CN116429858B

CN116429858B - Method and use for acclimatizing electrochemically active biofilms

Info

Publication number: CN116429858B
Application number: CN202310708143.7A
Authority: CN
Inventors: 戈燕红; 查凡; 余梅; 郭德音; 刘红; 易越; 刘桂雄
Original assignee: Guangdong Yingfeng Technology Co ltd
Current assignee: Guangdong Yingfeng Technology Co ltd
Priority date: 2023-06-15
Filing date: 2023-06-15
Publication date: 2023-09-19
Anticipated expiration: 2043-06-15
Also published as: CN116429858A

Abstract

The invention discloses a method for domesticating electrochemical active biological membranes and application thereof. The method for acclimating the electrochemical active biological film comprises the following steps: and simultaneously culturing a plurality of bioanode in the same electrolytic cell under the same culture solution environment and the same culture condition to obtain a plurality of electrochemical active biological membranes, wherein the bioanode comprises a biological membrane carrier attached with target bacteria. The method not only can rapidly domesticate a plurality of EAB biological films with high incubation consistency in batches, can shorten the incubation period to below 7 days, is beneficial to shortening the on-machine adaptation time in the later test or application, but also can avoid frequent liquid change operation, even can realize the effect of not needing to change the culture liquid in the domestication process, has high repeatability of the domesticated biological films, can provide an application basis for MEB water biotoxicity detection, and is suitable for large-scale production and application.

Description

Method and use for acclimatizing electrochemically active biofilms

Technical Field

The invention belongs to the field of biochemistry, and particularly relates to a method for domesticating an electrochemical active biological membrane and application thereof.

Background

The microbial electrochemical sensor (MEB) taking an electrochemical active microorganism (EAB) biomembrane as a core can directly convert substances to be detected in a water body into bioelectric signals, has the advantages of rapid detection, high sensitivity, low detection cost, strong anti-interference capability and the like, and has good application prospects in the fields of biomedicine and environmental monitoring. However, the EAB biological film domestication time is long, and a plurality of batches of culture solutions are often required to be replaced, so that dominant bacterial groups in a mixed bacterial system are unstable, the biological activity difference of the mature EAB biological film is large, the toxicity detection repeatability is low, and the practical application requirements cannot be met.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, the invention aims to provide a method and application for domesticating an electrochemical active biological film, which are used for solving at least one of the problems of low domestication efficiency, poor consistency of an EAB biological film obtained by incubation and the like.

In one aspect, the invention features a method of acclimating an electrochemically active biofilm, the method comprising: and simultaneously culturing a plurality of bioanode in the same electrolytic cell under the same culture solution environment and the same culture condition to obtain a plurality of electrochemical active biological membranes, wherein the bioanode comprises a biological membrane carrier attached with target bacteria.

In the invention, the method for domesticating the electrochemical active biological film has at least the following beneficial effects: in the domestication process, the environment of a nutrition system, temperature, pH value and the like where a plurality of biological anodes are positioned is good in consistency, and when a carbon source in a culture solution is enough, the culture solution is not required to be replaced in the culture process, so that the method is not only favorable for obtaining EAB biological membranes with high consistency, but also can avoid frequent liquid replacement operation, even can realize the effect of not replacing the culture solution in the whole domestication process, can greatly reduce the domestication workload and the requirements on operators, effectively avoid the biological membrane incubation control condition difference caused by the differences of a plurality of independent small electrolytic cell systems due to the flora, dissolved oxygen, nutritional ingredients, temperature and the like, further cause the problems of batch-to-batch difference, poor consistency and the like of the EAB biological membranes obtained by domestication, and also be favorable for shortening the on-machine adaptation time in the later test or application. In conclusion, the method not only can rapidly domesticate a plurality of EAB with high incubation consistency in batches, and can shorten the incubation period to below 7 days (for example, to 2-3 days), and is beneficial to shortening the on-machine adaptation time in the later test or application, but also can avoid frequent liquid change operation, even can realize the effect of not needing to change culture liquid in the domestication process, the domestication biological film has high repeatability, can provide an application foundation for MEB water biotoxicity detection, and is suitable for mass production application.

In some embodiments of the present invention, the electrolytic cell includes at least one electrode group, each electrode group includes a reference electrode, a counter electrode and at least one bioanode, the electrode groups are connected to potentiostats, during the culturing process, different electrode groups are connected to different potentiostats, the culturing voltages controlled by the different potentiostats are the same, and current changes of the channels during the culturing process are monitored in real time to determine whether to supplement carbon sources into the culture solution and whether to complete the culturing, wherein the type of the carbon sources is the same as the type of the carbon sources contained in the culture solution.

In some embodiments of the invention, the distances between the reference electrode and the counter electrode in different electrode sets are equal, or the difference in distance between the reference electrode and the counter electrode in different electrode sets is greater than 0 and less than or equal to 5% based on the distance between the reference electrode and the counter electrode in any one electrode set.

In some embodiments of the present invention, during the culturing process, as nutrients in the culture solution are consumed, the current of each channel tends to increase and decrease, and based on the peak value and the current stabilizing value of the current increase of any channel, whether the carbon source is fed into the culture solution and whether the culturing is completed are determined; if the carbon source is fed, judging whether to feed fresh culture solution or whether to finish the culture based on the current magnitude of any channel in a first preset culture time after the carbon source is fed and the current peak value before the carbon source is fed.

In some embodiments of the invention, the first preset incubation time is 20 minutes to 40 minutes.

In some embodiments of the invention, the current in any of the channels decreases to 10 as nutrients in the culture fluid are consumed during the culture ^-8 After A, supplementing a carbon source into the culture solution; after the carbon source is added, if the current peak value of each channel is more than or equal to 2.0X10 in the first preset culture time ^-3 The current stabilizing value of A or each channel is more than or equal to 1.5X10 ^-3 The end of the culture is judged if the current A is stable and less than or equal to 1.2 times of the current peak before the carbon source is fedAnd in the first preset culture time, the current of any channel is 1.2 times greater than the current peak value before the carbon source is fed in, and then the fresh culture solution is replaced for continuous culture.

In some embodiments of the invention, the biofilm carrier comprises at least one of carbon cloth, carbon paper, carbon felt, carbon fiber brush.

In some embodiments of the invention, the culturing is performed under stirring conditions by magnetic stirring or stirring with a stirring rod.

In some embodiments of the invention, the culturing is performed under sealed, light-protected conditions at less than or equal to 25 ℃.

In some embodiments of the invention, a plurality of biological membrane carriers are inoculated with a target strain in the same source liquid under the same inoculation condition to obtain a plurality of biological anodes.

In some embodiments of the invention, the molar concentration of the carbon source in the culture broth is less than or equal to 10mmol/L.

In some embodiments of the invention, the carbon source in the culture broth comprises a soluble acetate salt and/or a soluble lactate salt.

In some embodiments of the invention, the ratio of the total volume of the plurality of bioanodes to the volume of the culture broth during the culturing is less than or equal to 1/300.

In some embodiments of the invention, a method of acclimating an electrochemically active biofilm comprises scheme 1, scheme 1 comprising: (1) Installing a plurality of biological film carriers in the electrolytic cell, wherein the electrolytic cell comprises at least one group of electrode groups, each group of electrode groups comprises a reference electrode and a counter electrode, and at least one biological film carrier is correspondingly installed along each counter electrode in the electrolytic cell; (2) Injecting fresh culture solution and a seed source into the electrolytic cell, connecting a potentiostat with the electrode groups, connecting different electrode groups with different potentiostat channels, and performing adsorption inoculation culture under the same electrolytic adsorption voltage to obtain the biological anode; (3) And replacing the liquid in the electrolytic cell with fresh culture solution, and culturing the plurality of biological anodes under the same culture voltage to obtain a plurality of electrochemical active biological membranes.

In some embodiments of the invention, the method of acclimating an electrochemically active biofilm comprises scheme 2, scheme 2 comprising: (I) Installing a plurality of biological film carriers in the electrolytic cell, wherein the electrolytic cell comprises at least one group of electrode groups, each group of electrode groups comprises a reference electrode and a counter electrode, and at least one biological film carrier is correspondingly installed along each counter electrode in the electrolytic cell; and (II) injecting fresh culture solution and a seed source into the electrolytic cell, connecting a potentiostat with the electrode groups, connecting different electrode groups with different potentiostat channels, and culturing under the same adsorption voltage to obtain a plurality of electrochemical active biological membranes.

In some embodiments of the invention, scheme 1 satisfies at least one of the following conditions: in the step (2), the volume ratio of the seed source to the fresh culture solution is greater than or equal to 1/2; in the step (2), current changes of all channels in the culture process are monitored in real time, and after the current growth rate of all channels starts to be reduced, the adsorption inoculation culture is judged to be completed; the incubation voltage in step (3) is less than the electrowinning voltage described in step (2).

In some embodiments of the invention, scheme 2 satisfies the following condition: in the step (II), a fresh culture solution and a seed source are injected into the electrolytic cell, and the volume ratio of the seed source to the fresh culture solution is less than or equal to 1/2.

In some embodiments of the invention, the method of acclimating an electrochemically active biofilm is implemented with a system for acclimating an electrochemically active biofilm, the system comprising: the top of the electrolytic cell is provided with a cover body, the upper part of the electrolytic cell is provided with a liquid outlet, the lower part of the electrolytic cell is provided with a liquid inlet, and the cover body is provided with a pressure relief opening; the counter electrode group comprises a plurality of counter electrodes, and the counter electrodes are respectively and independently arranged on the side wall of the electrolytic cell at intervals and extend into the accommodating cavity of the electrolytic cell through the side wall of the electrolytic cell; the reference electrode group comprises a plurality of reference electrodes, the plurality of reference electrodes are respectively and independently arranged on the cover body at intervals and extend into the accommodating cavity of the electrolytic cell through the cover body, and the number of the reference electrodes is the same as that of the counter electrodes; the biological anode group comprises a plurality of biological film carriers, the biological film carriers are arranged on the side wall of the electrolytic cell at intervals along the counter electrode group, penetrate through the side wall of the electrolytic cell and extend into the accommodating cavity of the electrolytic cell, and each counter electrode is correspondingly provided with at least one biological film carrier. The system for domesticating the electrochemical active biological membranes is not only beneficial to rapidly domesticating in batches to obtain a plurality of EAB biological membranes with high incubation consistency, but also beneficial to avoiding frequent liquid change operation, and is suitable for large-scale production and application.

