CN107132227A - A kind of biomembrane film forming procedure online monitoring system and monitoring method - Google Patents
A kind of biomembrane film forming procedure online monitoring system and monitoring method Download PDFInfo
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Abstract
A kind of biomembrane film forming procedure online monitoring system and monitoring method, the microbe film forming process online monitoring system mainly includes electro-chemical systems unit and optical microscopy imaging unit, and the electro-chemical systems unit includes electrochemical reaction cell, electrochemical workstation and sampling system；Described electrochemical reaction tank main body is made up of lower cover, upper lid and pond body, the pond body bottom is hollow design, it is provided with slot, it is conductive glass carbon or the electro-conductive glass working electrode of surface modification in slot, the working electrode is connected with the working electrode binding post in pond body, and Ag/AgCl or saturated calomel reference electrode and platinum disk or platinum filament are respectively arranged with the upper lid to electrode.The microbe film forming process online monitoring system, which overcomes traditional biological film monitoring technology, can not realize electrochemical signals with optical imagery while the bottleneck monitored, the microbe film forming process online monitoring system of the present invention is stable, accurate, it is applied widely, expand excellent performance.
Description
Technical field
The present invention relates to field of microbial electromechanical technology, more particularly to a kind of biomembrane film forming procedure online monitoring system
And monitoring method.
Background technology
Biomembrane is the general name for constituting all films of cell, including is trapped among the plasma membrane of cytoplasm periphery and the internal membrane system of organelle
System.Biomembrane is wrapped in bacterium, with protection bacterium, provides the binding site of a variety of enzymes, provides cell recognition site, participates in shape
Into functions such as the cell surface specialised structures with difference in functionality.Monitor microbe film forming process, research nature or man-made environment
Interaction inside middle biomembrane between microorganism, not only for formation and the During Succession for understanding nature or artificial biomembrane
It is significant, and can also provide theoretical in terms of environmental quality lifting, microbial diversity are maintained for biomembrane
Instruct.
Microbe film forming process online monitoring system is shown by the electro-chemical systems unit matched completely and existing optics
Micro- imaging unit is constituted, so as to realize high stability, real time and on line monitoring, broad operating space and good expansion performance.
It is microbe survival environment that microbe film forming process online monitoring system, which is needed with visual electrochemical reaction cell, and electro-conductive glass is work
Make electrode, using electric current or potential as electrochemical monitoring signal, light microscope (including wide visual field light microscope, copolymerization Jiao show
Micro mirror, fluorescence microscope etc.) it is used for realtime graphic output, record.
Traditional microorganism monitoring system includes biofilm monitor monitoring, utilizes the instrument monitoring of light microscope.But
On-line monitoring due to realizing biomembrane film forming procedure needs the complicated electro-chemical systems of matching running to provide stable existence
Environment is to realize real-time in-situ monitoring, and traditional electrochemical reactor seal is poor, size compares that objective table is excessive, knot
Structure is light tight or can not form light-path, and is not suitable for existing monitoring instrument, it is impossible to realize electro-chemical systems and optical system
The matching running of system, it is impossible to realize microorganism the online, in situ of film forming procedure, Real Time Observation, record in electro-chemical systems.
During biomembrane film forming procedure on-line monitoring, Conventional electrochemical reaction tank can not be matched with electro-chemical systems and light microscope simultaneously
Imaging system, causes biomembrane film forming procedure on line real-time monitoring to realize, causes information loss and inaccurate, imperfect
Property.
The content of the invention
There is problem present invention aim to address above-mentioned there is provided a kind of microbe film forming process online monitoring system and prison
Prosecutor method.The invention be applied to microbe film forming process on-line monitoring when, can under anaerobism or aerobic condition stable operation it is same
When in real time the film forming situation of monitoring electrode surface microorganism and record in real time, output pattern data and electrochemical signals, not light
Limitation of the microscope to sample size to be seen, specification is learned, light reflex path is accurately formed, is a kind of brand-new efficient, steady
The microbe film forming process online monitoring system of fixed, the wide scope of application.
