 30 minutes to obtain the activated and resuscitated embedded EAB biofilm.

[0117] Step 10: Except that the working electrode directly uses the embedded EAB biofilm activated and revived in step 9 above, other operations are the same as step 4 of the comparative example, and 3 groups of MES are assembled; 40 mL of DM electrolyte is added to each group of MES, and the peristaltic pump is started to circulate at a speed of 10 mL / min for 10 minutes, and the constant potential instrument is started to collect the current signal I 01 ,I 02 ,I 03 After draining the electrolyte from the MES, add 40 mL of toxic DM electrolyte (containing 0.2 mg / L of toxic substance Cd 2+ ), start the peristaltic pump to circulate at a speed of 10 mL / min for 10 min, and start the constant potential instrument to collect the current signal I 11 ,I 12 ,I 13 .

[0118] DM electrolyte composition: Each liter of DM electrolyte contains 2.5g NaHCO3, 1.0g NH4Cl, 0.08g CaCl2·H2O, 0.2g MgCl2·6H2O, 10g NaCl, 7.2g HEPES, and 2mmol sodium lactate.

[0119] Toxic DM electrolyte: Each liter of solution contains 2.5g NaHCO3, 1.0g NH4Cl, 0.08g CaCl2·H2O, 0.2g MgCl2·6H2O, 10g NaCl, 7.2g HEPES, 2mmol sodium lactate, and 0.2mg Cd(NO3)2.

[0120] In non-toxic electrolyte, the MES current is I 01 =42μA, I 02 =55μA, I 03 =49μA, which is significantly higher than the current of the comparative example in the non-toxic electrolyte (the original current of the comparative example is 30μA; the current of the test group containing toxic substances is 25μA). It can be seen that compared with the comparative example 1, the embodiment 1 has a higher bioelectric signal. The higher bioelectric signal can reduce the test interference caused by other factors such as vibration and fluctuation of the signal acquisition system during the test process, and improve the data accuracy. In this embodiment, after adding 0.2mg / LCd 2+ In the electrolyte, the MES current is I 11 =28μA, I 12 =33μA, I 13 =34μA, the inhibition rates were 33.3%, 40.0%, and 30.6% respectively (significantly higher than the control test containing 0.5mg / LCd 2+ The inhibition rate of toxic substances was 16.67%), which shows that the embodiment of the present application significantly improves the inhibition of heavy metal toxic substances Cd 2+ sensitivity.

[0121] Example 2

[0122] The preparation method of this embodiment is basically the same as that of embodiment 1, except that:

[0123] The toxic substance 0.2 mg / L Cd 2+ Changed to 0.2mg / L Cu 2+ The other operations are the same as those in Example 1.

[0124] The composition of DM electrolyte is the same as that in Example 1.

[0125] Toxic DM electrolyte: Each liter of solution contains 2.5g NaHCO3, 1.0g NH4Cl, 0.08g CaCl2·H2O, 0.2g MgCl2·6H2O, 10g NaCl, 7.2g HEPES, 2mmol sodium lactate, and 0.2mg CuSO4.

[0126] Toxic substances from highly toxic Cd 2+ Changed to Cu with less toxicity 2+, three groups of MES were tested, and the inhibition rates were 22.4%, 25.7% and 23.6% respectively, which still had a relatively obvious toxic response, indicating that the biofilm prepared in this application can also respond to weakly toxic heavy metals.

[0127] Example 3

[0128] The preparation method of this embodiment is basically the same as that of embodiment 1, except that:

[0129] The DM electrolyte composition tested at the end of Example 1 was changed to the following concentration, and the other operations were the same as in Example 1.

[0130] DM electrolyte composition: each liter of solution contains 2.5g NaHCO3, 1.0g NH4Cl, 0.08g CaCl2·H2O, 0.2g MgCl2·6H2O, 10g NaCl, 7.2g HEPES, and 1mmol sodium lactate.

[0131] Toxic DM electrolyte: Each liter of solution contains 2.5g NaHCO3, 1.0g NH4Cl, 0.08g CaCl2·H2O, 0.2g MgCl2·6H2O, 10g NaCl, 7.2g HEPES, 1mmol sodium lactate, and 0.2mg Cd(NO3)2.

[0132] In non-toxic electrolyte, the MES current is I 01 =28μA, I 02 =35μA, I 03 =29μA, which is lower than that of Example 1 but still meets the test requirements, indicating that the biofilm prepared in this application uses low-concentration organic matter (2mmol / L sodium lactate in Example 1 and 1mmol / L sodium lactate in Example 3); when adding 0.2mg / L Cd 2+ In the electrolyte, the MES current is I 11 =13μA, I 12 =21μA, I 13 =18μA, the inhibition rates were 53.6%, 40.0%, and 37.9% respectively (the inhibition rates of some tests were higher than those of Example 1), indicating that the biofilm prepared in this application can further improve the resistance to heavy metal toxic substances Cd when tested with low concentrations of organic matter. 2+ sensitivity.

