In-situ real-time observation device and method for electroactive biofilm based on image recognition

Abstract

The present application relates to microbial fuel cell technology, aiming at providing an in-situ real-time observation device and method for electroactive biofilm based on image recognition. The device comprises an automatic liquid changing system, a laminated biofilm incubator, an OCT in-situ observation and control system; wherein the automatic liquid changing system comprises a culture solution conveying device and is connected to the laminated biofilm incubator through a pipeline; the laminated biofilm incubator has a multi-layer structure formed by stacking, and sequentially comprises a culture chamber back cover plate, a gasket, a first titanium sheet, a punched graphite sheet, an area control sheet, a culture chamber cavity, a carbon cloth, a second titanium sheet, a high-transparency cover sheet and an L-shaped Luggin capillary; the OCT in-situ observation and control system comprises a voltage control instrument, an optical module and an optical signal processing module, and is connected to an upper computer through cables respectively. The present application can obtain microstructure information of electroactive biofilm with high resolution, realize real-time dynamic observation of biofilm, and improve observation efficiency and data reliability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fuel cells, and particularly relates to an in-situ real-time observation device and method for electroactive biofilm based on image recognition. BACKGROUND

[0002] Microbial fuel cell technology and its potential as a renewable energy technology have attracted much attention. This technology uses microorganisms to release electrons during the metabolic process, oxidizes organic matter in wastewater to produce electricity, and achieves the dual benefits of waste treatment and energy production. In microbial fuel cells, electroactive biofilm plays a crucial role as the key interface between microorganisms and electrodes, directly affecting electron transfer efficiency and cell power generation performance. However, the microstructure of electroactive biofilm is difficult to observe directly, which limits the further development of microbial fuel cell technology.

[0003] Currently, the observation of electroactive microbial membrane still remains in the stage of low-precision in-situ observation or high-precision non-in-situ intermittent observation. For example, low-precision in-situ observation usually adopts a method of naked eye observation, which can only observe the approximate change process of thickness, color and other characteristics. Although the traditional high-precision observation method such as scanning electron microscope (SEM) can provide high-resolution images, it cannot realize in-situ and real-time observation, and needs to be destructive to the sample. The defects of the above observation techniques limit the further development and application of electroactive biofilm research. In addition, while constructing electroactive biofilm, how to intelligently control the potential to limit the occurrence of competing reactions such as water electrolysis is also a big difficulty.

[0004] Therefore, in order to further understand the principle of microbial fuel cell and improve its performance, it is necessary to develop an in-situ real-time observation device that can realize high-precision observation and continuous operation. SUMMARY

[0005] The technical problem to be solved by the application is to overcome the deficiencies in the prior art and provide an in-situ real-time observation device and method for electroactive biofilm based on image recognition.

[0006] To solve the technical problem, the solution of the application is:

[0007] An in-situ real-time observation device for electroactive biofilm based on image recognition is provided, comprising an automatic liquid changing system, a layered biofilm incubator, and an OCT in-situ observation and control system.

[0008] The automatic liquid changing system comprises a culture solution conveying device and is connected to the layered biofilm incubator through a pipeline.

[0009] The laminated biofilm culture device has a multi-layer structure formed by laminating, and sequentially comprises a culture chamber back cover plate, a gasket, a first titanium sheet, a punched graphite sheet, an area control sheet, a culture chamber cavity, a carbon cloth, a second titanium sheet, a high-transparency cover sheet and an L-shaped Luggin capillary; through holes are arranged in the middle of the gasket, the first titanium sheet, the punched graphite sheet, the area control sheet, the culture chamber cavity, the carbon cloth and the second titanium sheet; a liquid inlet channel, a liquid outlet channel and an exhaust channel are arranged in the radial direction of the through hole of the culture chamber cavity, and the inner ends of the channels are respectively connected to the internal through hole of the culture chamber cavity; the sizes and positions of the through holes on the area control sheet, the culture chamber cavity, the carbon cloth and the second titanium sheet are selected to meet the biofilm growth and observation requirements; the sizes and positions of the through holes of the culture chamber back cover plate, the gasket, the first titanium sheet and the punched graphite sheet are selected to enable the L-shaped Luggin capillary to be fixedly installed; a reference electrode is inserted into the vertical pipe of the L-shaped Luggin capillary, and the capillary opening at the bottom is communicated with the culture chamber cavity.