Technical scheme：
A kind of microbe film forming process online monitoring system, the microbe film forming process online monitoring system mainly includes
Electro-chemical systems unit and optical microscopy imaging unit, the electro-chemical systems unit include electrochemical reaction cell, electrochemistry work
Make station and sampling system (including syringe pump, syringe, water inlet)；Described electrochemical reaction tank main body is by lower cover, Shang Gai and pond
Body is constituted, and the pond body bottom and lower cover center are to be provided with hollow design, pond body bottom in slot, slot as conductive glass carbon
Or the electro-conductive glass working electrode of surface modification, the working electrode is connected with the working electrode binding post in pond body, the upper lid
It is upper to be provided with Ag/AgCl or saturated calomel reference electrode and platinum disk or platinum filament to electrode by electrode perforations respectively.The electrification
Learn and be additionally provided with inlet and leakage fluid dram in container of the reaction tank for volume 5-50mL polytetrafluoroethylmaterial material, the pond body,
Simultaneously provided with liquid injection port on upper lid, liquid injection port is provided with air plug.The area of working electrode is 1-20cm2.To the surface of electrode
Product is 1cm2。
The step of biomembrane film forming procedure on-line monitoring method, is as follows：
1) 1-20cm is placed to the electrode slot position of above-mentioned electrochemical reaction cell pond body bottom2Electro-conductive glass 8 work
Electrode, Ag/AgCl or saturated calomel reference electrode 1,1cm are inserted by the electrode perforations on upper lid into pond body2Platinum disk or
Platinum filament is to electrode 2 (referring to Fig. 1), and then into pond body, the pure bacterium of addition Anaerobic Treatment, sanitary sewage, anaerobic sludge, river bed are extra large
The water outlet of bottom sediment or the microbiological fuel cell mutually stablized through long-term domesticated organism, using concentration as 0.5-2g/L acetic acid
Sodium or glucose as substrate, using potentiostat chronoamperometry or when m- current method carry out microorganism continuous stream or
Stream of having a rest is cultivated；
2) time of running, light microscope is started, in real time record or use time sequential recording microbe film forming mistake
Journey, exports picture, graphical information in real time, defeated to output image computer for the film forming situation and rule of any incubation time
Go out light field, metallographic and the fluoroscopic image information of three-dimensional/two dimension.Realize the connection of electro-chemical systems unit and optical microphotograph imaging technique
Close operation.
A kind of application of microbe film forming process online monitoring system, for real-time, in situ, accurate, stable monitoring and
Record the electrochemical signals of microbe film forming process and the apparent or micro image information of biomembrane pattern.The monitoring system is being detested
Oxygen, aerobic, continuous stream, intermittent flow, it is various it is lighttight exist to electrode material (such as platinum electrode) in the environment of, it is ensured that monitoring knot
True, the validity of fruit.The system when external direct current, AC power, Monitoring Data, image still can intuitively, it is accurate
The truth of true reaction biomembrane.
Concrete application method is as follows：
1) after electrochemical reaction cell is cleaned by ultrasonic with 75% ethanol, drying is cleaned with distilled water；
2) added into electrochemical reaction cell by the pure bacterium of the separating-purifying of Anaerobic Treatment, preliminary sedimentation tank sewage or through length
The water outlet for the microbiological fuel cell that phase domesticated organism is mutually stablized, is that 0.5-2g/L sodium acetates or glucose are used as bottom using concentration
Thing, using potentiostat chronoamperometry or when m- current method carry out the culture of microorganism, connection light microscope is exported in real time
Feature image.
The present invention is with accurate (can be precisely controlled working electrode with the distance to electrode and accurately light reflex path) electricity
Chemical reaction pond (can match electro-chemical systems and optical microphotograph imaging technique simultaneously) is stable microbial film culture environment, is used
Potentiostat and the biological film forming procedure of the online in-situ monitoring of light microscope.
The beneficial effects of the invention are as follows：The microbe film forming process online monitoring system overcomes traditional biological film monitoring skill
Art can not realize electrochemical signals with optical imagery while the bottleneck monitored, microbe film forming process on-line monitoring system of the invention
System is stable, accurate, applied widely, expands excellent performance.
Brief description of the drawings
Fig. 1 is the structural representation of electrochemical reaction cell.
Fig. 2 is the overlooking structure figure of electrochemical reaction cell.
Fig. 3 is microbe film forming process online monitoring system.
Fig. 4 is fluorescence microscope and confocal images.Wherein, a wide-field microscopes image, b fluorescence microscopes are glimmering
Light image, c laser confocal microscope bright field images, d laser confocal microscope fluoroscopic images.In figure, 1 reference electrode, 2 disks
Shape platinum electrode, 3 air plugs, 4 working electrode binding posts are covered on 5,6 pond bodies, 7 lower covers, 8 electro-conductive glass, 9 leakage fluid drams, 10 feed liquors
Mouthful, 11 sampling systems (including syringe pump, syringe, water inlet), 12 electrochemical reaction cells, 13 light microscopes, 14 electrochemistry works
Stand, 15 output image computers.
