KR100337943B1 - Multi-channel device for continuously monitoring toxicity in water and method for monitoring toxicity in water using same - Google Patents

Abstract

The present invention relates to a multi-channel continuous underwater toxicity detection device for efficiently detecting toxicity in water and a method for detecting underwater toxicity using the same, the device according to the present invention comprises (a) a filtration means of a water sample, (b) (i) A method for culturing a recombinant microorganism having luminescent properties with respect to toxic substances, the first reactor equipped with an outlet of the culture medium and an optical fiber, and (ii) for detecting toxic substances, the microbial culture solution from the outlet of the first reactor. At least four two-stage reactors consisting of an inlet, an inlet of the water sample exiting the filtering means and a second reactor with an optical fiber, and (c) a photodetector connected respectively to the optical fibers of the first and second reactors. In the method of detecting underwater toxicity using the apparatus of the present invention, (A) after filtering the water sample, (B) the filtrate to toxicity After a year each of the different recombinant microorganism having a light-emitting property added to each of four or more reactors are continuously fed to, (C) includes a step of detecting the light emitted from each reactor.

Description

Multi-channel continuous underwater toxicity detection device and underwater toxicity detection method using the same {MULTI-CHANNEL DEVICE FOR CONTINUOUSLY MONITORING TOXICITY IN WATER AND METHOD FOR MONITORING TOXICITY IN WATER USING SAME}

The present invention relates to an underwater toxicity detection device for efficiently detecting various toxicity in water and a method for detecting underwater toxicity using the same.

Various toxic substances damaging genetic materials, cell membranes, proteins, etc., which are essential for living organisms, are being introduced into wastewater treatment plants, rivers, rivers, and water supplies. Therefore, detection of these toxic substances is very important to properly cope with them.

Conventionally, a method using water creatures such as daphnia and fish has been used for detection of toxic substances in water. This method is to investigate the death of aquatic organisms due to toxicity, but it does not only delay the time required for the detection of toxicity, but also does not distinguish the types of toxicity.

As a method without these disadvantages, a method using underwater microorganisms or recombinant microorganisms that emit light in response to specific toxicity has been attempted. This method is to investigate the luminescence response of the microorganisms due to toxicity, which has the advantage of fast detection speed and biological toxicity. However, this method has the disadvantage of being inefficient due to the killing of microorganisms or the inhibition of growth due to toxic substances.

In order to improve this disadvantage, the present inventors have developed and patented a two-stage reactor consisting of a first reactor for culturing recombinant microorganisms and a second reactor for detecting toxicity in water while continuously receiving microorganisms therefrom (patent Application No. 98-3621). However, the two-stage reactor has a limitation in that it cannot detect other toxicity by using only one reactor for detecting specific toxicity, so there is a problem that the effectiveness is less.

Accordingly, it is an object of the present invention to provide an apparatus for efficiently detecting various toxicities in water.

Another object of the present invention is to provide a method for detecting various toxicity in water using the device.

1 is a schematic diagram showing an example of the underwater toxicity detection apparatus according to the present invention.

2 is a schematic diagram showing a channel unit reactor used in the apparatus of the present invention.

3 (a) and 3 (b) are graphs showing the results of luminescence reaction for each channel of the device of the present invention by mitomycin C.

4 (a) and 4 (b) are graphs showing the results of luminescence reaction for each channel of the device of the present invention by cerulein.

5 (a) and 5 (b) are graphs showing the results of luminescence reaction for each channel of the device of the present invention by phenol.

<Explanation of symbols for main parts of the drawings>

101: nutrient medium inlet 102: culture medium outlet

103: air inlet 104: microorganism inlet

105 gas outlet 106 condenser

107: filter 108: hot water inlet

109: hot water outlet 110: stirring rod

111: agitator 112: glass bulkhead

113: attachment means 114: optical fiber

115: photodetector 116: hot water jacket

201: culture solution inlet 202: culture solution and water outlet

203: air inlet 204: water inlet

205: gas outlet 206: condenser

207: filter 208: hot water inlet

209: hot water outlet 210: stirring rod

211 stirrer 212 glass bulkhead

213: attachment means 214: optical fiber

215: photodetector 216: hot water jacket

According to the above object, in the present invention

(a) means for filtering water samples,

(b) (i) for culturing recombinant microorganisms having luminescent properties for toxic substances, the first reactor having an outlet of the culture medium and an optical fiber, and (ii) detecting toxic substances, wherein At least four two-stage reactors consisting of a second reactor equipped with an inlet of a microbial culture from an outlet, an inlet of a water sample discharged from said filtering means, and an optical fiber, and

and (c) a photodetector connected to the optical fibers of the first reactor and the second reactor, respectively.