In some embodiments of the present invention, a plurality of reference electrodes are uniformly spaced along the circumference of the cover, a plurality of counter electrodes are spaced along the circumference of the electrolytic cell, and distances between the counter electrodes and the reference electrodes in different electrode groups are equal.

In some embodiments of the invention, the counter electrode is positioned at a height that is in the middle of the electrolytic cell.

In some embodiments of the present invention, each of the counter electrodes is provided with a plurality of the biofilm carriers, which are arranged at intervals in the height direction of the electrolytic cell, and/or are arranged at intervals up and down the counter electrodes.

In some embodiments of the invention, the electrolytic cell is cylindrical or polygonal.

In some embodiments of the invention, the cover is removably disposed with the electrolytic cell.

In some embodiments of the invention, a gasket is also provided between the cover and the electrolytic cell.

In some embodiments of the invention, the system for acclimating an electrochemically active biofilm further comprises: a potentiostat comprising a plurality of channels, each of said channels being connected to an electrode set, each of said electrode sets comprising one of said counter electrodes, one of said reference electrodes and at least one of said biofilm carriers.

In some embodiments of the invention, the system for acclimating an electrochemically active biofilm further comprises: a magnetic stirring assembly or an electric stirring assembly, wherein the magnetic stirring assembly comprises a magnetic stirrer and a magnetic stirrer, the electrolytic cell is suitable for being placed on the magnetic stirrer, and the magnetic stirrer is suitable for being placed in the electrolytic cell; the electric stirring assembly comprises a rotating motor and a stirring rod, the rotating motor is arranged outside the electrolytic cell, one end of the stirring rod stretches into the electrolytic cell, and the other end of the stirring rod is connected with the rotating motor.

In some embodiments of the invention, the system for acclimating an electrochemically active biofilm further comprises: and the storage pool is communicated with the electrolytic cell and is provided with a storage space.

In some embodiments of the invention, the system for acclimating an electrochemically active biofilm further comprises: and the pumping device is used for providing power for the electrolyte supplementing or discharging of the electrolytic cell.

In a second aspect of the invention, the invention proposes the use of the method of acclimating an electrochemically active biofilm of the first aspect of the invention in a biotoxicity assay. The electrochemical active biological film prepared by the method for domesticating the electrochemical active biological film of the first aspect has close toxicity response sensitivity, high consistency and basically consistent current value, toxicity response capability and stability in the long-period toxicity detection process. In addition, the device has the advantages of short adaptation and stabilization time and the like.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

fig. 1 is a schematic diagram of a system for performing a method of acclimating an electrochemically active biofilm in accordance with one embodiment of the present invention.

Fig. 2 is a schematic diagram of a system for performing a method of acclimating an electrochemically active biofilm in accordance with yet another embodiment of the present invention.

Fig. 3 is a schematic diagram of a system for performing a method of acclimating an electrochemically active biofilm in accordance with yet another embodiment of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.

The existing EAB biomembrane domestication scheme has the problems of long domestication time, often being completed through multiple liquid exchange, and more factors (such as dissolved oxygen, temperature, pH and the like of a culture solution) which are difficult to control in the liquid exchange process, causing the difference of dominant strains of the mixed biomembrane, large workload, low working efficiency and the like. For example, there is a method for rapidly acclimatizing a bioanode by electrolysis, comprising: a) Preparing an anode culture solution, connecting a biological anode, a constant voltage power supply, a current monitoring element and a chemical cathode to form a microbial electrolytic cell loop, and starting a 1 st domestication period by adopting a constant voltage electrolysis mode; b) When the current monitoring element monitors that the electrolytic current value of the 1 st domestication period is reduced, a circuit is disconnected, a biological anode culture solution is replaced, and the voltage of the constant-voltage power supply is regulated to start the 2 nd domestication period; c) Repeating the operation of the step B) for 3 times to finish an initial domestication period; and then, selecting the repetition number of the operation of the B) according to the required power generation capacity to finish a stable domestication period, wherein the method needs to monitor the electrolysis current in real time, and the culture solution is replaced when the current intensity reaches a range threshold value. For another example, there are also Lorentwa-based The bacillus isShewanella loihica) According to the scheme, although the problems that a traditional microbial electrochemical sensor based on a mature biological film cannot detect in time can be solved, the problems that an EAB biological film is long in starting time, difficult to incubate and the like are solved, each set of system can only produce one group of microbial electrochemical sensors (MEB) at a time, adsorption state EAB is suitable for instant detection and cannot be used for long-term online monitoring, the microbial electrochemical sensors (MEB) needing stable signals are required to be monitored online for a long time, the stable mature EAB biological film is required to be adopted, and the adsorption state EAB can be separated from a medium due to insufficient adsorption and combination tightness degree of the EAB and an adsorption medium, so that bioelectric signal mutation in a detection process is caused, and interference is generated on the test.

Clearly, the existing EAB biofilm organisms have more or less the following problems: 1) The independent domestication system is easy to cause the problems of great difference of the biological activity and the like of the dominant bacterial group of the EAB biological film due to the difference of culture solutions; 2) The domestication process has the problems of complex operation and need of replacing the culture solution for many times, such as need of monitoring the electrolysis signal in real time and replacing the culture solution for many times when the electrolysis signal reaches a certain change degree, and is not suitable for large-scale production and application; 3) The problem of long domestication time of mature EAB biological membranes; 4) The EAB biomembrane water flow rate adaptation period is long.

In view of this, in one aspect, the present invention provides a method of acclimating an electrochemically active biofilm, the method comprising: and simultaneously culturing a plurality of bioanode in the same electrolytic cell under the same culture solution environment and the same culture condition to obtain a plurality of electrochemical active biological membranes, wherein the bioanode comprises a biological membrane carrier attached with target bacteria.

In the invention, the method for domesticating the electrochemical active biological film has at least the following beneficial effects: in the domestication process, the environment of a nutrition system, temperature, pH value and the like where a plurality of biological anodes are positioned is good in consistency, and when a carbon source in a culture solution is enough, the culture solution is not required to be replaced in the culture process, so that the method is not only favorable for obtaining EAB biological membranes with high consistency, but also can avoid frequent liquid replacement operation, even can realize the effect of not replacing the culture solution in the whole domestication process, can greatly reduce the domestication workload and the requirements on operators, effectively avoid the biological membrane incubation control condition difference caused by the differences of a plurality of independent small electrolytic cell systems due to the flora, dissolved oxygen, nutritional ingredients, temperature and the like, further cause the problems of batch-to-batch difference, poor consistency and the like of the EAB biological membranes obtained by domestication, and also be favorable for shortening the on-machine adaptation time in the later test or application. In conclusion, the method not only can rapidly domesticate a plurality of EAB with high incubation consistency in batches, but also is beneficial to shortening the on-machine adaptation time in the later test or application, and can shorten the incubation period to below 7 days (for example, to 2-3 days), and can also avoid frequent liquid change operation, even can realize the effect of not changing the culture solution in the domestication process, the domestication biological film has high repeatability, can provide an application foundation for MEB water biotoxicity detection, and is suitable for mass production application.

The method for acclimating an electrochemically active biofilm according to the above-described embodiment of the present invention is described in detail as follows.

In some embodiments of the present invention, the culture solution includes four parts of carbon source, salts, trace elements and vitamins, and in the preparation process, the mother solutions of the four parts may be prepared separately, and the mother solutions of the four parts may be mixed in a preset ratio and diluted with deionized water.

In some embodiments of the invention, the electrolytic cell may include at least one electrode group, each electrode group includes a reference electrode, a counter electrode and at least one bioanode, the electrode groups are connected with a potentiostat, during the culturing process, different electrode groups are connected with different potentiostat channels, the culturing voltages controlled by the different potentiostat channels are the same, and whether to supplement carbon sources into the culture solution and whether to complete the culturing are determined by monitoring the current changes of the channels during the culturing process in real time, wherein the type of the supplemented carbon sources is the same as the type of the carbon sources contained in the culture solution. The method is more beneficial to improving the consistency of the nutrient system, temperature, pH value and other environments of each biological anode in the domestication process, is beneficial to further improving the consistency of EAB biological membranes obtained by batch domestication, and avoids frequent liquid changing operation and abrupt changes of dissolved oxygen, temperature, pH value and the like of an incubation system caused by liquid changing.

Further, the distances between the reference electrode and the counter electrode in the different electrode groups are equal or substantially equal. In the present invention, the substantially equal means that the difference in distance between the reference electrode and the counter electrode in the different electrode groups is greater than 0 and less than or equal to 5% based on the distance between the reference electrode and the counter electrode in any one electrode group. The distances between the reference electrode and the counter electrode in different electrode groups in the domestication process can be equal or nearly equal, the resistance difference between the reference electrode and the counter electrode in different electrode groups is greatly reduced, and the consistency of the EAB biological film obtained by the biological anode in different electrode groups can be further improved.

Further, culturing a plurality of biological anodes under the same culture solution environment and the same culture condition, wherein in the culture process, along with the consumption of nutrient substances in the culture solution, the current of each channel is in a trend of rising and then reducing, based on the peak value of the rising of the current of any channel and the current stability value (the judgment standard of the current stability value can be flexibly selected according to actual needs, for example, when judging whether a certain current value is a stability value, the culture can be continued for 2 hours, whether the current change exceeds 20% in the 2-hour culture time is observed, if the current change range is between-20% and 20%, the current stability is indicated), and whether the carbon source is fed into the culture solution and the culture is completed is judged; if the carbon source is fed, judging whether the fresh culture solution is fed or whether the culture is finished or not based on the current magnitude of any channel in the first preset culture time after the carbon source is fed and the current peak value before the carbon source is fed. It should be noted that, the criterion for judging whether the cultivation is completed or not and whether the carbon source needs to be supplemented is not particularly limited, and those skilled in the art can flexibly select according to specific cultivation requirements, for example, during the cultivation, as nutrients in the cultivation liquid are consumed, the current of each channel will be in a trend of increasing and decreasing after increasing, if the current peak value or the current stabilizing value of each channel is larger than a certain value A threshold value (which can be flexibly selected according to practical needs, and which can be 2.0X10 when the completion of cultivation is judged by the current peak value ^-3 A, etc.; when judged by a current stable value, the threshold value may be 1.5X10 ^-3 A, etc.), it can be judged that the cultivation is completed without continuing the carbon source supply.