[0133] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0134] 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 embedded electrochemically active microbial membrane, characterized in that: include: (1) preparing an embedded electrochemically active microbial membrane, wherein the electrochemically active microorganisms in the embedded electrochemically active microbial membrane are a single species; (2) Screening of highly active embedded electrochemically active microbial membranes; (3) immersing the embedded electrochemically active microbial membrane in a protective agent solution to form a protective agent-coupled embedded electrochemically active microbial membrane; (4) subjecting the protective agent-coupled embedded electrochemically active microbial membrane to a gradient cooling to form a frozen embedded electrochemically active microbial membrane; (5) drying the frozen embedded electrochemically active microbial membrane by gradient temperature increase to form a dry embedded electrochemically active microbial membrane wrapped by a protective agent; (6) preserving the dry embedded electrochemically active microbial membrane; Step (1) comprises the following steps: (1-1) Cultivate fresh bacterial culture; (1-2) trapping the electrochemically active microorganisms in the fresh bacterial liquid in a porous conductive material; (1-3) trapping activated carbon powder in the porous conductive material; (1-4) Dense conductive material wrapping layers are respectively provided on both sides of the porous conductive material on which the electrochemically active microorganisms and the activated carbon powder are trapped, so as to form an embedded electrochemically active microorganism membrane.

2. The method according to claim 1, characterized in that The electrochemically active microorganisms in the embedded electrochemically active microbial membrane include one of Acinetobacter bayleyii BD4, Shewanella PV-4 and Shewanella MR-1.

3. The method according to claim 1, characterized in that The porous conductive material includes at least one of carbon cloth and carbon felt; And / or, the dense conductive material wrapping layer includes at least one of carbon paper and carbon cloth.

4. The method according to claim 1, characterized in that: In step (2), the screening of highly active embedded electrochemically active microbial membranes comprises: (2-1) assembling the original embedded electrochemically active microbial membrane into a microbial electrochemical sensor, and connecting the 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; (2-2) When the current of the microbial electrochemical sensor is lower than 10 -8 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 -8 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 the highly active embedded electrochemically active microbial membrane simultaneously meet the following requirements: (I2-I1) / (I3-I1)≥90%; (I4-I5) / (I3-I5)≥80%; I2, I3 and I4 are all greater than or equal to 5.0*10 -5 A.

5. The method according to claim 4, 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 lactate, sodium acetate and glucose; and / or, the organic substrate in the second culture solution includes at least one of sodium lactate, sodium acetate and glucose; And / or, in step (2-2), the second culture solution is pumped into the culture system at a flow rate of 10 mL / min to 50 mL / min.

6. The method according to claim 1, characterized in that In step (3), 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% to 10wt%, the content of trehalose in the protective agent solution is 2wt% to 10wt%, and the content of sodium chloride in the protective agent solution is 0.8wt% to 3wt%.

7. The method according to any one of claims 1 to 6, characterized in that: In step (4), the protective agent-coupled embedded electrochemically active microbial membrane is cooled from T1 to T2, T3, and T4 in sequence, and the protective agent-coupled embedded electrochemically active microbial membrane is maintained at the temperatures of T1, T2, and T3 for 20 min to 40 min, respectively, and maintained 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.; And / or, in step (5), the temperature of the frozen-embedded electrochemically active microbial membrane is raised to T'1, T'2, and T'3 in sequence; the frozen-embedded electrochemically active microbial membrane is maintained at the temperature of T'1 for 20 min to 40 min, and the vacuum degree is ≤5 Pa; the frozen-embedded electrochemically active microbial membrane is maintained at the temperature of T'2 for 1 h to 2 h, and the vacuum degree is ≤5 Pa; the frozen-embedded electrochemically active microbial membrane is maintained at the temperature of T'3 for 2 h to 3 h, and the vacuum degree is ≤5 Pa; the T'1 is equal to the eutectic point temperature T0-(0°C to 5°C) of the protective agent solution, the T'2 is 0°C to 5°C, and the T'3 is 20°C to 30°C.

8. The method according to claim 7, characterized in that The temperature of the frozen-embedded electrochemically active microbial film is increased from T4 to T'1 at a heating rate of 5°C / min to 10°C / min, the temperature of the frozen-embedded electrochemically active microbial film is increased from T'1 to T'2 at a heating rate of 1°C / min to 5°C / min, and the temperature of the frozen-embedded electrochemically active microbial film is increased 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 dry embedded electrochemically active microbial membrane preserved by the method according to any one of claims 1 to 8; (b) assembling an electrochemically active microorganism sensor using the activated and revived electrochemically active microorganisms.

10. The method according to claim 9, characterized in that In step (a), the dried embedded 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 lactate, sodium acetate and glucose.

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