KR100253760B1 - Small bioreactor with optical fiber and toxicity detection method using the same - Google Patents

Abstract

The present invention is capable of continuously and non-continuously detecting toxic substances introduced into plant wastewater, wastewater treatment plants, and effluents, streams, and water supplies using bacteria that are genetically synthesized to emit light according to the types of toxic substances. The present invention relates to a small bioreactor with an optical fiber and a toxic substance detection method using the same. The small volume of work allows for low batch consumption and easy operation and management to keep microorganisms growing over time. In addition, by combining the optical fiber with a small reactor, the microorganisms used for the detection of toxicity can transmit the light change emitted in the small bioreactor to the luminometer, which is a light measuring device, quickly and without loss of light. In addition, this small bioreactor equipped with optical fiber is composed of two stages, and in the first stage reactor, microorganisms are not mixed with toxic substances or exposed to radiation, and are introduced into the second stage reactor and mixed with toxic substances to measure the change of light emitted by the microorganisms. Thus, the invention is capable of detecting the harmfulness of toxic substances or radioactivity continuously over a long period of time despite the growth inhibition due to mixing with toxic substances or radioactivity.

Description

Small bioreactor with optical fiber and toxicity detection method using the same

The present invention relates to a small bioreactor equipped with an optical fiber capable of detecting toxicity and radioactivity contained in waste water, river water, river water, water supply, and the like, and a method for detecting water toxicity using the same.

The toxicity of conventional wastewater treatment plants, rivers, rivers and water supplies is measured using the survival of aquatic organisms such as daphnia or fish. The method of using aquatic organisms such as water fleas has the disadvantage of delaying the detection time and detecting various types of toxicity by determining the influence of toxic substances on the survival of aquatic life and judging the inflow and toxicity of toxic substances. In addition, it has the disadvantage of not being able to detect toxicity continuously. Another method is to use microorganisms to improve the shortcomings of the toxicity detection method using aquatic organisms, and there is a method of using genetically recombined bacteria to emit biological light according to the type of toxic substances. However, the conventional method using microorganisms consumes a large amount of nutrient medium to grow and maintain the recombinant bacteria that are used to detect toxic substances when detecting the presence of toxic substances in waste water treatment plants, rivers and water sources for a long time. The influx of substances inhibits the growth of microorganisms, which eventually stops the growth of microorganisms and makes it impossible to detect toxic substances.

The present invention uses microorganisms that have been genetically recombined to emit biological light according to the type of toxic substances, so that microorganisms can be grown for a long time despite the influx of toxic substances to detect toxic substances entering wastewater treatment plants, rivers, and water sources for a long time. It is to provide a small bioreactor and water toxicity detection method that can stably maintain the growth and minimize the consumption of nutrient medium and directly measure the biological light emitted from microorganisms.

1 is a detailed view of a small bioreactor equipped with an optical fiber for measuring light generated from microorganisms grown in a bioreactor for recombinant bacteria used for detection of toxicity and radioactivity.

2 is a schematic diagram of a water quality toxicity detection system consisting of two stages of a small bioreactor equipped with optical fibers for continuous and discontinuous detection of toxicity and radioactivity using recombinant bacteria.

Figure 3 shows the results of measuring the light emitted by DPD 2794 in the bioreactor of mitomycin.

4 is a result showing the stable growth of microorganisms in the bioreactor before the injection of mitomycin.

5 shows the BL value at the dilution rate of 0.74 / hr.

6 shows experimental results at a dilution rate of 1.03 / hr.

7 is a light specific result according to the amount of phenol injection by concentration in the two-stage reactor of phenol having a dilution rate of 0.829 / hr fixed in the two-stage reactor.

8 (a), (b), (c) and (d) are light measurement results according to the amount of phenol added for each time zone in FIG.