Further, during the culture, the current in any channel is reduced to 10 as nutrients in the culture solution are consumed ^-8 After A, supplementing a carbon source into the culture solution for continuous culture. Thus, it can be further ensured that each of the domesticated EAB biofilms is domesticated and matured. Preferably, the carbon source concentration in the culture broth after the carbon source is fed may be made the same or substantially the same as the carbon source concentration in the fresh culture broth.

Further, after the carbon source is fed, the current of each channel rises instantaneously, and if the current of each channel is close to the current peak value before the carbon source is fed after the first preset culture time, the culture can be considered to be completed. The first preset incubation time is not particularly limited, and a person skilled in the art can flexibly select the first preset incubation time according to actual needs, for example, the first preset incubation time may be 20 minutes to 40 minutes, specifically may be 30 minutes, and the like. In addition, the criterion that the current of each channel is close to the current peak before the carbon source is fed may be determined based on actual needs of those skilled in the art, for example, the current of each channel after the carbon source is fed may be regarded as being close to the current peak before the carbon source by stabilizing the current of each channel in the first preset incubation time and being 1.2 times or less than the current peak before the carbon source is fed.

As some specific examples, after the carbon source is fed, if the current of each channel is stable and less than or equal to 1.2 times of the current peak value before the carbon source is fed in the first preset culture time, the end of the culture can be judged; if the current of any channel is 1.2 times greater than the current peak value before the carbon source is fed in the first preset culture time, the fresh culture solution can be replaced to continue culture. Thus, it can be further ensured that each of the domesticated EAB biofilms is domesticated and matured.

In some embodiments of the present invention, the specific type of the biofilm carrier is not particularly limited, and a person skilled in the art may flexibly select according to actual needs, and may include, for example, but not limited to, at least one of carbon cloth, carbon paper, carbon felt, and carbon fiber brush. In addition, the size of the biological membrane carrier is not particularly limited, and a person skilled in the art can flexibly select the biological membrane carrier according to actual needs, and preferably the biological membrane carriers adopted by a plurality of biological anodes in batch domestication in the same electrolytic cell have the same size, volume and material.

In some embodiments of the invention, the culturing may be performed under agitation conditions, which may include, but are not limited to, magnetic stirring or stirring with a stirring rod. The stirring mode can be used for realizing uniform mixing of an incubation system, eliminating complex pipelines, avoiding the problem that the pipelines are blocked by biological films frequently occurring in a circulating external flow path, promoting mass transfer of microbial electrochemical reaction, simultaneously enabling the EAB biological films to adapt to water flow rate in the forming process, and greatly shortening the on-machine adaptation time in the later test or application. Alternatively, when magnetic stirring is adopted, the electrolytic cell can be placed on a magnetic stirrer, a magnetic stirrer is placed in the electrolytic cell, magnetic stirring is started, and a proper stirring speed is set; when stirring by adopting the stirring rod, the stirring rod can be additionally arranged in the incubation electrolytic cell, and the outside is connected with the rotating motor to realize the mixing of incubation culture solution.

In some embodiments of the invention, the culturing is performed under sealed, light-protected conditions at less than or equal to 25 ℃. Wherein, the control of the low temperature condition of less than or equal to 25 ℃ can reduce the microorganism propagation rate and reduce the growth of mixed bacteria, and the cultivation is preferably carried out under the constant temperature condition of less than or equal to 25 ℃; the sealing can prevent external air from entering the system and carrying in dissolved oxygen, thereby leading to the growth of aerobic bacteria and inhibiting the growth of target bacteria (anaerobic bacteria); the phototactic microorganism growth can be limited by light shading, the phototactic microorganism is prevented from growing on the side wall of the electrolytic cell and the pipeline in an aggregation way, and the problems that a locally formed biological film blocks the pipeline and the like are solved. Therefore, the method is further beneficial to inhibiting the growth of mixed bacteria such as aerobic bacteria and polarized bacteria in an incubation system and reducing the nutrition consumption speed of the system, thereby being further beneficial to the formation of a target bacterial group biological film in the initial stage of incubation.

In some embodiments of the invention, the molar concentration of the carbon source in the culture solution may be less than or equal to 10mmol/L, for example, 0.05mmol/L, 0.5mmol/L, 1mmol/L, 3mmol/L, 5mmol/L, 7mmol/L, 9mmol/L, etc., and may be selected to be 0.1mmol/L to 10mmol/L. Further, the carbon source in the culture medium may include a small molecule carbon source such as a soluble acetate and/or a soluble lactate, and sodium acetate and/or sodium lactate may be preferable, for example. The low concentration and the small molecular carbon source are also favorable for inhibiting the growth of mixed bacteria and promoting the formation of a target flora biomembrane.

In some embodiments of the invention, the ratio of the total volume of the plurality of bioanodes to the volume of the culture broth during the culturing process may be less than or equal to 1/300, for example, may be 1/350, 1/400, 1/500, 1/600, 1/700, 1/800, 1/900, etc., and preferably may be less than or equal to 1/500. By adopting the condition, the incubation culture solution of a large system can be used for providing sufficient nutrition for the formation of the EAB biological film, so that the incubation culture solution can be continuously used for a long time, thereby further effectively avoiding frequent liquid change operation, reducing the workload of production operators, and simultaneously avoiding the mutation problems of dissolved oxygen, temperature, pH and the like of the incubation system in the liquid change process.

In some embodiments of the present invention, a target strain may be inoculated to multiple biofilm carriers simultaneously in the same source liquid under the same inoculation conditions, resulting in multiple bioanode. This approach is more advantageous for ensuring the consistency of the initially formed target flora biofilm.

Further, in the process of domesticating the electrochemical active biological membrane, different inoculation modes can be selected according to different target strain concentrations in the inoculation process. Based on the different inoculation modes of the target strains, the method for domesticating the electrochemical active biological film can concretely comprise two modes of scheme 1 and scheme 2, wherein:

Scheme 1 includes:

(1) A plurality of biomembrane carriers are arranged in the electrolytic cell, so that the electrolytic cell comprises at least one group of electrode groups, each group of electrode groups comprises a reference electrode and a counter electrode, and at least one biomembrane carrier is correspondingly arranged along each counter electrode in the electrolytic cell. The distances between the reference electrode and the counter electrode in the different electrode groups are preferably made equal or substantially equal.

(2) And (3) injecting fresh culture solution and a seed source into the electrolytic cell, connecting the potentiostat with the electrode groups, connecting different electrode groups with different potentiostat channels, and carrying out adsorption inoculation culture under the same electrolytic adsorption voltage to obtain the biological anode. In this process, the level of the suspension formed by the fresh broth and the seed source should be such that it is above at least a portion of all of the counter electrodes, all of the biofilm carriers and each of the reference electrodes. In the process, the seed source of the target strain is provided in a liquid phase form, preferably, the volume ratio of the seed source to the fresh culture solution is greater than or equal to 1/2, for example, 1/1, 2/1 or 4/1, and the like, under the condition that the concentration of the target strain in the suspension is relatively large, under the action of the potentiostat voltage, the high-activity EAB in the system can be preferentially and rapidly enriched on a biological film carrier, thereby being beneficial to realizing the rapid formation of an initial target flora biological film, reducing the biomass of mixed bacteria in the culture system, and improving the utilization rate of nutrient substances in the culture system. Further, after the electrolytic adsorption culture is started, since the high-activity EAB in the system is preferentially and rapidly enriched on the biological film carrier, the adsorption electrolytic current of each channel is rapidly increased, the increase rate of the adsorption electrolytic current is increased firstly and then gradually reduced, in the process, the current change of each channel in the adsorption electrolytic culture process can be monitored in real time, and when the increase rate of the current of each channel starts to be reduced, the adsorption inoculation culture can be completed. After the adsorption inoculation culture is completed, the potentiostat control is disconnected.

(3) And replacing the liquid in the electrolytic cell with fresh culture solution, starting a potentiostat, and culturing the plurality of biological anodes under the same culture voltage to obtain a plurality of electrochemical active biological membranes. In this process, the liquid level of the fresh broth should also be over at least a portion of all of the counter electrodes, all of the biofilm carriers and each of the reference electrodes. In the process, the ratio of the total volume of the bioanode to the volume of the fresh culture solution can be made to be less than or equal to 1/300, such as less than or equal toAt 1/500, the culture voltage should be controlled to be smaller than the electrolytic adsorption voltage controlled in the step (2). After the culture process is started, the electrolytic current of each channel starts to gradually increase, when the current of each channel reaches the highest point (called the first saturation peak current), the current consumption of the system is reduced along with the decrease of the current consumption, and then whether the carbon source is fed into the culture solution and whether the culture is completed or not can be judged based on the first saturation peak current of each channel and the specific magnitude of the stable and stable value after the decrease of the current, for example, when the current of any channel is reduced to 10 ^-8 And (3) when the carbon source (such as a carbon source mother solution) is added into the electrolytic cell, so that the concentration of the carbon source in the system is consistent with that of the fresh culture solution, at the moment, the culture current of each channel is observed to rise to the vicinity of the first saturation peak current again in a short time, and as a specific example, after the fresh carbon source is added, if the culture current of each channel rises to the vicinity of the first saturation peak current again, the current growth speed in the subsequent culture time can be continuously counted, and if the culture is continuously carried out for a preset time (such as 20 minutes and the like) and the current growth speed in the preset time is smaller than a certain preset value (such as 0.005mA/s and the like), the culture can be considered to be completed.

