KR20000031934A - Automatic water toxicant measuring instrument using immobilized photogenic microorganism - Google Patents

Abstract

PURPOSE: An automatic water toxicant measuring instrument is provided which measures the light emission of photogenic microorganisms induced by toxicants, in which pretreatment of photogenic microorganisms is not involved so, the apparatus measures water toxicant easily and economically. CONSTITUTION: An instrument for measuring water toxicant using photogenic microorganisms comprises the following parts: 1) a stage driving part consisting of plural vials(21) which contains test samples and immobilized microorganisms, a movable X-Y stage(23), a sub-driver(24) connected to the stage, and a controller(25) connected to the sub-driver for controlling the position of the stage; 2) a sample supplying part consisting of a sample inlet(26), a test sample storage plant(28), of which one side is connected to an automatic sample collector(27) and other side is connected to an outlet of water(29), and a sample inlet controller(30) connected to the sample inlet; 3) a luminescence intensity measuring part; and an arithmetic and control part. Immobilized microorganisms are as follows; Photogenic microorganisms such as Photo bacterium phosphoreum, Vibrio fischeri, or recombinant microorganisms with lux gene are immobilized on porous matrix such as sodium alginate, strontium alginate, cappa-carrageenan, polyacrylamide, cellulose or agarose. Sample water is sequentially added to aligned vials containing immobilized photogenic microorganism and the difference of luminescence intensity is measured by luminescence dosimeter before and after the sample injection.

Description

Automatic Water Toxicity Measurement Device Using Immobilized Luminescent Microorganisms

The present invention relates to a method for measuring a substance by a biological method and an apparatus therefor, more particularly, a method for automatically measuring a toxic substance using a luminescent microorganism and an automatic early warning device therefor.

The method for measuring toxic substances according to the present invention and the automatic alarm device for the same are installed in a water supply protection zone in addition to operating as a rapid alarm system in an emergency water pollution accident, and cope with unauthorized discharge of private companies and facilities, and installed in a military protection zone. Quickly determine if hostile biochemical weapons are used. It can also regulate the biological toxicity of sewage and wastewater treatment plants and use it to quickly determine whether contaminants enter the drinking water intake. In particular, it can be an important means to prevent and solve the recent serious environmental pollution problem by detecting and alarming toxic substances in water system.

In order to improve the water pollution problem, which is one of various environmental problems, technologies related to the water environment have been developed a lot, but the pollution of water systems such as water supply, river water, lakes, wastewater, and sewage is serious, and outbreaks such as wastewater leakage The necessity of preparing for environmental accidents and monitoring of water quality is increasing day by day, and there is a great need for the development of a water toxicity measurement device that can prevent such damage.

Devices for early measurement and warning of wastewater outflows and other inflows of toxic substances in water have been developed by many researchers. Conventional methods for monitoring water toxicity include chemical water toxicity monitors and biological water toxicity monitors. Chemical water toxicity monitoring is limited by the number of substances that can occur in aquatic systems and can only identify a few substances, with very few substances being quantitatively analyzed. Such chemical analytical methods require the acquisition of quite expensive equipment and skilled related technologies. In addition, the flowing water in which the chemical conditions are constantly changing is less satisfactory because of the use and function of each type of water that can occur in a situation requiring rapid interference.

The development of biological water toxicity monitoring has continued to develop systems that complement chemical water toxicity monitoring. Representative biological water quality monitoring methods of the related art include water quality monitoring method using fish and water toxicity monitoring method using daphnia. In particular, biological alarm devices are installed in river water, sewage treatment plants, and wastewater treatment plants. In case of river water, it is installed to prevent the inflow of pollutants from the wastewater discharge facility and damage from sudden accidents and to monitor the river water.In the case of the sewage treatment plant, there are many pollutants. More serious. In addition, many sewage treatment plants are treating wastewater or leachate from landfills, so it is more necessary to monitor the water quality by installing a biological alarm system. Overseas, biological alarm systems are also installed in wastewater discharge facilities.

On the other hand, the water pollution monitoring network is established to establish an efficient water quality monitoring and water quality management system for maintaining and restoring lake and river water quality, and can be divided into automatic monitoring network and manual monitoring network. Passive monitoring network means a method to collect samples at a fixed location in a target water area for analysis in a laboratory, and automatic monitoring network continuously transfers collected samples to a measuring device and analyzes them automatically. Says how it is handled. The automatic water pollution monitoring network can automatically obtain continuous measurements on a large number of items, which is a great help in the rapid and reasonable response to sudden water pollution in the target water area. Automatic water quality measurement method for monitoring water pollution is necessary to accumulate data that can cope with water quality accidents with consistency, promptness, and continuous measurement, and to improve water quality management effect of water resources.