According to another object, in the present invention, after (A) the water sample is filtered, (B) the filtrate is added to each of four or more reactors continuously fed with each different recombinant microorganism having luminescent properties for toxicity, ( C) provides an underwater toxicity detection method comprising the step of detecting the light emitted from each reactor.

Hereinafter, the present invention will be described in detail.

The apparatus of the present invention is characterized by using a plurality of two-stage reactors continuously supplied with recombinant microorganisms having luminescent properties for a particular toxicity in order to efficiently detect all the various toxic substances contained in the water quality at the same time. By using such a plurality of reactors, multiplexing (multichannelization) of channels for detection of various toxic substances is possible.

Multichannelization of toxicity detection is achieved by configuring each reactor to emit luminescence at different toxicity and connecting photodetectors to each reactor.

Toxicity that can be detected by the device of the present invention, depending on the site damaged by the toxicity, toxicity that damages DNA (DNA toxicity), toxicity that damages the cell membrane (cell membrane toxicity), damage to proteins Toxicity (protein toxicity) and the like, but are not limited thereto.

For example, substances that cause such toxicity include mitomycin C and gamma rays as DNA toxic substances, cerulenin as a cell membrane toxic substance, ethanol, phenol and pentachlorophenol as protein toxic substances ( PCP), but the toxic substance in the present invention is not limited thereto.

Recombinant microorganisms having luminescent properties with respect to such toxicity include recombinant microorganisms which do not emit light under nontoxic conditions but those which emit light under specific toxic conditions and those which emit light under nontoxic conditions but decrease luminescence under toxic conditions.

Recombinant microorganisms that emit specific toxicity include, for example, E. coli DPD2794 (RecA :: luxCDABE, Dupont, USA) that emits DNA toxicity, and E. coli DPD2540 (FabA :: luxCDABE, Dupont, USA, which emits cell membrane toxicity ), And E. coli TV1061 (GrpE :: luxCDABE, ATCC 69315) which emits protein toxicity, but is not limited thereto. By using these recombinant microorganisms in appropriate combination, it is possible to determine the type of toxicity contained in the water.

Recombinant microorganisms whose luminescence is reduced by toxicity include, for example, E. coli GC2 (lac :: luxCDABE, obtained from Gwangju Institute of Science and Technology) and photobacterium phosphoreum (KCTC 2852). It is not limited to this. By using such recombinant microorganisms in appropriate combination with microorganisms that emit these toxicities, the level of toxicity to the microorganisms can be determined.

An example of a toxicity detection device according to the present invention is shown in FIG. 1, and with reference to FIG. 1, the device of the present invention comprises: (a) a filtration means for a water sample, and (b) a first method for culturing a recombinant microorganism. A two stage reactor consisting of a reactor and a second reactor for detecting toxic substances and (c) a photodetector, and (b) at least four as channel units.

As the filtration means of the water sample, a primary pre-filter and a micro filter can be used. Such filtering means removes suspended matter contained in the water sample to prevent loss of microbial activity by the suspended matter, for example, contamination of the nutrient medium by other microorganisms contained in the suspended matter and clogging of the tubing by the suspended matter. The filtered water sample is transported and distributed to the reactor of each channel through a tube connected to the reactor of each channel.

The two-stage reactors form a multi-channel for the detection of various toxicities, wherein the two-stage reactor that forms the channel unit detects toxic substances in water by using the first and cultivated recombinant microorganisms. Consisting of a second reactor. Reactors in channel units are described in published patent application No. 98-3621 filed by the inventors, which is shown in FIG. 2.

The first reactor may be divided into a top, a middle, a bottom, and a warm water jacket surrounding the top.

The upper part of the first reactor is equipped with a nutrient medium inlet 101 for continuously supplying a nutrient medium, a microorganism inlet 104 for providing microorganisms, and a filter 107 for providing air for cultivation of microorganisms. And a gas outlet 105 equipped with a condenser 106 using cooling water for removing the gas generated by the culture of the microorganisms. The tube forming the air inlet 103 may extend to the bottom of the reactor to provide air directly to the culture.