Scheme 2 includes:

(I) A plurality of biomembrane carriers are arranged in an electrolytic cell, the electrolytic cell comprises at least one group of electrode groups, each group of electrode groups comprises a reference electrode and a counter electrode, and at least one biomembrane carrier is correspondingly arranged along each counter electrode in the electrolytic cell. The distances between the reference electrode and the counter electrode in the different electrode groups are preferably made equal or substantially equal.

(II) injecting fresh culture solution and a seed source into the electrolytic cell, connecting a potentiostat with the electrode groups, connecting different electrode groups with different potentiostat channels, and culturing under the same adsorption voltage. In this process, the level of the suspension formed by the fresh broth and the seed source should be such that it is above at least a portion of all of the counter electrodes, all of the biofilm carriers and each of the reference electrodes. The culture mode is suitable for the condition of smaller concentration of target strains, the seed sources of the target strains are provided in a liquid phase form,preferably, the volume ratio of the seed source to the fresh culture medium may be less than or equal to 1/2, for example, 1/1, 1/2, or 1/4, etc., under which conditions the concentration of the target species in the suspension is relatively small, the target species grow slower at the initial stage of initiation of the culture, and a longer adaptation period may be provided compared to step (3) of scheme 1. After the culture process is started, the electrolysis current of each channel starts to gradually increase, when the current of each channel reaches the highest point (called as first saturation peak current), the current consumption of the system is reduced along with the decrease of the current consumption, and then whether the carbon source is fed into the culture solution and whether the culture is completed can be judged based on the first saturation peak current of each channel and the specific magnitude of the stability and the stability value after the current is reduced. As a specific example, when the current in any channel decreases to a predetermined value (e.g. 10 ^-8 A, etc.), supplementing a carbon source (mother liquor) to make the concentration of the carbon source in the system consistent with that of the fresh culture solution, wherein the culture current of each channel is observed to rise to the vicinity of the first saturation peak current again in a short time, and as a specific example, after the fresh carbon source is added, if the culture current of each channel rises to the vicinity of the first saturation peak current again, the current magnitude or the current increasing speed in the subsequent culture time can be continuously counted, and if the culture is continuously carried out for a preset time (such as 20-40 minutes) and the current is still close to the first saturation peak current in the preset time, or the current increasing speed is smaller than a certain preset value (such as 0.005 mA/s), the culture can be considered to be completed; if the current of any channel is greater than 1.2 times of the first saturation peak value within the preset time (20-40 minutes) for continuous culture, the culture is not completed, and the fresh culture solution needs to be replaced for continuous culture.

In the process of acclimating an electrochemically active biofilm according to the above embodiment of the present invention, the acclimating method according to scheme 1 generally does not require replacement of a fresh culture solution, and the acclimating method according to scheme 2 requires replacement of a fresh culture solution during actual operation with a probability greater than that of scheme 1. In addition, either the method of scheme 1 or the method of scheme 2 requires nitrogen exposure treatment of fresh culture solution or a suspension of fresh culture solution and seed source to remove dissolved oxygen before inoculating the target strain.

In some embodiments of the present invention, the method for acclimating an electrochemically active biofilm may be implemented by a system for acclimating an electrochemically active biofilm, and as understood with reference to fig. 1 to 3, the system may include: an electrolytic cell 10, a counter electrode set, a reference electrode set, and a bioanode set. Wherein, the top of the electrolytic cell 10 is provided with a cover 11, the upper part of the electrolytic cell 10 is provided with a liquid outlet 12, the lower part is provided with a liquid inlet 13, and the cover 11 is provided with a pressure relief opening 14; the counter electrode group comprises a plurality of counter electrodes CE which are respectively and independently arranged on the side wall of the electrolytic cell 10 at intervals and extend into the accommodating cavity of the electrolytic cell 10 through the side wall of the electrolytic cell 10; the reference electrode group comprises a plurality of reference electrodes RE which are respectively and independently arranged on the cover body 11 at intervals and extend into the accommodating cavity of the electrolytic cell 10 through the cover body 11, and the number of the reference electrodes is the same as that of the counter electrodes; the biological anode group comprises a plurality of biological film carriers WE, the biological film carriers WE are arranged on the side wall of the electrolytic cell 10 at intervals along the counter electrode group CE, and extend into the accommodating cavity of the electrolytic cell 10 through the side wall of the electrolytic cell 10, and each counter electrode CE is correspondingly provided with at least one biological film carrier WE.

Alternatively, the counter electrode set, the reference electrode set and the bioanode set may constitute a plurality of electrode sets, each of which may include one counter electrode CE, one reference electrode RE and at least one biofilm carrier WE. For example, as will be appreciated with reference to fig. 3, as some specific examples, a counter electrode set, a reference electrode set, and a bioanode set may constitute an 8-electrode set, which may include 8 counter electrodes CE, named CE1, CE2, CE3 … CE8 in order; the reference electrode group may include 8 reference electrodes RE, named RE1, RE2, RE3 … RE8 in order; the bioanode group may include 32 biofilm carriers, each counter electrode CE may correspond to 4 biofilm carriers, respectively, and the 32 biofilm carriers may be named WE1-1, WE1-2, WE1-3, WE1-4, WE2-1, WE2-2, WE2-3, WE2-4, WE3-1, WE3-2, WE3-3, WE3-4 … … WE8-1, WE8-2, WE8-3, WE8-4 in order. Wherein, only the reference numerals are used to show the composition of one electrode group in fig. 3, which comprises 1 counter electrode CE1, 1 reference electrode RE1 and 4 biofilm carriers WE1-1, WE1-2, WE1-3, WE1-4.

Alternatively, as will be appreciated in conjunction with fig. 1-3, the reference electrode RE may be disposed perpendicular to the cover 11, and the perpendicular mounting may avoid the electrode liquid backflow from affecting the service life and stability of the electrode.

According to the system for acclimating electrochemical active biological film, in the process of acclimating electrochemical active biological film, the liquid inlet 13 of the electrolytic cell is suitable for injecting fresh culture solution or suspension formed by fresh culture solution and seed source into the electrolytic cell 10 or reversely sucking out liquid in the electrolytic cell, in addition, the liquid inlet 13 is also suitable for introducing nitrogen into the electrolytic cell 10 so as to perform nitrogen aeration treatment on liquid phase in the electrolytic solution before loading bacteria on the biological film carrier and discharge dissolved oxygen in the liquid phase; the liquid outlet 12 of the electrolytic cell is suitable for discharging liquid phase in the electrolytic cell, such as nutrient-consumed culture solution or inoculation suspension after the inoculation of target strain by electroabsorption; the pressure relief opening is suitable for realizing pressure relief treatment in the culture process or adding carbon source mother liquor into the culture solution. The electrode groups formed by the counter electrode group, the reference electrode group and the biological anode group are suitable for being communicated with different channels of the potentiostat, so that the effect of simultaneously culturing a plurality of biological anodes in the same electrolytic cell under the same culture solution environment and the same culture condition can be realized, and therefore, the method is not only favorable for rapid batch domestication to obtain a plurality of EAB biological membranes with high incubation consistency, but also favorable for avoiding frequent liquid change operation, and is suitable for large-scale production and application.

In some embodiments of the present invention, the shape of the electrolytic cell 10 is not particularly limited, and those skilled in the art may choose according to practical needs, and may include, but not limited to, cylindrical or polygonal columns, etc., where the selection of a cylindrical or polygonal column shape for the electrolytic cell facilitates uniform or symmetrical distribution of multiple electrode sets, and also facilitates the equalization or substantial equalization of distances between reference electrodes and counter electrodes located in different electrode sets during the acclimation process, thereby further facilitating the improvement of uniformity of the culture system, and thus facilitating the batch incubation of EAB biofilms with high uniformity.

In some embodiments of the present invention, the cover 11 and the electrolytic cell 10 may be detachably disposed, wherein a specific manner of the detachable disposition is not particularly limited, and a person skilled in the art may flexibly select according to actual needs, for example, may be a bolt connection or a snap connection, etc., in which manner the installation and the detachment of the plurality of electrode groups are more facilitated.

In some embodiments of the present invention, as will be understood with reference to fig. 2, a sealing gasket 20 may be further disposed between the cover 11 and the electrolytic cell 10, and by providing the sealing gasket, the air tightness between the cover and the electrolytic cell after packaging may be further improved, so that the culturing process is performed in a better sealed environment, which is beneficial to inhibiting the growth of aerobic bacteria, polarized bacteria and other bacteria in the incubation system, reducing the nutrition consumption rate of the system, and further beneficial to the formation of a biological film of the target bacteria group in the initial stage of incubation.

In some embodiments of the present invention, as will be understood with reference to fig. 2 to 3, the plurality of reference electrodes RE may be uniformly spaced along the circumference of the cover 11, the plurality of counter electrodes may be spaced along the circumference of the electrolytic cell 10, and the distances between the counter electrodes CE and the reference electrodes RE in different electrode groups may be equal or substantially equal. Satisfying this arrangement can greatly reduce the difference in resistance between the reference electrode and the counter electrode in different electrode sets during the culture process, thereby facilitating further improvement in the uniformity of EAB biofilm obtained from the biofilm carriers in the different electrode sets.

In some embodiments of the present invention, as will be appreciated in connection with fig. 1-3, the placement height of the counter electrode CE may be located in the middle of the electrolytic cell 10. Further, each counter electrode CE is provided with a plurality of biofilm carriers WE, which may be arranged at intervals in the height direction of the electrolytic cell and/or at intervals up and down the counter electrode CE, respectively. For example, as a specific example, it is understood with reference to fig. 1 that each counter electrode CE may be correspondingly provided with 4 biofilm carriers WE, and the 4 biofilm carriers WE corresponding to each counter electrode CE may be symmetrically arranged at intervals on the side wall of the electrolytic cell 10 in the up-down direction of the counter electrode CE. The mode is more beneficial to improving the consistency of different biomembrane carrier culture systems in the culture process.