Biological methods of the conventional automatic automatic monitoring system for water pollution include an early warning device using fish and an early warning device using water fleas. Early warning device using a fish is a device that measures by using the property of going back to the water flow of the fish, that is, back oil. If harmful toxic substances are introduced through the water inlet (1), the fish will be damaged and the swimming will be reduced. As such, the fish that have lost swimming ability are pushed backwards without being able to overcome the flow rate, but because of the instincts of the fish, the tail fins are forced to move forward again. At this time, the tail fins touch the sensor (3), which is an electrical signal value. Appears and is recorded. Install a fish safety net (2) to prevent fish from escaping. This electrical signal value is measured in the water quality meter (4). In addition, a circulation pump 5 is used to regulate the flow of water through the water flow regulator (6). The water used in this way is discharged through the water outlet (8) and the water is controlled through the water separation pipe (7). It is used for alarm and flow rate control with the control device (9). This information is input and output via the monitor and keyboard 10. The fish used here are mainly carp and sulfur yellow gold fish.

However, the disadvantage of early warning devices using fish is that the size of the individual detecting the toxicity is large, so phenol takes about 8 hours when 10ppm is introduced. As described above, in the case of a biotoxic alarm using fish, the sensitivity is low, and the measurement time and the error range are large. Cultivation and selection of fish is also a prerequisite.

Early warning device using daphnia detects daphnia activity through infrared sensor. The water flea toxicity alarm is based on the flowability of the water flea, which will be described in detail with reference to FIG. 2, and the test chamber 12 made of glass or acrylic has water to be tested in and out, and there are 20 water fleas therein. When water flows in through the inlet 11, the water flea in the test chamber 12 reacts. The water flea shows a regular movement when the water is not contaminated with toxic substances, but the movement changes irregularly and rapidly when exposed to toxic substances. . The sharper the movement, the greater the number of times of blocking the infrared sensor 13, so that the electrical signal value increases. The temperature is always measured through the temperature sensing device 15 to control the infrared sensor through the electronic control device 16 and output the value through the output device 17 and the measured water is discharged through the water outlet 14. do. In the case of the early warning device using the water flea, since the size of the individual is smaller than the fish, sensitivity is superior to the device using the fish, but difficult to manage. In addition, it is necessary to wash or exchange various kinds of tubes for the test tank, influent, and effluent when replacing daphnia, and much effort is required for the cultivation of daphnia.It is necessary to make a culture medium and replace the culture medium 2-3 times a week. The focus is on separation. In addition, daphnia should be cultured in separate culture rooms, where no objects or equipment would interfere with the disinfection or cultivation of indoor spaces. In addition, there is a problem that the air of the chemical analysis room should not be transferred to the culture chamber.

In order to solve the above problems, the device of the present invention provides a device using a fixed light emitting microorganism capable of automatic early warning at any position, the breakthrough toxicity for fast, accurate measurement and low maintenance cost and easy maintenance It is to provide an automatic material early warning device. In addition, the biological analysis method using the light-emitting microorganisms to provide an effective method and apparatus for showing the biological toxicity of various trace toxic substances that are difficult to analyze even in physicochemical analysis.

1 is a water flea automatic water alarm using a conventional biological method.

Figure 2 is a fish water toxicity automatic alarm device using a conventional biological method.

Figure 3 shows an embodiment of a conventional toxic substance automatic early warning device according to the present invention.

Figure 4 is a detailed view of the sample input unit and the measurement unit of the automatic warning device for toxic substances according to the present invention.

** Description of Drawing Symbols **

1. Water inlet 2. Fish escape prevention net

3. Sensor 4. Water Quality Meter

5. Circulation Pump 6. Water Flow Controller

7. Water separation pipe 8. Water outlet

9. Control Unit 10. Monitor and Input Device

11. Water inlet 12. Test chamber

13. Infrared sensor 14. Water outlet

15. Temperature-sensing device 16. Electronic control device

17. Output device

21. Measuring Vials 22. Immobilized Luminescent Microorganisms

23. X-Y Stage 24. Servo Driver

25. Controller for Stage Position Control

26. Sample Feeder 27. Automatic Sampler

28. Sample reservoir 29. Water outlet

30. Sampler controller 31. Wash solution reservoir

35. Fiber Optic 36. Photometer

37. Computer 38. LCD Screen

The present invention relates to a method for measuring a toxic substance in an aqueous system by using a luminescent microorganism and an automatic measuring device therefor, and more particularly, to measure the change in the quantity of light of the immobilized luminescent microorganism by the toxic substance, and the toxicities are automatically controlled. It relates to a material automatic measuring device.

