CN103323433A - Shaking bed device of non-contact-type multichannel online detection fermentation liquid as well as testing method - Google Patents
Shaking bed device of non-contact-type multichannel online detection fermentation liquid as well as testing method Download PDFInfo
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- CN103323433A CN103323433A CN2013102464805A CN201310246480A CN103323433A CN 103323433 A CN103323433 A CN 103323433A CN 2013102464805 A CN2013102464805 A CN 2013102464805A CN 201310246480 A CN201310246480 A CN 201310246480A CN 103323433 A CN103323433 A CN 103323433A
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Abstract
The invention provides a shaking bed device of non-contact-type multichannel online detection fermentation liquid as well as a testing method. The shaking bed device of the non-contact-type multichannel online detection fermentation liquid is characterized in that a shaking bed is provided with n testing bottles filled with fermentation liquid; a laser emitter is arranged at one side of each testing bottle, and a laser receiver is arranged at the other side of each testing bottle; the output of each laser receiver is connected with a data collecting and processing module connected with a computer so as to form n testing channels; the computer draws n microorganism growth processes filled with different fermentation liquid respectively into curve lines, and monitors the growth process of microorganisms in each testing bottle in real time; the bottom of each testing bottle is provided with a cylindrical body. The shaking bed device of non-contact-type multichannel online detection fermentation liquid as well as the testing method have the advantages that each growth process of microorganisms in each testing bottle on the shaking bed can be observed visually through the curve line graphical representation conveniently in real time, and the scientific research and microorganism product production efficiency are improved greatly.
Description
Technical field
The present invention relates to the biochemical instrument technical field, particularly relate to a kind of n test bottle that fills fermentation liquor that in shaking table device, arrange, device and the method for testing of microorganism growth process in each test bottle of Real-Time Monitoring.
Background technology
At present, fermentation liquor microorganism growth process in the test bottle on the domestic monitoring shaking table all is to adopt the off-line test method.Way is to fermentation liquor timing sampling in the single test bottle, is transplanted in the spectrophotometer cell, then tests its transmittance and optical density (being commonly referred to as absorbance A in the spectrophotometer), and measured value and time relationship are drawn.This method is complete manual operations, can not detect in real time online, and is very time-consuming again.
Summary of the invention
For solving the problem of aforementioned existence, the invention provides shaking table device and method of testing that a kind of contactless hyperchannel detects fermentation liquor online, the shaking table device that hyperchannel detects fermentation liquor online is, be provided with n test bottle that fills fermentation liquor on the shaking table, each test bottle one side is provided with generating laser, opposite side is provided with laser pickoff, the output of laser pickoff is connected with the data acquisition and processing (DAP) module, the data acquisition and processing (DAP) module is connected with computing machine, consist of n test channel, computing machine is depicted as respectively curve with n microorganism growth process that fills different fermentations liquid, microbial growth process in each test bottle of Real-Time Monitoring.
One section right cylinder is arranged at the bottom of described test bottle.
The method of testing that hyperchannel detects the shaking table device of fermentation liquor online is:
(1) numbering of n passage is set, default n passage maximum optical density value, startup shaking table 50-250rps;
When (2) measurement laser is not lighted, N mean value of the minimum output voltage value of the laser pickoff of each passage;
(3) light generating laser;
(4) N mean value of the maximum voltage value of the output of the laser pickoff of n passage of collection measurement;
(5) gather the real-time voltage value of measuring n passage laser pickoff output;
(6) transmittance of microorganism in the calculating test bottle;
Transmittance=((real-time voltage value-minimum voltage mean value)/(the maximum voltage mean value-little mean value of voltage)) * 100;
(7) the optical density OD value of microorganism in the calculating test bottle;
Optical density (OD)=LOG(maximum transmission rate/transmittance);
Maximum transmission rate=100%;
(8) draw microorganism optical density temporal evolution curve in each lane testing bottle;
(9) reach default experimental period or optical density value, off-test, save data;
(10) do not reach default experimental period or optical density value, return step (5).