[0010] The OCT in-situ observation and control system comprises a voltage control instrument, an optical module and an optical signal processing module, which are respectively connected to an upper computer through cables; the voltage control instrument is connected to a first titanium sheet, a second titanium sheet and a reference electrode through wires; the optical module comprises a light source and a fiber Bragg grating interferometer, and the optical signal processing module comprises a scanning detector and an electrical signal processor, and the upper computer is configured with a man-machine dialogue interface; the voltage control instrument, the electrical signal processor and the fiber Bragg grating interferometer are connected to the upper computer through cables.

[0011] As a preferred scheme of the present application, the culture solution conveying device is a micro-injection pump or a peristaltic pump, the inlet end of which is connected to a culture solution storage tank through a pipeline, the outlet end is connected to a liquid inlet hose, and the other end of the liquid inlet hose extends into the liquid inlet channel of the culture chamber cavity.

[0012] As a preferred scheme of the present application, the waste liquid storage tank is connected to a liquid outlet hose, and the other end of the liquid outlet hose extends into the liquid outlet channel of the culture chamber cavity.

[0013] As a preferred scheme of the present application, the laminated biofilm culture device is placed on a height-adjustable platform, and the platform has an electric or manual height adjustment mechanism.

[0014] As a preferred scheme of the present application, the high-transparency cover sheet in the laminated biofilm culture device is arranged opposite to the fiber Bragg grating interferometer in the optical module and maintains a distance.

[0015] As a preferred scheme of the present application, the upper computer is internally provided with a timing photographing control module and an imaging and recognition module, the former is used for controlling the scanning detector to take OCT images at a timing, and the latter is used for analyzing and recognizing the acquired images.

[0016] As a preferred scheme of the present application, the culture chamber rear cover plate is a perforated cuboid with a length of 80 mm, a width of 60 mm, and a thickness of 10 mm, and the perforation diameter is 4 mm; the gasket has a length of 80 mm, a width of 60 mm, and a thickness of 2 mm; the perforated graphite sheet is a circle with a diameter of 35 mm and a thickness of 1 mm, and the perforation diameter is 1 mm; the area control sheet is a concentric circular ring sheet with an outer diameter of 35 mm and an inner diameter of 10 mm; the carbon cloth is a concentric circular ring sheet with an outer diameter of 30 mm and an inner diameter of 10 mm; and the high-transmittance cover sheet is a square with a side length of 40 mm and a thickness of 0.3-1 mm.

[0017] The present application further provides a method for in-situ real-time observation of electroactive biofilm based on image recognition by using the aforementioned observation device, which comprises the following steps:

[0018] (1) Anaerobic sludge treatment liquid taken from a sewage treatment plant is added to the culture chamber cavity through the liquid inlet channel to inoculate electroactive microorganisms;

[0019] (2) The culture liquid is input into the culture chamber cavity through the liquid inlet hose by using the culture liquid delivery device to provide the energy required for the construction of biofilm by electroactive microorganisms, and the waste liquid generated in the process is recovered into the waste liquid storage tank through the liquid outlet hose;

[0020] (3) The voltage control instrument is controlled by the voltage regulation module built in the upper computer to adjust the culture potential of the stacked biofilm incubator, and the cathode potential of the carbon cloth and the size of the generated current in the bioelectrochemical system are detected;

[0021] (4) The light source in the optical module generates a light beam, which is transmitted through the high-transmittance cover sheet to perform real-time observation on the electroactive biofilm generated on the perforated graphite sheet, and the sample light and the reference light interfere with each other through the fiber Bragg grating interferometer in the optical module; the scanning detector in the optical signal processing module converts the optical interference signal into an electrical signal, which is processed by the electrical signal processor to generate an image and transmitted to the upper computer;

[0022] (5) The timing imaging module built in the upper computer is used to control the fiber Bragg grating interferometer and the electrical signal processor to image at a set interval time; the imaging and recognition module built in the upper computer is used to analyze, recognize, analyze and store the obtained OCT image, and the result is displayed on the display screen in the man-machine dialogue interface; through the recognition and analysis of the OCT image, the number of bubbles generated on the perforated graphite sheet in real time can be obtained, and accordingly the growth of the electroactive biofilm under different culture liquid and potential conditions can be observed in-situ and in real time.