The middle of the first reactor is provided with a culture medium outlet 102 for continuously supplying the culture solution to the second reactor.

In the lower portion of the first reactor, the stirring rod 110 and the stirrer 111 for stirring the culture solution, the glass partition 112 for the transmission of light in the culture solution, and the attachment for transferring the light to the photodetector 115. The optical fiber 114 is fixed to the reactor by means 113. In particular, the stirrer 111 may use a multi-magnetic stirrer capable of simultaneously stirring the multi-channel first reactors.

The first reactor is equipped with a hot water jacket 116 having a hot water inlet 108 and an outlet 109 for maintaining the culture temperature of the microorganism at 30 to 37 ° C.

The first reactor can be made into a small reactor, for example having a volume of 10 ml.

The second reactor also has a structure similar to that of the first reactor.

In the upper part of the second reactor, a filter 207 similar to that of the first reactor, together with a water inlet 204 through which the filtered water sample is continuously introduced, and a culture solution inlet 201 for continuously supplying the microbial culture solution. Is equipped with an air inlet 203 equipped with a gas outlet 205 equipped with a condenser 206.

The middle of the second reactor is provided with a mixed solution outlet 202 for discharging the mixed solution of the culture solution and the water sample.

The lower portion of the second reactor is equipped with an agitating rod 210 similar to that of the first reactor, an agitator 211, a glass partition 212, and an optical fiber 214 fixed to the reactor by an attachment means 213. Light sensed in the second reactor is measured by the photodetector 215.

The second reactor is equipped with a hot water jacket 216 having a hot water inlet 208 and an outlet 209 similar to that of the first reactor.

The second reactor can be made into a small reactor, for example having a volume of 20 ml.

Four or more reactors having this two-step structure forms a multi-channel for detecting various toxicity. The number of reactors is preferably 4 to 100, more preferably 4 to 10. Multi-channel can be used to combine the recombinant microorganisms that emit light with a specific toxicity and the recombinant microorganisms that reduce light emission by toxicity, for example, E. coli TV1061, DPD2794, DPD2540 and GC2 in each channel Is to use.

The method of detecting toxicity in water using the apparatus of the present invention is as follows.

First, the water sample is filtered to remove suspended solids that may lose the activity of microorganisms. The filtrate is then transported to a second reactor in each channel. In this case, the recombinant microorganisms cultured in the first reactor are continuously supplied to the second reactor, and in each channel, a recombinant microorganism that emits light of different toxicity or a recombinant microorganism that reduces light emission by toxicity may be used. In the second reactor, the light emitted by the toxicity in the water is received through the optical fiber and delivered to a photodetector such as a luminometer. Data from photodetectors can be analyzed and stored in real time using the LABVIEW computer program.

The toxic emission reaction can be expressed as the ratio (relative bioluminescence) of the second reactor emission after the injection of the toxic substance to the second reactor emission before the injection of the toxic substance, based on the relative bioluminescence values of each channel. The type and level of toxicity can be determined.

The apparatus and method of the present invention can simultaneously determine various types and levels of toxicity by using a multi-channel reactor, and use a two-stage reactor using recombinant microorganisms as a channel unit to rapidly and rapidly detect toxicity in water for a long time. There is an advantage.

Hereinafter, the effect of the apparatus of the present invention will be described by the following test example. The apparatus used in the test example is the apparatus of FIG. 1 having four channels, each of which contains E. coli TV1061 (ATCC69315), DPD2794 (Dupont, USA), DPD2540 (Dupont, USA) and GC2 (Dept. of Environmental Engineering, Gwangju Institute of Science and Technology). Obtained) was used.

Test Example 1: DNA Toxicity Test

After injecting 0.05 ppm of mitomycin C into the water inlet of the apparatus of FIG. 1, the luminescence reaction was investigated in the second reactor of each channel.

The results are shown in Figures 3 (a) and 3 (b). As shown in Figures 3 (a) and 3 (b), the relative bioluminescence increased only in the channel using E. coli DPD2794, there was no change in the relative bioluminescence in the channel using E. coli DPD2540 and TV1061. In particular, luminescence did not decrease in the channel using E. coli GC2, which reduced luminescence in response to all toxicity, indicating that 0.05 ppm of mitomycin was not toxic to cells.