Specifically, the present invention includes a plurality of measurement vials 21 including the injected sample and the immobilized microorganisms on the upper surface, and a stage 23 made of a transparent substrate that is movable so that the sample can be introduced into the next measurement vials. A stage driver including a servo driver 24 connected to the stage for moving the stage, and a controller 25 for controlling the position of the stage connected to the servo driver;

A sample injector 26 located above the stage, an automatic sampler on one side, and a water outlet 29 on the other side, a sample reservoir 28 for delivering a sample to the sample injector, and a sample injector controller connected to the sample injector ( A sample supply unit consisting of 30);

An optical measuring unit positioned below the stage and configured to measure a change in quantity of light in the measuring vial;

An operation and control unit for controlling the stage position control controller 24 and the sample input controller 30 so that samples are sequentially input to a plurality of measurement vials, and calculating concentrations of toxic substances due to light quantity change results of an optical measuring unit;

The present invention relates to an apparatus for automatically measuring the concentration of toxic substances using an immobilized luminescent microorganism.

In addition, if the measured concentration of the toxic substance is above the set value may include a method for alarm using the alarm device.

The present invention includes an immobilized luminescent microorganism in the interior of a measurement vial, and the immobilized microorganism includes a photobacterium phosphoreum, Vibrio fischeri or a gene encoding a luminescent enzyme thereof. It may be a recombinant strain introduced into the host microorganism.

The immobilized luminescent microorganism may be an immobilized luminescent microorganism immobilized on a porous substrate selected from sodium alginate, strontium alginate, kappa-carrageenan, polyacrylamide, cellulose, agarose. Alginates are preferred as immobilization substrates.

The toxic substance concentration automatic measuring device of the present invention is further provided with a device for alarming when the measured toxic substance concentration is above the set value.

The optical measuring device of the automatic toxic substance concentration measuring device according to the present invention may be an optical amplifier or a photodiode, and the change in the amount of light in the measuring vial may be transmitted to the optical measuring device or directly measured by the optical measuring device. . In other words, the measurement of the toxic substance uses a photometer to convert the change in the amount of light into the amount of charge and send it to the calculation means, the calculation means analyzes and stores the amount of charge sent. At this time, the change of the charge amount can be analyzed by gamma method, specific velocity method, and bioluminescence method to determine the concentration of toxic substances.

Measuring and analyzing the change in the amount of light may be converted to the amount of charge by using a photometer to the amount of charge and analyzed and stored by the calculation means.

The measured values are analyzed by EC ₅₀ , specific rate method, bioluminescence intensity method, etc. and displayed by performing storage function and calculation function in the calculation means. These values are output through an output means such as a liquid crystal display, a printer port or a communication port.

Herein, the difference in emission amount change is calculated through a specific velocity method, a bioluminescence method, a gamma method, and the like, and the value calculated here has a value close to a straight line. At this time, the calculation method is as follows.

a) using specific rates

As a method of measuring the amount of bioluminescence generated over a period of time,

μ = (lnL ₂ -lnL ₁ (t ₂ -t ₁ )

μ: specific velocity

t ₁ : initial measurement time

t ₂ : second measurement time

L ₁ : first bioluminescence measurement

L ₂ : second measured bioluminescence

b) bioluminescence intensity

I _o / I = I + Ks [Q]

Io: Bioluminescence without toxic substances

I: Bioluminescence without toxic substances

Ks: constant

[Q]: Toxic Concentration

c) gamma value

The ratio of the amount of light reduced to the amount of light remaining

γ (t, T): Gamma effect during t hours at T ° C

R (t): This is obtained by dividing the ratio of the bioluminescence value of the sample (blank) in which no toxic substance is not introduced for t hours and the value of the first measurement of the bioluminescence value of the sample that is not toxic substance introduced after t hours.

L (0): bioluminescence of the sample at the first measurement

L (t): Bioluminescence after t hours.

The γ values at each concentration can be obtained and the concentrations and γ values of the toxic substances can be expressed in a log-log graph and can be expressed in a straight line. At this time, the concentration of the toxic substance at γ = 1 is the EC ₅₀ value. The value at this time is expressed differently according to each type of toxic substance. Using these characteristics, the value is obtained and an alarm is issued or displayed when the value exceeds EC ₅₀ .