[0023] As a preferred scheme of the present application, the culture liquid contains glucose or sodium acetate, the organic matter concentration is 1000 mg COD / L, contains 50 mM PBS buffer, 12.5 mL·L -1 of mineral solution, and 5 mL·L-1 The vitamin solution is washed with nitrogen gas with a purity of 99.99% for 20 minutes before use to remove dissolved oxygen and ensure an anaerobic environment for the bioelectrode; the temperature for culturing the electroactive microorganism is 30 DEG C.

[0024] As a preferred scheme of the present application, the wavelength tuning range of the light source in the optical module is 100KHz-200kHz, the center wavelength is 1325nm, the scanning line speed is 10kH-146kHz, the sensitivity is 109dB, the imaging depth in a solution environment is 3.5um, and the longitudinal resolution is 9.0um; the timing shooting module sets the photographing interval time to be 10-3600s; the voltage control instrument controls the potential range to be-60-60V, the time to be 0.01S-100000h, and the time interval for recording the current to be 0.01S-100000h; the receiving rate of the scanning detector in the optical signal processing module is 10-1000M.

[0025] Compared with the prior art, the present application has the beneficial effects that:

[0026] (1) The device of the present application adopts the optical coherence tomography (OCT) technology, and its non-invasive feature enables high-resolution acquisition of the microstructure information of the electroactive biofilm without damaging the biofilm structure, realizes real-time dynamic observation of the biofilm, and improves the observation efficiency and data reliability.

[0027] (2) The device of the present application is based on an intelligent actuator, and can realize automatic liquid replacement of the reaction chamber cavity. This not only ensures the freshness and stability of the culture solution, but also avoids the disturbance to the construction of the electroactive biofilm caused by manual liquid replacement, thereby ensuring the accuracy and repeatability of the experimental results.

[0028] (3) The device of the present application integrates the OCT and image recognition technologies, and can realize real-time identification and analysis of the bubble generation in the OCT image; the voltage control instrument is controlled by the upper computer, and the culture voltage of the electroactive microorganism can be intelligently adjusted to prevent the influence of bubbles on the experimental results.

[0029] (4) The present application can automatically realize whole-process monitoring and control of the experimental process, thereby reducing the work burden of researchers and improving the efficiency and stability of the experiment. Meanwhile, the device also has certain flexibility and expandability, and can be customized and optimized according to different experimental requirements to meet the application requirements in different fields.

[0030] (5) The present application has the characteristics of high efficiency, intelligence, accuracy and controllability, and provides important technical support and innovative ideas for the further development and application of the electroactive biofilm research field. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The figure is a schematic diagram of the connection relationship of the in-situ real-time observation device for the electrically active biofilm based on image recognition.

[0032] Figure 2 The figure is an exploded view of the bioelectrochemical device in the application.

[0033] Figure 3 The figure is a left view of the connection of the bioelectrochemical device in the application with the Luggin capillary and the reference electrode.

[0034] Figure 4 The figure is a front view of the connection of the reaction chamber cavity with the exhaust hose, the liquid inlet hose and the liquid outlet hose in the application.

[0035] Figure 5 The figure is an OCT online monitoring image in the embodiment, wherein the imaging and identification module and the voltage control instrument automatically identify bubbles and regulate the voltage.

[0036] Figure 6 The figure is an OCT online monitoring image in the embodiment, wherein the imaging and identification module is combined with the timing shooting module to realize real-time in-situ observation and record the OCT image of the construction of the electrically active biofilm.

[0037] The figure is a schematic diagram of the connection relationship of the in-situ real-time observation device for the electrically active biofilm based on image recognition. DETAILED DESCRIPTION

[0038] The application will be described in detail below with specific embodiments and in conjunction with the drawings, but the application is not limited to the following embodiments, and all variations should be included in the technical scope of the application without departing from the content and scope of the application.

[0039] First part of the implementation scheme of the application

[0040] 1. Structure of the observation device

[0041] The in-situ real-time observation device for the electrically active biofilm based on image recognition provided by the application comprises an automatic liquid changing system, a stacked biofilm incubator and an OCT in-situ observation and control system.

[0042] The automatic liquid exchange system comprises a culture solution delivery device 1-1 connected to a stacked biofilm incubator through a pipeline. The culture solution delivery device 1-1 can be a microsyringe pump or a peristaltic pump.