Embodiment
Embodiment 1：
First, microbe film forming process online monitoring system
As shown in figure 3, microbe film forming process online monitoring system shows to electro-chemical systems unit is mainly included with optics
Micro- imaging unit, the electro-chemical systems unit includes electrochemical reaction cell 12, electrochemical workstation 14 and the (bag of sampling system 11
Include syringe pump, syringe and water inlet).The electrochemical reaction cell 12 of the present invention can not only be connected to electrochemical workstation 14, also
Light microscope 13 can be connected to, so as to realize the microbe film forming process on-line monitoring that can not be carried out in the past.
As depicted in figs. 1 and 2, electrochemical reaction tank main body is by lower cover 7, upper lid 5 and pond body 6 for described electrochemical reaction cell
Constitute, can be connected through a screw thread each other；Electrochemical reaction tank main body uses diameter 35mm, high 35mm polytetrafluoroethylene (PTFE)
The container of material is constituted, and the bottom of pond body 6 and the center of lower cover 7 are hollow design, and hole diameter is 20mm, and the bottom of pond body 6 is simultaneously designed
For slot pattern, interior slot is working electrode, and working electrode is 1cm2Etched features electro-conductive glass 8 (real work area is
0.25cm2), electro-conductive glass 8 (working electrode) is connected with the working electrode binding post 4 on the side wall of pond body 6；Pass through upper lid 5 simultaneously
On electrode perforations reference electrode 1 (being 4MAg/AgCl electrodes) inserted into container and be 1cm to electrode2Plate-like platinum filament electricity
Simultaneously provided with liquid injection port on pole 2, the upper lid 5 of electrochemical reaction cell, so as to fluid injection and ventilation, liquid injection port is provided with air plug 3, pond
Body is provided with inlet 10 and leakage fluid dram 9.
Electrochemical reaction cell 12 is placed on the objective table of light microscope 13, is led to by light microscope wide field, fluorescence
The switching in road directly carries out eyepiece observation, also can carry out Real Time Observation and record by output image computer 15.Electrochemistry work
The reference electrode 1 of electrochemical reaction cell 12, plate-like platinum electrode 2 and electro-conductive glass 8 can be directly connected to by making station 14, anti-to electrochemistry
Answer pond 12 to apply constant output current potential etc., and pass through the record data image of output image computer 15.Sampling system (including injection
Pump, syringe, water inlet) 11 inlets 10 for being connected to electrochemical reaction cell 12, flow velocity can be adjusted by sampling system.
2nd, microbe film forming process on-line monitoring method
Added into reaction tank and expose the Microbial fuel electricity that the long-term domesticated organism of warp of nitrogen 10min Anaerobic Treatments is mutually stablized
The water outlet in pond, using 1g/L sodium acetates as substrate, with syringe pump it is constant enter flow velocity be 0.2mL/h, use potentiostat timing electricity
Stream method carries out the culture of microorganism, records real-time biological film pattern with wide visual field light microscope time series, monitored results are such as
Shown in Fig. 4, wherein, a wide-field microscope images, b fluorescence microscope fluoroscopic images, c laser confocal microscope bright field images, d
Laser confocal microscope fluoroscopic image.
Embodiment 2：
First, microbe film forming process online monitoring system
Concrete structure be the same as Example 1, electrochemical reaction cell therein uses diameter 70mm, high 50mm polytetrafluoroethylene (PTFE) material
The container of material is constituted, and pond body bottom is hollow design, and hole is diameter 32mm, and is designed as slot pattern, and working electrode is
10cm2Electro-conductive glass, into container inserting reference electrode is 4M Ag/AgCl electrodes and is 1cm to electrode2Platinum electrode,
Electrochemical reaction cell top is provided with liquid injection port, is easy to fluid injection and ventilation, pond body is provided with liquid in-out mouthful, and all electrodes can be accurate
Condition relative distance.
2nd, microbe film forming process on-line monitoring method
The Geobacter sulfurreducens bacterium solutions that purification is tamed under the conditions of strictly anaerobic are added into reaction tank
1mL, using 1g/L sodium acetates as substrate, with syringe pump it is constant enter flow velocity be 1mL/h, carried out using potentiostat chronoamperometry
The culture of microorganism, video recording operation is carried out with fluorescence microscope, in real time record biomembrane pattern.