In some embodiments of the invention, the system for acclimating an electrochemically active biofilm may further comprise: potentiostat (not shown), the potentiostat may comprise a plurality of channels, for example, may comprise 8 channels, each of which may be connected to one electrode set, each of which may comprise a counter electrode, a reference electrode and at least one biofilm carrier. By adopting the mode, the consistency of the culture parameters and the culture system of each electrode group can be further regulated and controlled, and the difference between the performances of the prepared EAB biological film can be further reduced.

In some embodiments of the invention, the system for acclimating an electrochemically active biofilm may further comprise: a magnetic stirrer assembly or an electric stirrer assembly (not shown), wherein the magnetic stirrer assembly may comprise a magnetic stirrer and a magnetic stirrer, the electrolytic cell 10 being adapted to be placed on the magnetic stirrer, the magnetic stirrer being adapted to be placed inside the electrolytic cell; the electric stirring assembly can comprise a rotating motor and a stirring rod, wherein the rotating motor is arranged outside the electrolytic cell, one end of the stirring rod extends into the electrolytic cell, and the other end of the stirring rod is connected with the rotating motor. By adopting the mode, the EAB biological film culture can be realized under the stirring condition, so that the uniform mixing of an incubation system can be realized, the problem that the biological film blocks the pipeline frequently occurring in a circulating external flow path is solved, the mass transfer of the microbial electrochemical reaction is promoted, the self-adaptive water flow rate of the EAB biological film in the forming process can be realized, and the on-machine adaptation time in the later test or application is greatly shortened.

In some embodiments of the invention, the system for acclimating an electrochemically active biofilm may further comprise: a reservoir (not shown) may be in communication with the electrolytic cell 10 and may have a storage space, wherein the reservoir may be utilized to store electrolyte or a suspension containing a target species that is not contained in the electrolytic cell, whereby a large reaction system ratio incubation, in which the ratio of the total volume of the plurality of bioanode to the volume of the culture solution during the culture is small, such as may be less than or equal to 1/300, preferably may be less than or equal to 1/500, may be achieved even though the capacity of the electrolytic cell is small. Further, the storage pool may include an outlet and an inlet, the outlet of the storage pool may be connected to the liquid inlet 13 of the electrolytic cell 10, the inlet of the storage pool may be connected to the liquid outlet 12 of the electrolytic cell 10, a circulation pump may be disposed between the outlet of the storage pool and the liquid inlet 13 of the electrolytic cell 10 and at least one of the inlet of the storage pool and the liquid outlet 12 of the electrolytic cell 10, and by adopting this arrangement, the communication of the electrolyte or the suspension containing the target strain in the electrolytic cell and the storage pool may be further facilitated, and further the consistency of the culture system may be further realized, and the consistency of a plurality of EAB biofilms obtained by batch incubation may be improved.

In some embodiments of the invention, the system for acclimating an electrochemically active biofilm may further comprise: pumping means (not shown) which may be used to power the electrolyte make-up or draining of the cell.

In summary, the method for acclimating an electrochemically active biofilm according to the above embodiment of the present invention may have the following advantages:

(1) The batch type integrated multi-electrode synchronous incubation can be realized, a batch of biological films are in a common large-system electrolytic cell, the difference of biological film incubation control conditions caused by the differences of bacterial groups, dissolved oxygen, nutritional ingredients, temperature and the like of a plurality of independent small electrolytic cell systems is avoided, and the EAB biological film with high consistency is quickly and efficiently domesticated;

(2) The large reaction system ratio can be adopted, and the large system incubation culture solution can provide sufficient nutrition for the formation of the EAB biological film, so that the EAB biological film can be continuously used for a long time, frequent liquid change operation is avoided, the workload of production operators is reduced, and meanwhile, mutation such as dissolved oxygen, temperature, pH and the like of the incubation system in the liquid change process is avoided.

(3) The method can incubate under the conditions of low concentration carbon source, temperature less than or equal to 25 ℃ and sealing and light shielding, and can inhibit the growth of aerobic flora, polarized flora and other miscellaneous bacteria in the incubation system by adopting the given conditions, thereby reducing the nutrition consumption speed of the system and being more beneficial to the formation of target flora biomembrane in the initial incubation period.

(4) The domestication culture can be carried out under the stirring condition, the incubation system can be uniformly mixed under the combination of the stirring condition, the problem that a complex pipeline is prevented from blocking the pipeline by a biological film frequently occurring in a circulating external flow path is removed, the mass transfer of the microbial electrochemical reaction is promoted, meanwhile, the water flow rate can be self-adapted in the EAB biological film forming process, and the on-machine adaptation time of the later test or application is shortened.

(5) The target strain can realize the rapid formation of the initial target flora biomembrane by an electrolytic adsorption method, and simultaneously reduce the biomass of mixed bacteria in a culture system and improve the utilization rate of nutrient substances in the culture system.

(6) The incubation period can be shortened to below 7 days (for example, 2-3 days), the domesticated biological film has high repeatability, can provide an application foundation for MEB water biotoxicity detection, and is suitable for mass production application.

In a second aspect of the invention, the invention provides the use of the method of acclimating an electrochemically active biofilm of the first aspect of the invention in a biotoxicity assay, e.g. the biotoxicity assay may be performed using an electrochemically active biofilm produced by the method of acclimating an electrochemically active biofilm of the first aspect of the invention. It should be noted that the features and effects described for the method for acclimating an electrochemically active biofilm according to the first aspect of the present invention are equally applicable to the use, and are not described here again. In general, the electrochemical active biological film prepared by the method for domesticating the electrochemical active biological film of the first aspect has close toxicity response sensitivity, high consistency and basically consistent current value, toxicity response capability and stability in the long-period toxicity detection process. In addition, the device has the advantages of short adaptation and stabilization time and the like.

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Comparative example 1

And (3) adopting independent domestication systems, wherein each set of system domesticates 1 biomembrane. The procedure is described by taking acclimated 8 biofilms as an example.

Step S101, 8 acclimatized small electrolytic cells, named small electrolytic cell 1, small electrolytic cell 2, &..the small electrolytic cell 8, respectively, were assembled. Each small cell contains 1 reference electrode RE, 1 counter electrode CE, and 1 bioanode WE; the shape of the small electrolytic cell is a cube structure, and the inside of the small electrolytic cell is a hollow cylinder; the device comprises a tank body and an upper cover, wherein the tank body is used for containing domestication culture solution, the upper cover is used for assembling electrodes RE, CE and WE, and the tank body and the upper cover are tightly sealed by fastening screws through threads; the bottom of one side of the tank body is provided with a water inlet, and the upper side of the opposite side is provided with a water outlet. Assembling an electrode, a water inlet and a water outlet.

Step S102, preparing fresh culture solution. The culture solution comprises four parts of salts, trace elements, vitamins and carbon sources. The four parts of mother liquor are prepared separately, 50mL of salt mother liquor, 10mL of trace element mother liquor, 10mL of vitamin mother liquor and 10mL of carbon source mother liquor are respectively taken, and diluted to 1L by deionized water. Sodium acetate is used as a carbon source, and the mass concentration of sodium acetate substances in the culture solution is 5mmol/L. Placing the mixture in a constant temperature incubator at 22.5+/-0.5 ℃, introducing nitrogen with the purity of more than 99.5% at the rate of 500mL/min for 10min, and keeping the temperature while introducing nitrogen after the nitrogen is introduced, wherein the content of dissolved oxygen in the solution is less than or equal to 1 mg/L.

Step S103, connecting a potentiostat. Opening eight channels of a potentiostat, and respectively connecting 8 small electrolytic cells; wherein, the first channels RE, CE and WE of the potentiostat are respectively connected with the small electrolytic cell 1 in the step S101; the second channels RE, CE and WE of the potentiostat are respectively connected with the small electrolytic cell 2, … …, and the eighth channels RE, CE and WE of the potentiostat are respectively connected with the small electrolytic cell 8.

Step S104, inoculating target strains. And (3) mixing the fresh culture solution obtained in the step S102 with seed source effluent at a ratio of 1:1 to obtain an inoculation bacterial suspension.

Step S105, starting culture. The inoculum suspension of S104 was added equally to 8 small electrolytic cells, filled with containers, sealed and protected from light. Each small electrolytic cell bioanode takes 0.1cm multiplied by 1cm carbon cloth as a biomembrane carrier, and the volume of the bioanode is about 0.1mL; 40mL of inoculum suspension was added, culture broth: bioanode volume ratio = 400. Setting a potentiostat culture voltage V2 = 0V, starting the potentiostat culture, and monitoring the electrolytic current in the culture process of each channel in real time.

Step S106, a liquid changing flow. When the culture is started, the culture current of each channel of the new culture system gradually increases, and the later period gradually decreases along with nutrition consumption, and the current reaches the lowest point (10 ^-8 A) After stabilization, 20mL of fresh culture solution is slowly injected from the water inlet, and 20mL of original culture solution is replaced from the water outlet. After each liquid change, the current is reduced to 10 after reaching the peak value ^-8 And (3) replacing once after the step A, repeating the operation for 4 to 6 times, and when the current growth speed from 20min to 40min of the next liquid replacement is smaller than 0.005 mA/s, considering that the EAB biological film is incubated (or the difference of the peak value of the culture current in the two adjacent liquid replacement processes is not more than +/-10 percent, and also considering that the EAB biological film is incubated to be mature and the culture is completed).

The test data are as follows:

in comparative example 1, 8 channels (designated channel 1, channel 2,.. The term "channel 8") each cultured 1 sheet of EAB biofilm, with channel 5 and channel 7 biofilm culturing currents always being less than 10 ^-8 A, unsuccessful culture; of the remaining 6 channels, 3 channels (here channel 1, channel 2, channel 3) were randomly selected for on-board testing of EAB biofilms. The water quality biotoxicity on-line monitor (hereinafter referred to as instrument) is adopted for testing, each instrument of the 3 instruments is provided with a test channel, and the test channels are placed in the same space, and the same test sequence is set for automatically running the test.

The instrument is set to be in a 24-hour automatic whole-point operation mode, the first day and the second day of the EAB biofilm loading are loading adaptation periods, the automatic calibration is carried out once every 12 hours, and a blank water test (shown as dz in the table) is started every whole point thereafter; on the third, fourth and fifth days, the EAB biological film is suitable for detecting the water flow rate of toxicity, stabilizing the signal, and starting the toxicity response sensitivity and stability test; the final concentration of Cu is 0.3mg/L ²⁺ The standard solution was used as a simulated toxic substance solution, calibrated twice, started once with a blank water test (as identified in table dz) and started once with a toxic substance test (as identified in table sc 1).