The optical maser wavelength of generating laser output is generally selected 600nm or 660nm, because itself do not absorb luminous energy in the many microorganisms of the light wave of this wavelength, only reflects or diffuse lightwave.Also can be different according to microorganism property, select the excitation source of other wavelength, so this experiment is the turbidity of having measured microorganism, but getting used to optical density OD, the microbe research personnel represent, be identical physical concept with absorbance A that spectral analysis is used.
Optical density does not have dimensional unit, is a logarithm value, and optical density is the logarithm of incident light and transmitted light ratio or perhaps the logarithm of light penetration inverse.Computing formula is OD=log10(incident light/transmitted light) or the OD=log10(1/ transmittance);
The optical maser wavelength of generating laser output is generally selected 600nm or 660nm, because itself do not absorb luminous energy in the many microorganisms of the light wave of this wavelength, only reflects or diffuse lightwave.Also can be different according to microorganism property, select the excitation source of other wavelength, so this experiment is the turbidity of having measured microorganism, but getting used to optical density OD, the microbe research personnel represent, be identical physical concept with absorbance A that spectral analysis is used.
Advantage of the present invention is, can illustrate by curve, each process of growth of microorganism in each test bottle on the shaking table directly perceived, in real time, conveniently, the experimental provision of robotization can be provided for microbe research, also can provide optimization data and automatically control information for microniological proudcts production.Scientific research and microniological proudcts production efficiency have greatly been improved.
Description of drawings
Accompanying drawing 1 is microorganism fungus kind increment curve.
Accompanying drawing 2 is block schemes of the present invention.
Accompanying drawing 3 is test bottle stereographic maps of the present invention.
Accompanying drawing 4 is schematic diagram (arrow signal laser sees through test bottle among the figure) that fermentation liquor is arranged in the test bottle of the present invention.
Accompanying drawing 6 shows it is switching regulator laser diode driver circuit figure.
Accompanying drawing 7 is three kinds of fermentation process optical density curve figure.
Embodiment
See also shown in the accompanying drawing 2, be provided with n test bottle 2 that fills fermentation liquor on the shaking table, each test bottle 2 one side is provided with generating laser 1, and generating laser 1 adopts laser diode, and wavelength is 600nm(or 660nm); Opposite side is provided with laser pickoff 3, adopts silicon photoelectric diode, and wavelength is 550-750nm or joins narrow band pass filter; The output of laser pickoff 3 is connected with data acquisition and processing (DAP) module 4, data acquisition and processing (DAP) module 4 is connected with computing machine 5, consist of n test channel, computing machine 5 is with n microorganism growth process that fills different fermentations liquid, be depicted as respectively curve, microbial growth process in each test bottle of Real-Time Monitoring.
In biotechnology, shaking table is research tool, also is the tool of production.Change in the various chemical physics parameters that need to monitor sample aspect growth of microorganism sweat and the biological medicine development, and wherein, spectrodensitometry is exactly an emphasis, because it is the variation of biomass in the monitoring biological growth sweat, show and judge the feature of the stages of biological respinse, seek the important evidence of best biochemical reaction pattern and optimization industry mathematical model; Example as shown in Figure 1, fermentation process has four-stage, number in the figure is:
-viable count ... total bacteria count
A-lag phase; B-increased logarithmic phase;
C-stationary phase; D-decline phase;
In scientific research, by the concentration change in the Real-Time Monitoring growth of microorganism sweat, just can declare and distinguish the residing stage of microbial fermentation, seek the biochemical reaction route.
On producing, in order to improve productive rate, in conjunction with the measurement of other the physical-chemical parameters, set up and optimize Biochemical Model, in order to instruct and the production control process.