[0043] The stacked biofilm incubator 3 has a multi-layer structure formed by stacking, sequentially comprising a culture chamber back cover plate 1-6, a gasket 1-7, a first titanium sheet 1-8, a punched graphite sheet 1-9, an area control sheet 1-10, a culture chamber cavity 1-3, a carbon cloth 1-11, a second titanium sheet 1-12, a high-transmittance cover sheet 1-13, and an L-shaped Lugan capillary tube; the middle part of the gasket 1-7, the first titanium sheet 1-8, the punched graphite sheet 1-9, the area control sheet 1-10, the culture chamber cavity 1-3, the carbon cloth 1-11, and the second titanium sheet 1-12 are provided with through holes; the liquid inlet channel, the liquid outlet channel, and the exhaust channel are arranged in the radial direction of the through hole of the culture chamber cavity 1-3, and the inner ends of the channels are respectively connected to the internal through hole of the culture chamber cavity; the size and position of the through holes on the area control sheet 1-10, the culture chamber cavity 1-3, the carbon cloth 1-11, and the second titanium sheet 1-12 are selected to meet the needs of biofilm growth and observation; the size and position of the through holes of the culture chamber back cover plate 1-6, the gasket 1-7, the first titanium sheet 1-8, and the punched graphite sheet 1-9 are selected to enable the L-shaped Lugan capillary tube 1-14 to be fixedly installed; a reference electrode is inserted into the vertical pipe of the L-shaped Lugan capillary tube 1-4, and the capillary opening at the bottom is connected to the culture chamber cavity 1-3.

[0044] The liquid inlet hose 1-17 is connected to the outlet end of the culture solution delivery device 1-1, and its inlet end is connected to the culture solution storage tank through a pipeline; the liquid outlet hose 1-18 is connected to the waste liquid storage tank 1-5 for recycling waste liquid. The exhaust hose 1-16 is inserted into the exhaust channel for exhausting waste gas. The stacked biofilm incubator 3 is placed on a height-adjustable platform 1-4, which has an electric or manual height adjustment mechanism. The high-transmittance cover sheet 1-13 is arranged opposite to the fiber Bragg grating interferometer in the optical module 2-1 and maintains a distance.

[0045] As optional example parameters: the rear cover plate 1-6 of the culture chamber is a perforated cuboid with a length of 80mm, a width of 60mm, and a thickness of 10mm, and the perforation diameter is 4mm; the gasket 1-7 is 80mm long, 60mm wide, and 2mm thick; the first titanium sheet 1-8 is a concentric ring sheet with an outer diameter of 38mm, an inner diameter of 5mm, and a thickness of 0.2mm, connected to a cuboid assembly with a width of 5mm, a length of 20mm, and a thickness of 0.2mm as a terminal; the perforated graphite sheet 1-9 is a circle with a diameter of 35mm and a thickness of 1mm, and the perforation diameter is... The diameter is 1mm; the area control sheet 1-10 is a concentric ring sheet with an outer diameter of 35mm and an inner diameter of 10mm; the carbon cloth 1-11 is a concentric ring sheet with an outer diameter of 30mm and an inner diameter of 10mm; the second titanium sheet 1-12 is a concentric ring sheet with an outer diameter of 30mm, an inner diameter of 5mm, and a thickness of 0.2mm, and a cuboid with a width of 5mm, a top width of 5mm, a length of 28mm, and a thickness of 0.2mm, which is used as a terminal; the high-transparency cover sheet 1-13 is a square with a side length of 40mm and a thickness of 0.3-1mm. The flow rate of the culture medium delivery device 1-1 ranges from 0.1 to 2000 mL / h, and the flow direction can be adjusted in both forward and reverse directions; the height-adjustable platform 1-4 can be adjusted within the range of 10 to 100 cm; the waste liquid storage tank 1-5 can be a beaker, waste liquid cylinder, conical flask, etc., with a storage volume of less than 500 mL; the materials for the culture chamber cavity 1-3 and the rear cover plate 1-6 of the culture chamber can be photosensitive resin, polymethyl methacrylate (PMMA), polypropylene (PP), etc.; the area control sheet 1-10 is made of... The materials can be insulating plastics such as polymethyl methacrylate (PMMA) and polypropylene (PP); the high-transparency cover sheet 1-13 can be made of optical glass, polymethyl methacrylate (PMMA), polypropylene (PP), or other materials with a light transmittance greater than 80%; the reference electrode 1-15 can be Ag / AgCl reference electrode, saturated calomel electrode, etc.; the exhaust hose 1-16, liquid inlet hose 1-17, and liquid outlet hose 1-18 can be made of flexible materials such as polyvinyl chloride (PVC), polyethylene (PE), and silicone.