In the toxic material automatic measuring device according to the present invention, the conventional method for activating free cells has a complicated and inconvenient operation, and uses a fixed luminescent microorganism to solve this problem. Toxic substances can be measured more sensitively and quickly by using immobilized microorganisms than when water toxic substances are measured in free cells.

Microorganisms usable for the measurement of toxic substances according to the present invention are luminescent microorganisms, specifically marine microorganisms that emit light. In this, as well as luminescent microorganisms, which are wild strains, recombinant strains in which lux genes are introduced into host microorganisms such as Escherichia coli can be used, and recombinant strains can be prepared by conventional methods. Luminescent microorganisms that can be used include Photobacterium phosphoreum and Vibrio fischeri.

Luminescence is usually achieved by the interaction of luciferases, reduced flavins and long chain aldehydes, which are part of the cell's electron transport system in the presence of oxygen. Flavin mononucleotide (FMN) is reduced by NADH, which reacts with luminase and emits light. Since the luminescence depends on the flow of electrons, the change in luminescence indicates a change in metabolic activity and the health state of the living body.

FMNH ₂ + O ₂ + RCHO → Luminous + FMN + H ₂ O + RCOOH

Luminescence is a light emission spectrum of Photobacterium Force Forum 4, which is one of light emitting microorganisms, ranging from 421 nm to 630 nm and exhibiting a maximum value at 490 nm, which is a visible light region. In fact, the color emitted is a light blue to green color. As mentioned earlier, such luminescence occurs in connection with aerobic respiration metabolism, but ATP production does not occur because energy is emitted by light. If any toxic substance acts on this process, the amount of emitted light decreases. Using this principle, the toxicity of toxic substances can be measured. Unlike most other microorganisms that cannot grow at low temperatures, Photobacterium Force Forum (4) has the advantage of being able to grow at low temperatures of 4 ° C, allowing long-term storage at low temperatures and monitoring of relatively low temperature water quality. can do.

At this time, the microorganisms were immobilized to add mobility and accuracy. The immobilization method can be any conventional immobilization method, but is particularly preferably a method of capturing microorganisms inside the porous gel to limit free movement of cells. By immobilization, it is possible to maintain a stable and active biocatalyst for a long time, increase the reaction speed, and increase productivity per unit volume.

In order to add mobility and accuracy, in the present invention, immobilized light emitting microorganisms are used, and as for the method of immobilizing luminescent microorganisms, all conventionally known immobilization methods can be used, and in particular, microorganisms are trapped inside the porous gel to restrict free movement of cells. By immobilization, it is possible to maintain stable and active biocatalyst for a long time, increase the reaction speed, and increase productivity per volume. For this purpose, the choice of immobilization substrate is important, and immobilization substrate should be insoluble, not chemically react with toxic substances or decompose, have good dispersibility, low cost and excellent permeability. Examples of immobilized substrates satisfying such requirements include starch, alginate, carrageenan, and the like, and alginate is most preferred. Alginate is relatively simple to immobilize, the cells survive in the immobilized material and are harmless to the human body so that they can be used as food additives. It also helps to maintain the luminescence metabolism rather than harm it.

In order to cure alginate, calcium ions, potassium ions, strontium ions, etc. are used, and the principle of hardening is that alginate is composed of D-mannuronic acid and L-gluronic acid group, and L-glunic acid is calcium ion. And strontium ions.

Sodium alginate immobilization method incubated cells incubated for 12 to 14 hours after inoculation in an appropriate ratio (100-10.000 times) in saline solution of 2.5% (W / V), followed by microbial dilution and 5.0% (W / V). Sodium alginate of) in a ratio of 1 to 8 to immobilize.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings.

Water automatic toxicity measuring apparatus of the present invention is shown in Figure 3,

XY stage 23 made of a transparent substrate which is provided with a plurality of measurement vials 21 including the injected sample and immobilized microorganisms on the upper surface and which can be moved to the next measurement vials, and the stage is moved. A stage driver including a servo driver 24 connected to the stage and a stage position control controller 25 connected to the servo driver;

A sample injector 26 located above the stage, an automatic sampler 27 on one side and a water outlet 29 on the other side, and a sample reservoir 28 for delivering the sample to the sample injector, a sample connected to the sample injector. A sample supply unit comprising an input controller 30;

An optical measuring unit located below the stage and connected to the computer 37 for measuring a change in quantity of light in the measuring vial;

Toxicity consisting of a computer that controls the stage position control controller 25 and the sample input controller 30 so that samples are sequentially introduced into a plurality of measurement vials, and calculates the concentration of the toxic substance due to the light quantity change result of the light measuring unit. It is a material automatic measuring device.