Embodiment 3：
First, microbe film forming process online monitoring system
Concrete structure be the same as Example 1, electrochemical reaction cell therein uses diameter 70mm, high 30mm polytetrafluoroethylene (PTFE) material
The container of material is constituted, and pond body bottom is hollow design, and hole is diameter 32mm, and is designed as slot pattern, and working electrode is
5cm2Electro-conductive glass, into container inserting reference electrode is 4M Ag/AgCl electrodes and is 1cm to electrode2Platinum electrode, electricity
Chemical reaction pond top is provided with liquid injection port, is easy to fluid injection and ventilation, and pond body is provided with liquid in-out mouthful, and all electrodes can accurate condition
Relative distance.
2nd, microbe film forming process on-line monitoring method
The preliminary sedimentation tank municipal wastewater for exposing nitrogen 30min Anaerobic Treatments is added into reaction tank, using 2g/L sodium acetates as substrate,
Liquid in-out mouthful is closed, the culture of microorganism is carried out using potentiostat chronoamperometry, is taken pictures in real time with fluorescence microscope
The operation of (light field, fluoroscopic image) record, records biomembrane pattern in real time.
Claims (4)
1. a kind of microbe film forming process online monitoring system, including electro-chemical systems unit and optical microscopy imaging unit, its
It is characterised by that the electro-chemical systems unit includes electrochemical reaction cell, electrochemical workstation and sampling system；Described electrification
Learn reaction tank main body to be made up of lower cover, upper lid and pond body, the pond body bottom and lower cover center are on hollow design, pond body bottom
It is provided with slot, slot on the electro-conductive glass working electrode for conductive glass carbon or surface modification, the working electrode and pond body
Working electrode binding post is connected, and electrode perforations are provided with Ag/AgCl or saturated calomel reference electrode respectively on the upper lid
With platinum disk or platinum filament to electrode.
2. microbe film forming process online monitoring system according to claim 1, it is characterised in that described electrochemistry is anti-
Answer and be additionally provided with inlet and leakage fluid dram in the container for the polytetrafluoroethylmaterial material that tank main body is volume 5-50mL, the pond body,
Simultaneously provided with liquid injection port on upper lid, liquid injection port is provided with air plug.
3. microbe film forming process online monitoring system according to claim 1 or 2, it is characterised in that the working electrode
Area be 1-20cm2；Surface area to electrode is 1cm2。
4. a kind of microbe film forming process on-line monitoring method, it is characterised in that this method includes：
1) 1-20cm is placed to the electrode slot position of the electrochemical reaction cell pond body bottom described in claim 12Electro-conductive glass
Working electrode, Ag/AgCl or saturated calomel reference electrode, 1cm are inserted by the electrode perforations on upper lid into pond body2Platinum disk
Or platinum filament is to electrode, the pure bacterium of Anaerobic Treatment, sanitary sewage, anaerobic sludge, river bed bottom sediment are then added into pond body
Or the water outlet for the microbiological fuel cell mutually stablized through long-term domesticated organism, using concentration as 0.5-2g/L sodium acetate or grape
Sugar as substrate, using potentiostat chronoamperometry or when m- current method carry out continuous stream or the intermittent flow culture of microorganism；
2) time of running, light microscope is started, in real time record or use time sequential recording microbe film forming process, it is real
When output picture, graphical information, for the film forming situation and rule of any incubation time, to output image computer export three
Light field, metallographic and the fluoroscopic image information of dimension/two dimension, realize the joint fortune of electro-chemical systems unit and optical microphotograph imaging technique
OK.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107831108A (en) * | 2017-11-24 | 2018-03-23 | 北京科技大学 | A kind of electrochemical test method and device based on automatic film covering Yu micro- liquid pool |
| CN111751334A (en) * | 2020-07-28 | 2020-10-09 | 广州大学 | Photoelectrochemistry detection pool |
| CN112067853A (en) * | 2020-08-28 | 2020-12-11 | 中国科学院城市环境研究所 | Microorganism modification scanning photoelectrochemical microscope system and imaging method thereof |
| CN113621494A (en) * | 2021-07-13 | 2021-11-09 | 浙江工商大学 | Visual biomembrane culture reactor and in-situ observation method |
| CN114414645A (en) * | 2022-01-19 | 2022-04-29 | 河北工业大学 | Device and method for monitoring bacteria/cell growth |
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Application publication date: 20170905 |