Test results:

(1) The EAB biofilm is subjected to on-machine adaptation period test. The relative current is qualified between +/-10% and excellent between +/-5%. The channel 1 EAB biomembrane has larger relative current jumping in two-day adaptation period test, which is far beyond +/-10%, and is a typical immature biomembrane, and is unqualified; the EAB biomembrane of the channel 2 is basically stable in the second calibration period, but a small amount of abnormal values appear until the last calibration period meets the requirements, and the adaptation period is 36h; the channel 3 EAB biomembrane starts to be stable in the 9 th test of the first calibration period, and the test data are qualified and adapt to the period of 9h. The adaptation period of the three channels on-line is greatly different.

(2) Toxicity response test. The toxicity is detected when the relative current is less than or equal to-20%, and the smaller the relative current is, the more sensitive the toxicity response is. Channel 1 EAB biomembrane, toxicity response is most sensitive, the relative current is less than-40% in last several times, but the total value of the current integral in the detection process is rapidly reduced in the toxicity test process of 3 days, which is probably that the biomembrane is not incubated and matured, has weak tolerance to toxic substances and dies gradually; after 4 toxic impacts, the channel 2 EAB biomembrane can detect toxicity in each toxicity test, the relative current is once lower than-50% and the other relative current is basically between-25% and-20%, and most data are stable and have a small amount of abnormal values; in the two-day test process, the channel 3 EAB biomembrane only detects the non-toxic response of other toxicity tests by two toxicity tests, and the total current integral value in the detection process is not changed obviously, thus the membrane is a typical aging and stress-resistant biomembrane.

Comprehensive analysis can be seen: the EAB biomembrane on-machine adaptation stable time of 3 channels exceeds 12 hours; channel 1 biofilm is not mature in incubation, cannot withstand long-term toxicity test, and has poor long-term test stability; the biological membrane of the channel 3 is excessively mature in incubation and tends to age, so that the toxicity detection rate is low, and the biological membrane cannot be used for toxicity detection; the values of the current, the toxicity response capability and the stability are greatly different, and the repeatability of the domesticated biological film is low. The detailed test data are shown in the following table.

Table 1 table of on-press test data for EAB biofilm in comparative example 1

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Note that: data identification dz, expressed as blank water test; data marker sc1, indicating that Cu was used at a concentration of 0.3mg/L ²⁺ The standard solution is used as a simulated toxic substance solution for toxicity response test; calibration refers to testing with blank water, collecting a current baseline, and calculating relative currents by taking the current of the current latest calibration as the baseline.

Example 1

The batch type efficient domestication method S10 of the electrochemical active biological film for water quality biotoxicity detection comprises the following steps:

step S101, assembling a large biological membrane domestication electrolytic cell. Referring to fig. 3, a 4×8 (8 groups of 4 biofilm) patch of biofilm is exemplified. The inside of the large electrolytic cell can be filled with about 2500mL of culture solution; eight groups, each group comprising 1 reference electrode RE, 1 counter electrode CE and 4 bioanode WE; uniformly selecting eight mounting hole sites on the upper cover, and assembling 1 reference electrode RE on each hole site, wherein the reference electrode RE is named RE1, RE2 and RE3 … RE8 in sequence; at the 1/2 height of the side wall of the cell body, eight mounting hole sites are uniformly selected, 1 counter electrode CE is assembled in each hole site, and the counter electrodes CE are sequentially named as CE1, CE2 and CE3 … CE8, so that the actual distances and the relative positions of eight groups of electrodes of RE1 and CE1, RE2 and CE2, and RE3 and CE3 … are the same; two mounting hole sites are respectively selected on the upper side and the lower side of the counter electrode CE in the vertical direction, the distance between two adjacent hole sites is 2cm, and each hole site is assembled with 1 bioanode WE, which are sequentially named as WE1-1, WE1-2, WE1-3, WE1-4, WE2-1, WE2-2, WE2-3, WE2-4, WE3-1, WE3-2, WE3-3, WE3-4 … … WE8-1, WE8-2, WE8-3 and WE8-4; each biological anode adopts 0.1cm multiplied by 1cm carbon cloth as a biological membrane carrier; the water inlet is connected with a water inlet pipe, and a three-way valve capable of being selectively opened and closed manually is connected to the water inlet pipe; the water outlet is connected with a water outlet pipe.

Step S102, preparing fresh culture solution. The culture solution comprises four parts of salts, trace elements, vitamins and carbon sources. The four parts of mother liquor are prepared separately, 50mL of salt mother liquor, 10mL of trace element mother liquor, 10mL of vitamin mother liquor and 10mL of carbon source mother liquor are respectively taken, and diluted to 1L by deionized water. Sodium acetate is used as a carbon source, and the mass concentration of sodium acetate substances in the culture solution is 5 mmol/L. Placing the mixture in a constant temperature incubator at 22.5+/-0.5 ℃, introducing nitrogen with the purity of more than 99.5% at the rate of 500mL/min for 10min, and keeping the temperature while introducing nitrogen after the nitrogen is introduced, wherein the content of dissolved oxygen in the solution is less than or equal to 1 mg/L.

Step S103, connecting a potentiostat. Opening eight channels of the potentiostat, and respectively connecting eight groups of electrodes; wherein, the first channels RE, CE and WE of the potentiostat are respectively connected with the large electrolytic cells RE1, CE1 and WE1 (WE 1-1, WE1-2, WE1-3 and WE1-4 are connected in parallel) in the step S101; the second channels RE, CE and WE of the potentiostat are respectively connected with the large electrolytic cells RE2, CE2 and WE2 (WE 2-1, WE2-2, WE2-3 and WE2-4 are connected in parallel), and the eighth channels RE, CE and WE of the … … potentiostat are respectively connected with the large electrolytic cells RE8, CE8 and WE8 (WE 8-1, WE8-2, WE8-3 and WE8-4 are connected in parallel).

Step S104, inoculating target strains. Mixing 500mL of fresh culture solution in the step S102 with 2000mL of seed source outlet water at a ratio of 1:4 to obtain inoculation bacterial suspension, wherein the liquid level is over all electrodes (the liquid level is over all counter electrodes, biological anodes and microporous ceramics at the bottom of a reference electrode); placing a magnetic stirrer in the electrolytic cell, placing the whole large electrolytic cell on a magnetic stirrer, starting magnetic stirring, and setting the rotating speed to 2000rpm; setting adsorption voltage V1 = 0.3V of a potentiostat, starting an electrolytic adsorption process, monitoring adsorption electrolytic current of each channel in real time, and under the action of the potentiostat voltage, preferentially and rapidly enriching high-activity EAB in a system onto a biological anode carrier, wherein the adsorption electrolytic current can be rapidly increased; the increase rate of the adsorption electrolysis current is increased firstly and then gradually decreased; after electrolytic adsorption for 1-3 h, early EAB biomembrane is formed on the biological anode, and single-channel adsorption current reaches 10 ^-6 Class a; when the adsorption current increases at a rate of onsetWhen the strain is lowered, the process of electrolytic adsorption inoculation of the target strain is completed; and (3) disconnecting the potentiostat control and sucking out the inoculated bacterial suspension from the water inlet in a back suction mode.

Step S105, starting culture. And (3) adding the fresh culture solution in the step S102 into the large electrolytic cell inoculated with the target strain in the step S104, filling the large electrolytic cell with the container, sealing the large electrolytic cell and keeping the large electrolytic cell away from light. The biological anode takes 0.1cm multiplied by 1cm carbon cloth as a biological membrane carrier, and the total volume of 32 biological anodes is about 3.2mL; 2500mL of fresh culture solution was added, culture solution: bioanode volume ratio = 781. The magnetic stirrer is placed in the electrolytic cell, the whole large electrolytic cell is placed on the magnetic stirrer, the magnetic stirring is started, and the rotating speed is set to 2000rpm. Setting a potentiostat culture voltage V2 = 0.15V (V2 is between 0 and 0.3V, V2 cannot be a negative value, is more than or equal to 0V and is less than an adsorption voltage V1), starting the potentiostat culture, and monitoring the electrolytic current in the culture process of each channel in real time. Thereafter, it can be observed that the electrolytic current of each channel is from 10 ^-6 The starting level of A gradually grows to 1.0A-3.0A when the final culture is completed, and the growth curve is S-shaped; the current increasing speed increases and then decreases, and is in an inverted V shape; when the culture current reaches the highest point (called the first saturation peak current), the current consumption of the system nutrition is reduced, and when the current is reduced to 10 ^-8 And A, adding a carbon source mother solution through an air pressure balance port of the domestication electrolytic cell to enable the concentration of the system carbon source to be consistent with that of the fresh culture solution, wherein the culture current is observed to rise to be close to a first peak current within 20min, and counting the current growth speed of the fresh carbon source from 20min to 40min, wherein if the current growth speed is smaller than 0.005 mA/s, the EAB biological film is considered to be completely incubated.

The test data are as follows:

in example 1, 4 EAB biofilms were cultured for each of 8 channels (designated channel 1, channel 2,.. The channels 8.) and the 8 channels were substantially identical in current; 3 channels (channel 1, channel 2 and channel 3 are selected in the embodiment) are selected randomly from 8 channels, and 1 EAB biological film is selected randomly for on-machine test. The specific test methods, steps, and data analysis were consistent with the comparative examples.

Test results:

(1) The EAB biofilm is subjected to on-machine adaptation period test. The EAB biological films of the 3 channels are stable in the first calibration period, all blank water tests are qualified from the 3 rd blank water test, and the adaptation period is less than or equal to 3h.