Referring to the operation of the automatic toxic substance measuring device, in order to measure the toxic substances introduced into the water system, first, the sample is taken from the water system in the automatic sample retaker 27 to take a sample from the water source such as water supply and river water. . The introduced water exits through the sample reservoir 28 to the water outlet 29. In addition, at this time, a part of the sample is collected once every 30 minutes and is input to the measuring vial 21 through the sample injector 26. The measuring vial is fixed to the X-Y stage, and the X-Y stage 23 moves horizontally once every 30 minutes, and once the lines are sampled, the measuring vial moves vertically to move the next measuring vial under the sample feeder. The measuring vial is 48 rows vertically and 20 rows on the X-Y stage, allowing up to 20 days of unmanned operation. Since the X-Y stage is driven by the servo motor, it is controlled by the servo driver 24 and the servo driver is driven by the position control controller 25. Further, the adjustment of the sample feeder causes the command of the personal computer 37 to drive the sample feeder controller 30. All settings here are controlled via software on the personal computer.

When the sample is put into the measuring vial 21, the light starts to decrease when the toxic substance is introduced by the immobilized light emitting microorganism 22. The bottom plate of the X-Y stage is a transparent plate that allows light to pass through. The light thus emitted enters the optical measuring device through the optical fiber. The amount of light measured in this way is determined to determine the inflow of toxic substances.

In addition, if the measured amount of light drops more than 20% in 1 minute, it is an toxic substance introduced and an alarm is issued.

In this case, the difference of emission amount according to the inflow of water toxic substance is analyzed by specific velocity method, bioluminescence emission method and gamma method, and is displayed by performing storage function and arithmetic function in microprocessor. These values are output on the LCD screen and output through the printer port or the communication port.

The present invention provides a method for measuring a toxic substance using an immobilized luminescent microorganism and an automatic toxic substance measuring device. In particular, by using an immobilized luminescent microorganism, a separate pretreatment step for a luminescent microorganism is not required, so it is quick and easy to use and economical toxicity It is an advantage to provide a material analysis apparatus and method. In addition, it is an effective device and method showing the biological toxicity of various trace toxic substances that are difficult to analyze in physicochemical analysis.

Claims (7)

A stage 23 made of a transparent substrate having a plurality of measurement vials 21 including an injected sample and an immobilized light emitting microorganism 22 on an upper surface thereof and movable to allow the sample to be introduced into the next measurement vials; A stage driver including a servo driver 24 connected to the stage for moving the stage, and a controller 25 for controlling the position of the stage connected to the servo driver; A sample injector 26 located above the stage, an automatic sampler 27 on one side and a water outlet 29 on the other side, and a sample reservoir 28 for delivering the sample to the sample injector, a sample connected to the sample injector. A sample supply unit comprising an input controller 30; An optical measuring unit positioned below the stage and configured to measure a change in quantity of light in the measuring vial; A control unit for controlling the stage position control controller 24 and the sample input controller 30 so that the samples are sequentially input to a plurality of measurement vials, and calculating the concentration of the toxic substance due to the light quantity change result of the light measuring unit; 37); Automatic measuring device of concentration of toxic substances using immobilized luminescent microorganisms. The apparatus of claim 1, further comprising a device for alarming when the measured concentration of the toxic substance is above a set value. The method of claim 1, wherein the immobilized luminescent microorganism is a recombinant bacterium introducing a photobacterium phosphoreum, Vibrio fischeri or a lux gene encoding a luminescent enzyme thereof into a host microorganism. Automatic concentration measuring device of toxic substance characterized in that. The method of claim 3, wherein the immobilized luminescent microorganism is an immobilized luminescent microorganism immobilized on a porous substrate selected from sodium alginate, strontium alginate, kappa-carrageenan, polyacrylamide, cellulose, agarose. Automatic measuring device for concentration of toxic substance. The apparatus of claim 1, wherein the photometer is an optical amplifier or photodiode. 6. The apparatus of claim 5, further comprising an optical fiber for transmitting a change in quantity of light in the measuring vial to the photometer. The apparatus of claim 1, wherein the control operation unit calculates the concentration of the toxic substance by using the gamma method, the specific velocity method, and the bioluminescence method.