Biological products are that 600nm or 660nm place do not have absorption spectrum at wavelength mostly, so usually use this in twos optical density of a wavelength detection microorganism.Owing to during shaking table work, being in the ceaselessly motion (vibration or rotation), therefore photo densitometry having been increased difficulty: 1. under vibration condition, guarantee the mechanical stability of whole optical system; 2. reduce the container tube wall to the refraction of incident light source; 3. reduce the interior liquid fluctuating of container to reflection and the refraction of light; 4. overcome fluid to be measured inner cell particulate to the scattering of light; 5. the focusing of light source device, power selection and temperature stability control; 6. the sensitivity homogeneity of the stability of electrooptical device and light-sensitive surface; So in real time long-term work under this environment, to have optical density testing apparatus stable, accurate, good reproducibility be very important.
For this reason, when using shaking table to carry out Fermentation Experiment or producing, generally adopt the commodity conical flask to hold nutrient solution.In order to guarantee the dissolved oxygen amount of microorganism in the growth sweat, can only in bottle, add a small amount of nutrient solution (in 30%).So there are the following problems: the 1. optical density (OD600 or OD660) in order to measure nutrient solution, just must put the horizontal position of LASER Light Source very low, otherwise at liquid level constantly in the fluctuation, the light liquid level of will overflowing, cause change in optical path length, produce the detection signal fluctuation.2. excessively low when the laser light source horizontal position, light just may be in the curved surface position at the bottom of the conical flask, so after light incident, refraction or scattering through this position glass defocus, and pass to the detecting device end face and have just become a bar shaped speckle.If 3. add again sample, in the container of sample in rocking, can produce liquid fluctuating, just as mirror, the light scattering four directions, this strip light spots more will enlarge, and exceeds the receiving plane of detecting device, causes larger detection signal fluctuation.Therefore, for above-mentioned problem and shortage, the present invention has developed a kind of Novel tapered bottle.Fig. 3 is the structure of conical flask of the present invention, and one section right cylinder is arranged at the bottom of bottle among the figure, and laser sees through from this right cylinder, can not produce the refraction of light.Fig. 4 has a small amount of nutrient solution in the bottle, laser sees through from the bottom nutrient solution of bottle, and this Novel tapered bottle does not reflect or dispersion effect laser.
Generating laser is the light source of a branch of single wavelength, utilizes micro lens to focus on, and is that 2-4mm is adjustable at the focus diameter from light source outlet 100mm; Microbiological specimens absorbs, stops or scattering in the laser beam process conical flask, and the luminosity that arrives at last silicon photoelectric diode 3 is converted into electric current.
If laser luminosity=Iin
By photometry luminosity=Iout
Absorbed light=Ia
Luminosity=the Id of scattered light and the light that is blocked
So Iout=Iin-(Ia+Id)
Select the laser of 600nm and 660nm wavelength as exciting light, Ia=0 then is so stopped by microorganism or the luminosity of scattering is Id.According to the photometer computing formula,
OD=Log (Iout/Iin) light path is 10mm
Consider the impact of light path, OD=f(Iout, Iin, S) rate
S-light path
In general spectroscopic instruments, light path mostly is greatly 10mm.And the light path of Real-Time Monitoring shaking table is relevant with the shaking flask capacity, is as the criterion with the 250mL shaking flask, and its light path is about 100mm, and this will increase by 10 times of exciting light power.Bring the laser tube heating and increase power, reduce and continue the stable of service time and luminosity.Therefore, must select the laser drive circuit with power stability.Perhaps, adopt switch control LASER Light Source.Fig. 5 is the laser diode drive schematic diagram of power stability; Fig. 6 switching regulator laser diode driver circuit.
Triangular flask is not only the microbial reaction container and has been become a optical device in the whole optical system, a chamfering curved surface is arranged at general commercial triangular flask bottom, just in time be in incident beam into and out of the zone, this curved surface is to the refraction of light beam, when adding cradle vibrate, the reflection of microorganism liquid level or refraction, to cause photoelectric detecting signal seriously to be beated, Fig. 3, the 4th, the improved test bottle of the present invention, one section right cylinder is arranged at the bottle bottom, laser sees through from cylinder, can not produce the refraction of light.