[0046] The OCT in-situ observation and control system includes a voltage controller 2-3, an optical module 2-1, and an optical signal processing module 2-4, which are connected to a host computer 2-5 via cables. The voltage controller 2-3 is connected to the first titanium sheet 1-8, the second titanium sheet 1-12, and the reference electrode 1-15 via wires. The optical module 2-1 includes a light source and a fiber optic interferometer, and the optical signal processing module 2-4 includes a scanning detector and an electrical signal processor. The host computer 2-5 is equipped with a human-machine interface 2-2. The voltage controller 2-3, the electrical signal processor, and the fiber optic interferometer are connected to the host computer 2-5 via cables.

[0047] The timing photographing control module and the imaging and identification module are built in the host computer, the former is used for controlling the scanning detector to photograph the OCT image at a certain time, and the latter is used for analyzing and identifying the obtained image. In order to realize the control of the voltage control instrument 2-3 to adjust the culture potential, the voltage control module is built in the host computer 2-5. The several built-in modules are functional modules realized by software programs. The applicant believes that, after carefully reading the application file and accurately understanding the implementation principle and the purpose of the application, the skilled in the art can realize the related modules by using the software programming skills mastered by them in combination with the prior art. Therefore, the application does not repeat the implementation of the software functional modules of the application.

[0048] 2. In-situ real-time observation method

[0049] By using the foregoing observation device, an in-situ real-time observation method of the electroactive biofilm based on image recognition can be realized; the method specifically includes the following steps:

[0050] (1) The anaerobic sludge treatment liquid taken from the sewage treatment plant is added to the culture chamber cavity 1-3 through the liquid inlet channel, to inoculate the electroactive microorganisms;

[0051] (2) The culture liquid 1-2 is input into the culture chamber cavity 1-3 through the liquid inlet hose 1-17 by using the culture liquid conveying device, to provide the energy required for the electroactive microorganisms to construct the biofilm, and the waste liquid generated in the process is recovered into the waste liquid storage tank 1-5 through the liquid outlet hose 1-18;

[0052] (3) The voltage control instrument 2-3 is controlled by the voltage control module built in the host computer to adjust the culture potential of the stacked biofilm culture device, and the cathode potential of the carbon cloth 1-11 and the size of the generated current in the bioelectrochemical system are detected;

[0053] (4) The light beam is generated by using the light source in the optical module 2-1, the electroactive biofilm generated on the punched graphite sheet 1-9 is observed in real time by penetrating the high-transmittance cover sheet 1-13, the sample light and the reference light interfere with each other in the fiber grating interferometer in the optical module 2-1; the scanning detector in the optical signal processing module 2-4 converts the optical interference signal into an electrical signal, and generates an image after being processed by the electrical signal processor and transmits the image to the host computer 2-5;

[0054] (5) The timing imaging module built-in the host computer 2-5 controls the fiber-optic grating interferometer and the electric signal processor to take pictures at the set interval time; the imaging and recognition module built-in the host computer 2-5 analyzes, recognizes, analyzes and stores the OCT images obtained, and displays the results on the display screen in the man-machine dialogue interface 2-2; through the recognition and analysis of the OCT images, the number of bubbles generated on the perforated graphite sheet 1-9 in real time can be obtained, and the growth of the electroactive biofilm under different culture solutions and potential conditions can be observed in situ in real time.

[0055] As an example of the image processing method, the imaging and recognition module built-in the host computer 2-5 converts the A-scan mode spectrum collected by the detector into a B-scan mode spectrum, and adjusts and edits the imaging scanning parameters according to the settings; and based on mature image recognition technology, the number of bubbles in the image is automatically recognized, so that the progress of the bubbles generated on the biofilm on the perforated graphite sheet 1-9 is recorded based on the time node, and the growth of the electroactive biofilm is evaluated.