Test Example 2: Cell Membrane Toxicity Test

After injecting 5 ppm of cerulein into the water inlet of the apparatus of FIG. 1, the luminescence reaction was investigated in the second reactor of each channel.

The results are shown in Figures 4 (a) and 4 (b). 4 (a) and 4 (b), the luminescence reaction was observed only in the channel using E. coli DPD2540, the luminescence reaction was not seen in the channel using E. coli DPD2794 and TV1061. Luminescence was also reduced in the channel using E. coli GC2, indicating that the cerulein used is toxic to cells.

Test Example 3: Protein Toxicity Test

100 ppm of phenol was injected into the water inlet of the apparatus of FIG. 1 and the luminescence reaction was investigated in the second reactor of each channel.

The results are shown in Figs. 5 (a) and 5 (b). 5 (a) and 5 (b), the luminescence reaction was observed only in the channel using E. coli TV1061, and the luminescence reaction was not observed in the channel using E. coli DPD2794 and DPD2540. In addition, luminescence was reduced in the channel using E. coli GC2, and it can be seen that the phenol used therein is toxic to cells.

The apparatus of the present invention is highly effective unlike the conventional apparatus because it efficiently detects not only the type of toxicity but also the type of toxicity contained in the water by using a multi-channelized two-stage reactor. In addition, the two-stage reactor used in the device of the present invention is economical because it detects toxicity in water continuously for a long time, reacts quickly to the introduced toxicity, and can be miniaturized.

The apparatus of the present invention can be usefully applied to detect toxic substances introduced into water resources such as rivers, rivers and water supplies as well as toxic substances in waste water.

Claims (8)

(a) means for filtering water samples, (b) (i) for culturing recombinant microorganisms having luminescent properties for toxic substances, the first reactor having an outlet of the culture medium and an optical fiber, and (ii) detecting toxic substances, wherein At least four two-stage reactors consisting of a second reactor equipped with an inlet of a microbial culture from an outlet, an inlet of a water sample discharged from said filtering means, and an optical fiber, and and (c) a photodetector connected to the optical fibers of the first reactor and the second reactor, respectively. The method of claim 1, The first reactor is equipped with a warm water jacket for maintaining the culture temperature of the microorganisms on the outside, and has a nutrient medium inlet, a microorganism inlet, an air inlet and a gas outlet at the top, and a outlet of the culture solution at the middle, It is equipped with a glass partition wall for light transmission, an optical fiber and a stirrer for transmitting this light to the photodetector, and the second reactor is equipped with a warm water jacket for maintaining the culture temperature of the microorganisms on the outside and the top of the first reactor. It is equipped with an inlet of the microbial culture solution from the outlet, an inlet of the water sample discharged from the filtration means, an air inlet port and a gas outlet port, and an outlet of the mixed solution of the microbial culture solution and the water sample in the middle part and a glass partition wall for light transmission at the bottom part. And an optical fiber and agitator for transmitting this light to the photodetector. The method of claim 1, The recombinant microorganism is E. coli DPD2794, which emits toxic to DNA damage, E. coli DPD2540, which emits toxic to cell membrane damage, E. coli TV1061, which emits toxic damage to protein, or E. coli GC2, which reduces luminescence by toxicity. Apparatus using different microorganisms in each of the two stage reactors. The method of claim 3, wherein The toxic substance causing damage to the DNA is mitomycin C or gamma rays. The method of claim 3, wherein The toxic substance causing damage to the cell membrane is cerulenin. The method of claim 3, wherein Wherein the toxic substance causing damage to the protein is ethanol, phenol or pentachlorophenol. After (a) filtering the water sample, (b) the filtrate is added to each of four or more reactors continuously fed with different different recombinant microorganisms having luminescent properties for toxicity, and (c) the light emitted from each reactor. Including the step of detecting, Toxic toxicity detection method. The method of claim 7, wherein E. coli DPD2794, the recombinant microorganism emits toxic to DNA damage, E. coli DPD2540, luminescent to damage the cell membrane, E. coli TV1061 to the toxicity causing damage to the protein, or E. coli GC2 reduced luminescence by toxicity How to be.