(2) Toxicity response test. After two toxic impacts, the relative currents of the biological membranes of the 3 channels are between-46% and-25%, and the toxicity detection rate is 100%; in the two-day toxicity test process, the total current integral value of the 3-channel biological film detection process is relatively stable, no obvious gradual increase or decrease and abnormal jump occur, and the stability is high; the average relative currents of the biological membranes of the channels 1, 2 and 3 EAB in the adaptation period of two toxic impact before rejection are respectively-36.82%, -36.11%, -38.38%, and the consistency is high.

Comprehensive analysis can be seen: the adaptation and stabilization time of the EAB biomembrane of the 3 channels is 3h; the toxicity response sensitivity is close, and the consistency is high; the magnitude of the current value, the toxicity response capability and the stability are basically consistent in the long-period toxicity detection process, and the biological film domesticated by the scheme has high repeatability. The detailed test data are shown in the following table.

Table 2 table of EAB biofilm on-board test data in example 1

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Note that: data identification dz, expressed as blank water test; data marker sc1, indicating that Cu was used at a concentration of 0.3mg/L ²⁺ The standard solution is used as a simulated toxic substance solution for toxicity response test; calibration ofThe method is to test with blank water, collect a current baseline, and calculate relative current by taking the current which is currently calibrated at the latest time as the baseline.

Example 2

Completely consistent with the operation of example 1, only an integrated domestication electrolytic cell with a reduced inner diameter was used; the height of the electrolytic cell is unchanged, the inner diameter is two thirds of that of the electrolytic cell of the embodiment 1, and about 1000mL of culture solution can be filled in the electrolytic cell, wherein the inner diameter is equal to the inner diameter of the electrolytic cell: bioanode volume ratio = 312.5.

The test data are as follows:

in example 2, 4 EAB biofilms were cultured for each of 8 channels (designated channel 1, channel 2,.. The channels 8.) and the 8 channels were substantially identical in current; 3 channels (channel 1, channel 2 and channel 3 are selected in the embodiment) are selected randomly from 8 channels, and 1 EAB biological film is selected randomly for on-machine test. The specific test methods, procedures and data analysis were identical to the comparative examples.

Test results:

(2) Toxicity response test. After two toxic impacts, the relative currents of the biological membranes of the 3 channels are between-35% and-20%, and the toxicity detection rate is 100%; in the two-day toxicity test process, the total current integral value of the 3-channel biological film detection process is relatively stable, no obvious gradual increase or decrease and abnormal jump occur, and the stability is high; the average relative currents of the biological membranes of the channels 1, 2 and 3 EAB in the adaptation period of two toxic impact before elimination are respectively-26.83%, -26.68%, -27.7%, and the consistency is high.

Table 3 table of EAB biofilm on-machine test data in example 2

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And (3) injection: data identification dz, expressed as blank water test; data marker sc1, indicating that Cu was used at a concentration of 0.3mg/L ²⁺ The standard solution is used as a simulated toxic substance solution for toxicity response test; calibration refers to testing with blank water, collecting a current baseline, and calculating relative currents by taking the current of the current latest calibration as the baseline.

Example 3

The procedure was essentially as in example 1, except that the target strain was inoculated. The domestication method S30 includes the following steps:

step S301, assembling a large biological membrane domestication electrolytic cell. Referring to example 1, the procedure is consistent with step S101.

Step S302, preparing fresh culture solution. Referring to embodiment 1, the process corresponds to step S102.

Step S303, connecting a potentiostat. Referring to example 1, the procedure is consistent with step S103.

Step S304, inoculating target strains and starting culture. 2000mL of the fresh culture solution prepared in the step S302 and 500mL of seed source water are added into a domestication electrolytic cell according to the ratio of 4:1, and the domestication electrolytic cell is filled with a container, sealed and protected from light; the biological anode takes 0.1cm multiplied by 1cm carbon cloth as a biological membrane carrier, and the total volume of 32 biological anodes is about 3.2mL; culture solution: biological anodeVolume ratio = 781 magnetic stirrer inside the electrolytic cell, the whole large electrolytic cell is placed on the magnetic stirrer, magnetic stirring is started, and the rotating speed is set to 2000rpm. Setting a potentiostat culture voltage V3 = 0.15V (the culture voltage V3 is between 0 and 0.3V and cannot be negative), starting the potentiostat culture, and monitoring the electrolytic current in the culture process of each channel in real time. Thereafter, it can be observed that the electrolytic current of each channel is from 10 ^-8 The starting level of A gradually grows to 1.0A-3.0A when the final culture is completed, and the growth curve is S-shaped; however, the target strain grew slower at the initial stage of the initiation of the culture, and had a longer adaptation period than in example 1. When the culture current reaches the highest point (called the first saturation peak current), the current consumption of the system nutrition is reduced, and when the current is reduced to 10 ^-8 And A, adding a carbon source mother solution through an air pressure balance port of the domestication electrolytic cell to enable the concentration of the system carbon source to be consistent with that of the fresh culture solution, wherein the culture current is observed to rise to be close to a first peak current within 20min, and counting the current growth speed of the fresh carbon source from 20min to 40min, wherein if the current growth speed is smaller than 0.005 mA/s, the EAB biological film is considered to be completely incubated.

The test data are as follows:

in example 3, 4 EAB biofilms were cultured for each of 8 channels (designated channel 1, channel 2,.. The channels 8.) and the 8 channels were substantially identical in current; 3 channels (channel 1, channel 2 and channel 3 are selected in the embodiment) are selected randomly from 8 channels, and 1 EAB biological film is selected randomly for on-machine test. The specific test methods, steps, and data analysis were consistent with the comparative examples.

Test results:

(1) The EAB biofilm is subjected to on-machine adaptation period test. The EAB biofilms of the 3 channels had stabilized in the first calibration period, and all blank water tests were acceptable starting from the 4 th blank water test, with an adaptation period of 4h.

(2) Toxicity response test. After two toxic impacts, the relative currents of the biological membranes of the 3 channels are between-48% and-20%, and the toxicity detection rate is 100%; in the two-day toxicity test process, the total current integral value of the 3-channel biological film detection process is relatively stable, no obvious gradual increase or decrease and abnormal jump occur, and the stability is high; the average relative currents of the biological membranes of the channels 1, 2 and 3 EAB in the adaptation period of two toxic impact before rejection are respectively-35.47%, -33.39%, -32.55%, and the consistency is high.

Comprehensive analysis can be seen: the adaptation and stabilization time of the EAB biomembrane with 3 channels is 4h; the toxicity response sensitivity is close, and the consistency is high; the magnitude of the current value, the toxicity response capability and the stability are basically consistent in the long-period toxicity detection process, and the biological film domesticated by the scheme has high repeatability. The detailed test data are shown in the following table.

Table 4 table of EAB biofilm on-machine test data in example 3

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Example 4

The same domestication electrolytic cell as in the embodiment 2 is adopted, a circulating solution storage bottle with the volume of 1500mL is additionally arranged, the water inlet and the water outlet of the domestication electrolytic cell are completely sealed and are respectively connected with the water outlet and the water inlet of the domestication electrolytic cell, and a liquid pump is additionally arranged in the middle of the domestication electrolytic cell, so that the circulating replacement of the culture solution between the domestication electrolytic cell and the circulating bottle is realized without magnetic stirring. The flow speed of the solution in the domesticated electrolytic cell relative to the bioanode is about 3 mL/min-7 mL/min.

A domestication method S40 comprising the steps of:

step S401, assembling a biological membrane domestication electrolytic cell. Referring to example 1, the procedure is consistent with step S101.

Step S402, preparing fresh culture solution. Referring to embodiment 1, the process corresponds to step S102.

Step S403, connecting a potentiostat. Referring to example 1, the procedure is consistent with step S103.

Step S404, inoculating target strains. Referring to embodiment 1, the process corresponds to step S104.

Step S405, connecting a circulating bottle and a pump, filling culture solution, and starting culture. Acclimating Chi Zhongzhuang mL of electrolysis, filling 1500mL in a circulating bottle, adding 2500mL of fresh culture solution, filling a container, sealing and avoiding light; the total volume of the bioanode is about 3.2mL; culture solution: bioanode volume ratio = 781. The solution circulation speed between the domestication electrolytic cell and the circulation bottle is 30mL/min. Setting a constant potential rectifier culture voltage V4 = 0.15V (the culture voltage is between 0 and 0.3V), starting the constant potential rectifier culture, and monitoring the electrolytic current in the culture process of each channel in real time. When the culture current reaches the highest point (called the first saturation peak current), the current consumption of the system nutrition is reduced, and when the current is reduced to 10 ^-8 And A, adding a carbon source mother solution through an air pressure balance port of the domestication electrolytic cell to enable the concentration of the system carbon source to be consistent with that of the fresh culture solution, wherein the culture current is observed to rise to be close to a first peak current within 20min, and counting the current growth speed of the fresh carbon source from 20min to 40min, wherein if the current growth speed is smaller than 0.005 mA/s, the EAB biological film is considered to be completely incubated.

The test data are as follows:

in example 4, 4 EAB biofilms were cultured for each of 8 channels (designated channel 1, channel 2,.. The channels 8.) and the 8 channels were substantially identical in current; 3 channels (channel 1, channel 2 and channel 3 are selected in the embodiment) are selected randomly from 8 channels, and 1 EAB biological film is selected randomly for on-machine test. The specific test methods, steps, and data analysis were consistent with the comparative examples.

Test results:

(1) The EAB biofilm is subjected to on-machine adaptation period test. The EAB biofilms of the 3 channels were stable in the first calibration period, and all blank water tests were qualified from the 5 th, 6 th and 8 th blank water tests, and the adaptation period was 9h.

(2) Toxicity response test. After three toxic impacts, the 3 channel biological membranes start toxicity alarm, the relative current is between-45% and-20%, and the toxicity detection rate is 100%; in the two-day toxicity test process, the total current integral value of the 3-channel biological film detection process is relatively stable, no obvious gradual increase or decrease and abnormal jump occur, and the stability is high; the average relative currents of the biological membranes of the channels 1, 2 and 3 EAB in the adaptation period of two toxic impact before elimination are respectively-34.53%, -33.85%, -32.76%, and the consistency is high.