[0056] As an example of the optional parameters, the culture solution 1-2 contains glucose or sodium acetate, the organic matter concentration is 1000 mg COD / L, contains 50 mM PBS buffer, 12.5 mL·L -1 of mineral solution and 5 mL·L -1 of vitamin solution; before use, it needs to be flushed with nitrogen gas with a purity of 99.99% for 20 min to remove dissolved oxygen and ensure the anaerobic environment of the bioelectrode; the temperature during the culture of electroactive microorganisms is 30°C. The wavelength tuning range of the light source in the optical module (2-1) is 100 KHz-200 kHz, the center wavelength is 1325 nm, the scanning line speed is 10 kH-146 kHz, the sensitivity is 109 dB, the imaging depth in the solution environment is 3.5 um, and the longitudinal resolution is 9.0 um; the photographing interval time of the timing imaging module is set to 10-3600 s; the voltage control instrument 2-3 controls the potential range of-60-60 V, the time is 0.01 S-100000 h, and the time interval for recording the current is 0.01 S-100000 h; the receiving rate of the scanning detector in the optical signal processing module 2-4 is 10-1000 M.

[0057] A specific example of the second part

[0058] As Figure 1As shown, an in-situ real-time observation device for electroactive biofilm based on image recognition includes an automatic liquid changing system and an OCT in-situ observation and voltage control system. In the automatic liquid changing system, culture solution 1-2 enters the culture chamber cavity 1-3 from the liquid inlet hose 1-17 through the culture solution delivery device 1-2, providing a source of organic matter for the electroactive microorganisms on the punched graphite sheet 1-9, and the generated waste liquid enters the waste liquid storage tank 1-5 through the liquid outlet hose 1-18.

[0059] The punched graphite sheet 1-9 is in close contact with the first titanium sheet 1-8, the carbon cloth 1-11 is in close contact with the second titanium sheet 1-12, and the Lugugin capillary 1-14 is embedded with the reference electrode 1-15, which are connected to the voltage control instrument 2-3 through wires. The voltage control module built-in the upper computer 2-5 is used to control the potential of the punched graphite sheet 1-9 and monitor the potential of the carbon cloth 1-11. The optical module 2-1 and the light signal processing module 2-4 are used to realize in-situ and continuous observation of the constructed electroactive biofilm. The timing shooting module, the control imaging and recognition module are used to record, recognize and store the OCT images.

[0060] In this example, the culture solution delivery device 1-1 uses a micro-injection pump with a flow rate of 0.5 mL / h, and the forward delivery glucose concentration is 1000 mgCOD / L, containing 50 mM PBS buffer, 12.5 mL·L -1 of mineral solution and 5 mL·L -1 of vitamin solution, and the culture solution 1-2 at a temperature of 30℃ enters the culture chamber cavity 1-3, and the culture solution 1-2 needs to be pretreated by flushing with 99.99% pure nitrogen for 20 min; the culture chamber cavity 1-3 and the culture chamber rear cover plate 1-6 are made of photosensitive resin 3D printing, the waste liquid storage tank 1-5 uses a 500 mL conical flask, the high-transmission cover sheet 1-13 is made of optical glass with a thickness of 0.5 mm, the reference electrode 1-15 is an Ag / AgCl reference electrode, and the exhaust hose 1-16, the liquid inlet hose 1-17 and the liquid outlet hose 1-18 are polyvinyl chloride (PVC) hoses; the height-adjustable platform 1-4 is fixed at 50 cm, so that the light source of the optical module 2-1 can enter the culture chamber cavity 1-3 through the 0.5 mm high-transmission cover sheet 1-13, thereby observing the process of constructing electroactive microbial membrane on the punched graphite sheet 1-9.

[0061] As shown in Figures 1 to 6 , a method for operating an in-situ real-time observation device for electroactive biofilm based on image recognition includes the following steps:

[0062] (1) Assemble the device;

[0063] (2) Inoculation: Take 5 mL of anaerobic sludge treatment effluent from a sewage plant and add it to the culture chamber cavity 1-3 to inoculate the electroactive microorganisms;

[0064] (3) Automatic liquid exchange: configure the culture solution 1-2 containing glucose concentration of 1000 mg COD / L, 50 mM PBS buffer, 12.5 mL·L -1 of mineral solution and 5 mL·L -1 of vitamin solution, and flush with 99.99% pure nitrogen for 20 min to complete the anaerobic pretreatment, and then add to the culture chamber cavity 1-3 through the liquid inlet hose 1-17 by using the culture solution delivery device 1-1 with a flow rate of 0.5 mL / h, and the waste liquid generated by the culture chamber cavity 1-3 is stored in the waste liquid storage tank 1-5 through the liquid outlet hose 1-18;