Laser pickoff uses silicon photoelectric diode, and its dark current is little, and the range of linearity is wide, good stability; For fear of additional detection voltage drift, only need the good equipment circuit of performance to follow output; And in order to prevent the interference of shaking table work tool on opportunity vibration, select to detect surface area large, the uniform silicon photoelectric diode of the light sensitivity of light-sensitive surface.
Data acquisition unit will be selected the A/D collector of 12 above resolution at least.During shaking table work, because the microorganism base fluid fluctuates in container, cause the optical density detection signal to rise and fall, therefore can not adopt the high-speed AD circuit, and should use double integrator or delta-cgma low speed analog to digital converter (current employing MSP-020102,24 bit analog to digital converter);
Concrete test findings:
Further illustrate summary of the invention below in conjunction with embodiment, but these examples do not limit protection scope of the present invention.
1) select shaking table: intelligence city analytical instrument Manufacturing Co., Ltd produces ZWY-100H
2) temperature: 37 ℃
3) rotating speed: 150-240rps
4) microbiological specimens: Escherichia coli; Agrobacterium and Shanghai Communications University's Onlly microbiological specimens
5) nutrient culture media: the unified identical goods nutrient culture media that uses
6) port number: 1-4
For optimizing the microbial growth fermentation condition, choose suitable nutrient culture media, cultivate three microorganism fungus kinds with apparatus of the present invention by triple channel: Escherichia coli, Agrobacterium and Onlly bacterial classification.Incubation time is 20 hours, optical density Real-Time Monitoring result in three growth sweats, process data acquisition and transport module collection also is transferred to terminal, carrying out data analysis processes, the optical density data that obtain at last microorganism real-time growth sweat in nutrient solution see Table-1, and the optical density temporal evolution curve of drafting is seen Fig. 7.
The optical density of showing in-1 three kind of microorganism fungus kind sweat changes (automatically generating excel spreadsheet):
| Time (dividing) | |
|
|
| 11.4 | 0 | 0.003 | -0.001 |
| 47.4 | 0 | 0.019 | -0.001 |
| 83.5 | -0.001 | 0.047 | -0.001 |
| 95.5 | -0.001 | 0.06 | -0.001 |
| 131.5 | -0.001 | 0.111 | -0.001 |
| 167.6 | -0.001 | 0.19 | -0.001 |
| 203.6 | -0.001 | 0.313 | -0.001 |
| 239.7 | 0 | 0.501 | -0.001 |
| 275.8 | 0.001 | 0.754 | -0.001 |
| 311.8 | 0.047 | 1.031 | -0.001 |
| 347.9 | 0.334 | 1.27 | -0.001 |
| 383.9 | 0.719 | 1.405 | 0.001 |
| 420 | 1.034 | 1.502 | 0.033 |
| 456 | 1.207 | 1.586 | 0.203 |
| 492.1 | 1.324 | 1.644 | 0.471 |
| 528.1 | 1.416 | 1.695 | 0.8 |
| 564.2 | 1.494 | 1.738 | 1.124 |
| 600.2 | 1.557 | 1.776 | 1.312 |
| 636.3 | 1.611 | 1.801 | 1.424 |
| 672.3 | 1.659 | 1.827 | 1.513 |
| 708.4 | 1.706 | 1.848 | 1.582 |
| 744.4 | 1.749 | 1.874 | 1.64 |
| 780.5 | 1.785 | 1.896 | 1.687 |
| 816.5 | 1.819 | 1.921 | 1.73 |
| 852.6 | 1.852 | 1.942 | 1.77 |
| 888.7 | 1.877 | 1.952 | 1.803 |
| 924.7 | 1.905 | 1.965 | 1.836 |
| 960.8 | 1.917 | 1.966 | 1.858 |
| 996.8 | 1.927 | 1.96 | 1.875 |
| 1032.9 | 1.938 | 1.963 | 1.892 |
| 1068.9 | 1.926 | 1.927 | 1.883 |
| 1105 | 1.926 | 1.92 | 1.89 |
| 1141 | 1.932 | 1.918 | 1.899 |
| 1177.1 | 1.94 | 1.922 | 1.912 |
| 1201.1 | 1.94 | 1.921 | 1.918 |
The clear optical density of microorganism in shaking table growth sweat that shown of this experimental result changes (that is microorganism concn variation), the feature that has reflected well the Main Stage of microbial fermentation, for research and production provide guidance and reference function, also simplified simultaneously operating process, saving the experimenter needs tens hours timing samplings (general one hour collecting sample once), then tests OD600 at spectrophotometer.This has not only improved work efficiency, has reduced researchist's working strength, particularly importantly, can provide optimized mathematical model for the industrialization of biotechnology research achievement, for the continuous automatic control of microbial fermentation or biochemical reaction lays the foundation.