Comprehensive analysis can be seen: the on-machine adaptation and stabilization time of the EAB biological films of the 3 channels is not more than 4 hours; the toxicity response sensitivity is close, and the consistency is high; the magnitude of the current value, the toxicity response capability and the stability are basically consistent in the long-period toxicity detection process, and the biological film domesticated by the scheme has high repeatability. The detailed test data are shown in the following table.

Table 5 table of EAB biofilm on-machine test data in example 4

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And (3) injection: data identification dz, expressed as blank water test; data marker sc1, indicating that Cu was used at a concentration of 0.3mg/L ²⁺ The standard solution is used as a simulated toxic substance solution for toxicity response test; calibration refers to testing with blank water, collecting current base line, and calculating relative current by taking current of the latest calibration as base lineA kind of electronic device.

Comparative example 1 and examples 1 to 4 data were compared and analyzed:

1. EAB biofilm acclimation process

TABLE 6 comparative analysis Table of EAB biofilm acclimation procedure in comparative example 1 and examples 1-4

2. EAB biofilm on-machine test performance

Table 7 on-press testing of EAB biofilm in comparative example 1

Table 8 EAB biofilm on-board testing in example 1

Table 9 EAB biofilm on-board testing in example 2

Table 10 EAB biofilm on-board testing in example 3

Table 11 EAB biofilm on-board testing in example 4

Results and conclusions:

as is clear from comprehensive comparison, the domestication method provided by the embodiment of the invention has the advantages that the incubation period is shortened, the total liquid change times, the first peak current value and the time consistency from the first peak current value to the first peak current value are high, the on-machine test adaptation of EAB biological films prepared by different channels is short, the on-machine adaptation period and the toxicity response test consistency of EAB biological films prepared by different channels are good, and the continuous toxicity impact current is stable, so that the toxicity response sensitivity of EAB biological films prepared in batches by the domestication method provided by the embodiment of the invention is close, the consistency is high, the current value, the toxicity response capability and the stability are basically consistent in the long-period toxicity detection process, and the on-machine adaptation stability time is short.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A method of acclimating an electrochemically active biofilm, comprising:

Simultaneously culturing a plurality of bioanode in the same electrolytic cell under the same culture solution environment and the same culture condition to obtain a plurality of electrochemical active biological membranes, wherein the bioanode comprises a biological membrane carrier attached with target strain,

wherein a plurality of the bioanode are cultured under the same culture voltage, and the culture is performed under stirring conditions; in the culture process, the ratio of the total volume of a plurality of biological anodes to the volume of the culture solution is less than or equal to 1/300,

the electrolytic cell comprises at least one group of electrode groups, each group of electrode groups comprises a reference electrode, a counter electrode and at least one biological anode, the electrolytic cell is provided with a containing cavity, all the reference electrode, the counter electrode and the biological anode extend into the containing cavity, the electrode groups are connected with a potentiostat, different electrode groups are connected with different potentiostat channels in the culture process, the culture voltages controlled by the different potentiostat channels are the same, whether the carbon sources are fed into the culture solution or not and whether the culture is completed or not is judged by monitoring the current changes of the channels in the culture process in real time, wherein the types of the fed carbon sources are the same as the types of the carbon sources contained in the culture solution, and the following conditions are met:

(a) The distances between the reference electrode and the counter electrode in different electrode groups are equal, or the distance difference between the reference electrode and the counter electrode in different electrode groups is more than 0 and less than or equal to 5% based on the distance between the reference electrode and the counter electrode in any electrode group;

(b) In the culture process, along with the consumption of nutrient substances in the culture solution, the current of each channel is in a trend of rising first and then reducing second, and whether the carbon source is fed into the culture solution or not and whether the culture is completed or not are judged based on the peak value of the rising current of any channel and the current stabilizing value; if the carbon source is fed, judging whether to feed fresh culture solution or whether to finish the culture based on the current magnitude of any channel in a first preset culture time after the carbon source is fed and the current peak value before the carbon source is fed.

2. The method of claim 1, wherein in condition (b), the first preset incubation time is 20 minutes to 40 minutes; and/or the number of the groups of groups,

in the culture processWhen any channel current is reduced to 10 along with the consumption of nutrient substances in the culture solution ^-8 After A, supplementing a carbon source into the culture solution; after the carbon source is added, if the current peak value of each channel is more than or equal to 2.0X10 in the first preset culture time ^-3 The current stabilizing value of A or each channel is more than or equal to 1.5X10 ^-3 And A, judging that the culture is finished, and if the current of any channel is 1.2 times greater than the current peak value before the carbon source is fed in the first preset culture time, replacing the fresh culture solution to continue the culture.

3. The method of claim 1, wherein at least one of the following conditions is satisfied:

the biomembrane carrier comprises at least one of carbon cloth, carbon paper, carbon felt and carbon fiber brush;

the stirring mode is magnetic stirring or stirring rod stirring;

the culture is carried out under the sealed and light-proof condition at the temperature of less than or equal to 25 ℃;

simultaneously inoculating target strains to a plurality of biological film carriers in the same source liquid under the same inoculation condition to obtain a plurality of biological anodes;

the molar concentration of the carbon source in the culture solution is less than or equal to 10mmol/L;

the carbon source in the culture solution comprises soluble acetate and/or soluble lactate.

4. A method according to any one of claims 1 to 3, comprising one of schemes 1 and 2:

scheme 1 includes:

(1) Installing a plurality of biological film carriers in the electrolytic cell, wherein the electrolytic cell comprises at least one group of electrode groups, each group of electrode groups comprises a reference electrode and a counter electrode, and at least one biological film carrier is correspondingly installed along each counter electrode in the electrolytic cell;

(2) Injecting fresh culture solution and a seed source into the electrolytic cell, connecting a potentiostat with the electrode groups, connecting different electrode groups with different potentiostat channels, and performing adsorption inoculation culture under the same electrolytic adsorption voltage to obtain the biological anode;

(3) Changing the liquid in the electrolytic cell into fresh culture solution, culturing a plurality of biological anodes under the same culture voltage to obtain a plurality of electrochemical active biological membranes,

scheme 2 includes:

(I) Installing a plurality of biological film carriers in the electrolytic cell, wherein the electrolytic cell comprises at least one group of electrode groups, each group of electrode groups comprises a reference electrode and a counter electrode, and at least one biological film carrier is correspondingly installed along each counter electrode in the electrolytic cell;

and (II) injecting fresh culture solution and a seed source into the electrolytic cell, connecting a potentiostat with the electrode groups, connecting different electrode groups with different potentiostat channels, and culturing under the same adsorption voltage to obtain a plurality of electrochemical active biological membranes.

5. The method of claim 4, wherein the step of determining the position of the first electrode is performed,

scheme 1 satisfies at least one of the following conditions:

In the step (2), the volume ratio of the seed source to the fresh culture solution is greater than or equal to 1/2;

in the step (2), current changes of all channels in the culture process are monitored in real time, and after the current growth rate of all channels starts to be reduced, the adsorption inoculation culture is judged to be completed;

the culture voltage in the step (3) is smaller than the electrolytic adsorption voltage in the step (2);

scheme 2 satisfies the following conditions: in the step (II), a fresh culture solution and a seed source are injected into the electrolytic cell, and the volume ratio of the seed source to the fresh culture solution is less than or equal to 1/2.

6. The method of claim 1 or 5, wherein the method is performed using a system for acclimating an electrochemically active biofilm, the system for acclimating an electrochemically active biofilm comprising:

the top of the electrolytic cell is provided with a cover body, the upper part of the electrolytic cell is provided with a liquid outlet, the lower part of the electrolytic cell is provided with a liquid inlet, and the cover body is provided with a pressure relief opening;

the counter electrode group comprises a plurality of counter electrodes, and the counter electrodes are respectively and independently arranged on the side wall of the electrolytic cell at intervals and extend into the accommodating cavity of the electrolytic cell through the side wall of the electrolytic cell;

The reference electrode group comprises a plurality of reference electrodes, the plurality of reference electrodes are respectively and independently arranged on the cover body at intervals and extend into the accommodating cavity of the electrolytic cell through the cover body, and the number of the reference electrodes is the same as that of the counter electrodes;

the biological anode group comprises a plurality of biological film carriers, the biological film carriers are arranged on the side wall of the electrolytic cell at intervals along the counter electrode group, and extend into the accommodating cavity of the electrolytic cell through the side wall of the electrolytic cell, and each counter electrode is correspondingly provided with at least one biological film carrier;

a magnetic stirring assembly or an electric stirring assembly, wherein the magnetic stirring assembly comprises a magnetic stirrer and a magnetic stirrer, the electrolytic cell is suitable for being placed on the magnetic stirrer, and the magnetic stirrer is suitable for being placed in the electrolytic cell; the electric stirring assembly comprises a rotating motor and a stirring rod, the rotating motor is arranged outside the electrolytic cell, one end of the stirring rod stretches into the electrolytic cell, and the other end of the stirring rod is connected with the rotating motor.

7. The method of claim 6, wherein the system for acclimating an electrochemically active biofilm satisfies at least one of the following conditions:

The plurality of reference electrodes are uniformly arranged at intervals along the circumferential direction of the cover body, the plurality of counter electrodes are arranged at intervals along the circumferential direction of the electrolytic cell, and the distances between the counter electrodes and the reference electrodes in different electrode groups are equal;

the setting height of the counter electrode is positioned in the middle of the electrolytic cell;

each counter electrode is correspondingly provided with a plurality of biological film carriers, and the biological film carriers are arranged at intervals along the height direction of the electrolytic cell and/or are arranged at intervals up and down along the counter electrode;

the electrolytic cell is cylindrical or polygonal;

the cover body and the electrolytic cell are detachably arranged;

a sealing gasket is arranged between the cover body and the electrolytic cell;

further comprises: a potentiostat comprising a plurality of channels, each of said channels being connected to an electrode set, each of said electrode sets comprising one of said counter electrodes, one of said reference electrodes and at least one of said biofilm carriers;

further comprises: a storage pool, which is communicated with the electrolytic cell and has a storage space;

further comprises: and the pumping device is used for providing power for the electrolyte supplementing or discharging of the electrolytic cell.

8. Use of the method of any one of claims 1-7 in a biotoxicity assay.