[0065] (4) Real-time observation: the optical module 2-1 of the voltage control system in situ observes the electroactive biofilm on the punched graphite sheet (1-9) through the high-transmittance cover sheet 1-13, the light source of the optical module 2-1 generates a light beam, the sample light and the reference light interfere through the fiber grating interferometer, the optical interference signal is converted into an electrical signal through the optical signal processing module 2-4 scanning detector, and the electrical signal processor further processes the electrical signal, and the obtained signal is displayed on the display screen after imaging, analysis and identification processing, the imaging is controlled by the timing shooting module at intervals of 15 min, and the results are recorded and stored as real-time OCT photos;

[0066] (5) Voltage regulation: through the voltage control module built-in the upper computer 2-5, the voltage control instrument 2-3 is used to set the potential of the punched graphite sheet 1-9 relative to the reference electrode 1-15 as -0.1 V, and the interval time is consistent with the interval time set by the timing shooting module 2-2; as shown in Figure 5 (a-c), if it is identified that there is a bubble in the OCT real-time image, the potential is controlled by the voltage control instrument 2-3 without change; as shown in Figure 5 (d), if it is identified that there is no bubble in the OCT real-time image, the potential is controlled by the voltage control instrument 2-3 to be lowered by 0.01 V; the voltage is adjusted to -0.3 V and remains unchanged after being stable.

[0067] Experimental results: as shown in Figure 6 , Figure 6 (a) is the initial state of the punched graphite sheet 1-9, at this time the surface is smooth; as shown in Figure 6 (b-e), the gradual construction process of the electroactive microbial membrane can be observed. It is illustrated that the system has the in-situ real-time observation ability of the electroactive biofilm, and can realize the high automation of voltage regulation and liquid exchange.

[0068] The present application is not limited to the above best embodiment, and anyone can derive other various forms of an in-situ real-time observation device and method for electroactive biofilm based on image recognition under the inspiration of the present application. Any equivalent changes and modifications made within the scope of the present application patent application shall be included in the scope of the present application.

Claims

1. An in-situ real-time observation device for electroactive biofilm based on image recognition, characterized in that, The application relates to an automatic liquid changing system, a laminated biofilm culture device and an OCT in-situ observation and control system. The automatic liquid changing system comprises a culture liquid conveying device and is connected to the laminated biofilm culture device through pipelines. The laminated biofilm culture device has a multi-layer structure formed by stacking and comprises, in sequence, a culture chamber back cover plate, a gasket, a first titanium sheet, a perforated graphite sheet, an area control sheet, a culture chamber cavity, carbon cloth, a second titanium sheet and a high-transparency cover sheet and an L-shaped Luggin capillary tube. The middle parts of the gasket, the first titanium sheet, the perforated graphite sheet, the area control sheet, the culture chamber cavity, the carbon cloth and the second titanium sheet are provided with through holes. The culture chamber cavity is provided with a liquid inlet channel, a liquid outlet channel and an exhaust channel in the radial direction of the through hole of the culture chamber cavity, and the inner ends of the channels are communicated with the inner through hole of the culture chamber cavity.

2. The viewing device of claim 1, wherein, The sizes and positions of the through holes on the area control sheet, the culture chamber cavity, the carbon cloth and the second titanium sheet are selected to meet the biofilm growth and observation requirements.

3. The viewing device of claim 1, wherein, The sizes and positions of the through holes of the culture chamber back cover plate, the gasket, the first titanium sheet and the perforated graphite sheet are selected to enable the L-shaped Luggin capillary tube to be fixedly installed.

4. The viewing device of claim 1, wherein, A reference electrode is inserted into the vertical pipe of the L-shaped Luggin capillary tube, and the capillary opening at the bottom is communicated with the culture chamber cavity.

5. The viewing device of claim 1, wherein, The OCT in-situ observation and control system comprises a voltage control instrument, an optical module and an optical signal processing module, which are connected to an upper computer through cables.

6. The viewing device of claim 1, wherein, The voltage control instrument is connected to the first titanium sheet, the second titanium sheet and the reference electrode through wires.