Claims (3)
1. a contactless hyperchannel detects the shaking table device of fermentation liquor online, it is characterized in that, be provided with n test bottle that fills fermentation liquor on the shaking table, each test bottle one side is provided with generating laser, opposite side is provided with laser pickoff, the output of laser pickoff is connected with the data acquisition and processing (DAP) module, the data acquisition and processing (DAP) module is connected with computing machine, consist of n test channel, computing machine is with n microorganism growth process that fills different fermentations liquid, be depicted as respectively curve, microbial growth process in each test bottle of Real-Time Monitoring.
2. detect online the shaking table device of fermentation liquor by contactless hyperchannel claimed in claim 1, it is characterized in that, one section right cylinder is arranged at the bottom of described test bottle.
3. detect online the method for testing of the shaking table device of fermentation liquor by contactless hyperchannel claimed in claim 1, may further comprise the steps:
(1) numbering of n passage is set, default n passage maximum optical density value, startup shaking table 50-250rps;
When (2) measurement laser is not lighted, N mean value of the minimum output voltage value of the laser pickoff of each passage;
(3) light generating laser;
(4) N mean value of the maximum voltage value of the output of the laser pickoff of n passage of collection measurement;
(5) gather the real-time voltage value of measuring n passage laser pickoff output;
(6) transmittance of microorganism in the calculating test bottle;
Transmittance=((real-time voltage value-minimum voltage mean value)/(the maximum voltage mean value-little mean value of voltage)) * 100;
(7) the optical density OD value of microorganism in the calculating test bottle;
Optical density (OD)=LOG(maximum transmission rate/transmittance);
Maximum transmission rate=100%;
(8) draw microorganism optical density temporal evolution curve in each lane testing bottle;
(9) reach default experimental period or optical density value, off-test, save data;
(10) do not reach default experimental period or optical density value, return step (5).
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120073992A1 (en) * | 2010-09-24 | 2012-03-29 | Kim Jae-Ho | Biosensor based on carbon nanotube-electric field effect transistor and method for producing the same |
| CN102519898A (en) * | 2011-12-29 | 2012-06-27 | 上海智城分析仪器制造有限公司 | Device utilizing single light source to detect fermentation liquid of shake table |
| CN102539366A (en) * | 2011-12-29 | 2012-07-04 | 上海智城分析仪器制造有限公司 | Device for detecting swinging bed fermentation liquid by double light sources |
| CN202415551U (en) * | 2011-12-29 | 2012-09-05 | 上海智城分析仪器制造有限公司 | Shake flask for photoelectric detection of fermentation liquor on rocking device |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120073992A1 (en) * | 2010-09-24 | 2012-03-29 | Kim Jae-Ho | Biosensor based on carbon nanotube-electric field effect transistor and method for producing the same |
| CN102519898A (en) * | 2011-12-29 | 2012-06-27 | 上海智城分析仪器制造有限公司 | Device utilizing single light source to detect fermentation liquid of shake table |
| CN102539366A (en) * | 2011-12-29 | 2012-07-04 | 上海智城分析仪器制造有限公司 | Device for detecting swinging bed fermentation liquid by double light sources |
| CN202415551U (en) * | 2011-12-29 | 2012-09-05 | 上海智城分析仪器制造有限公司 | Shake flask for photoelectric detection of fermentation liquor on rocking device |
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