7. The viewing device of claim 1, wherein, The optical module comprises a light source and a fiber Bragg grating interferometer, and the optical signal processing module comprises a scanning detector and an electrical signal processor.

8. A method for in-situ real-time observation of electroactive biofilm based on image recognition using the observation device of claim 1, characterized in that, The upper computer is configured with a man-machine dialogue interface. The voltage control instrument, the electrical signal processor and the fiber Bragg grating interferometer are connected to the upper computer through cables. The culture liquid conveying device is a micro-injection pump or a peristaltic pump, the inlet end of which is connected to a culture liquid storage tank through a pipeline, the outlet end is connected to a liquid inlet hose, and the other end of the liquid inlet hose is inserted into the liquid inlet channel of the culture chamber cavity. A waste liquid storage tank is connected to a liquid outlet hose, and the other end of the liquid outlet hose is inserted into the liquid outlet channel of the culture chamber cavity. The laminated biofilm culture device is placed on a height-adjustable platform which has an electric or manual height adjusting mechanism. The high-transparency cover sheet in the laminated biofilm culture device is oppositely arranged with the fiber Bragg grating interferometer in the optical module and is kept at a distance. The upper computer is internally provided with a timing photographing control module and an imaging and identification module. The culture chamber back cover plate is a perforated cuboid with a length of 80 mm, a width of 60 mm and a thickness of 10 mm, and the perforation diameter is 4 mm. The gasket has a length of 80 mm, a width of 60 mm and a thickness of 2 mm. The perforated graphite sheet is a circle with a diameter of 35 mm and a thickness of 1 mm, and the perforation diameter is 1 mm. The area control sheet is a concentric circular ring sheet with an outer diameter of 35 mm and an inner diameter of 10 mm. The steps comprise the following steps. (1) by the liquid inlet channel to the culture chamber cavity, sewage treatment plant for the addition of anaerobic sludge treatment liquid, for inoculation of electroactive microorganisms; (2) using the culture solution delivery equipment by the liquid inlet hose into the culture chamber cavity, for providing the energy required for the construction of biofilm electroactive microorganisms, the waste liquid generated in the process is recycled to the waste liquid storage tank through the liquid outlet hose; (3) using the voltage control module built-in upper computer to control the voltage control instrument to adjust the culture potential of the stacked biofilm incubator, and detect the cathode potential of the carbon cloth in the bioelectrochemical system and the size of the generated current; (4) using the light source in the optical module to generate a light beam, and observing the electroactive biofilm generated on the perforated graphite sheet in real time through the high-transparency cover sheet; sample light and reference light interfere with each other through the fiber grating interferometer in the optical module; the scanning detector in the optical signal processing module converts the optical interference signal into an electrical signal, which is processed by the electrical signal processor to generate an image and transmitted to the upper computer; (5) using the timing shooting module built-in upper computer, control fiber grating interferometer and electrical signal processor according to the set interval time timing imaging; using the imaging and recognition module built-in upper computer, the acquired OCT image is analyzed, recognized and stored, and the results are displayed on the display screen in the man-machine dialogue interface; through the identification and analysis of OCT image, the number of bubbles generated on the perforated graphite sheet in real time can be obtained, and the growth of electroactive biofilm under different culture liquid and potential conditions can be observed in situ in real time.

9. The method of claim 8, wherein, The culture solution contains glucose or sodium acetate, the organic matter concentration is 1000 mg COD / L, contains 50 mM PBS buffer, 12.5 mL·L -1 of mineral solution and 5 mL·L -1 of vitamin solution; before use, it needs to be flushed with nitrogen gas with a purity of 99.99% for 20 min to remove dissolved oxygen, so as to ensure the anaerobic environment of the bioelectrode; the temperature during the cultivation of the electroactive microorganism is 30°C.

10. The method of claim 8, wherein, The wavelength tuning range of the light source in the optical module is 100KHz-200kHz, the center wavelength is 1325nm, the scanning line speed is 10kH-146kHz, the sensitivity is 109dB, the imaging depth in solution environment is 3.5um, and the longitudinal resolution is 9.0um; the timing shooting module sets the shooting interval time to 10-3600s; the voltage control instrument controls the potential range of-60-60V, the time is 0.01S-100000h, and the time interval of recording current is 0.01S-100000h; the receiving rate of the scanning detector in the optical signal processing module is 10-1000M.

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