* Explanation of symbols for main parts of the drawings

1: nutrition medium inlet 2: nutrition medium outlet

3: air inlet port 4: inoculation port

5 exhaust gas outlet 6 condenser

7: air filter 8: water inlet

9: water outlet 10: stirring rod

11 stirring device 12 glass

13: optical fiber attachment device 14: optical fiber

15: 1st stage bioreactor 16: 2nd stage bioreactor

1 is a detailed view of a small bioreactor attached to an optical fiber, each having a nutrient medium inlet (1) and an outlet (2) for maintaining the growth of microorganisms, and removing the microorganisms in the air inlet (3) and the incoming air. There is a filter (7) for microbial inoculation and toxic material inlet (4), a small amount of bubbles and carbon dioxide generated in a small bioreactor with an optical fiber is condensed through the condenser (6) through the outlet (5) Discharged. There are inlets (8) and outlets (9) through which 30 ° C of water is circulated into a double jacket in the small bioreactor to maintain the temperature of the small bioreactor with the optical fiber attached thereto (10) and ( 11) has a stirrer (magnetic bar 10) and a magnetic stir plate (11) for stirring in a small bioreactor. The optical fiber connector 13 is fixed to the reactor in order to deliver light emitted by the microorganisms to the optical fiber 14 (Turner Designs, Fiber Optic Probe 20-040) through the glass 12. In a small bioreactor with fiber optics, the fiber is delivered quickly and continuously with no loss of light to a luminometer (Turner Designs, Moedel 20e Luminometer) that measures the biological light emitted by the microorganisms growing for the detection of toxicity. In order to economically detect the toxicity, the operating volume of 38m1, 20m1, and 10m1 was reduced to the size of the bioreactor-attached bioreactor, and the water quality toxicity detection device and method were invented.

2 is a schematic diagram of a continuous toxicity detection system using a small bioreactor equipped with a two-stage optical fiber. The small bioreactor 15 with a first stage optical fiber of 10m1 and the second stage optical fiber of 20m1 of a volume of operation The bioreactor 16 is shown. The nutrient medium 17 necessary for the growth of microorganisms in the first stage reactor is introduced into the first stage reactor and the microorganisms using the medium continue to grow. Microorganisms 18, which are constantly grown and maintained without contact with toxic substances in the first stage reactor, are introduced into the second stage reactor. The microorganisms introduced from the first stage reactor into the second stage reactor are mixed with the wastewater treatment plant, the inflow and discharge water, the river, the water supply source, and the nuclear power plant 19, which are introduced into the second stage reactor. When the introduced microorganism is a bacterium that is genetically recombined to emit light according to the type of the toxic material, the light is emitted according to the type of the toxic material introduced into the second stage reactor. The change of light emitted by the microorganisms in the two-stage reactor is transmitted to the luminometer, which is a light measuring device, through the optical fiber 21 connected to the two-stage reactor, so that the amount of light change is continuously measured and the light changes over time. It detects the presence of toxic substances and the degree of toxicity. This system, which continuously detects toxicity by growing and maintaining microorganisms that are genetically engineered to emit light according to the type of toxicity of the two-stage reactor, has a small operating volume, so that the consumption of the nutrient medium required to maintain the growth of microorganisms is not only small but overall. The small size of the system reduces the manufacturing cost of the reactor and facilitates operation and management. If the microorganisms used for the detection of toxicity are mixed with the toxic substances, the growth is inhibited and cannot be used for the detection of the toxicity for a long time. However, in the continuous toxicity detection system using the small-sized optical reactor with optical fiber of the second stage, the microorganisms are not mixed with the toxic substances and maintained in the first stage reactor and are introduced into the second stage reactor. Toxicity can be detected continuously. In addition, the two-stage reactor is connected to the optical fiber, so that the change of light is immediately transmitted to the light measuring device without loss of light according to the time when microorganisms are mixed with the toxic substances, and therefore the presence of toxic substances into the wastewater treatment plant, rivers, water supplies, etc. (B) It is possible to detect the presence of radioactive contamination of nuclear power wastewater and the effects of radioactivity around nuclear power plants quickly and continuously. Table 1 shows the types of genetically engineered bacteria (manufactured by DuPont, USA) used to detect toxic substances.

Second degree. Toxicity Detection Zones

Example 1

Mito, a substance that destroys the genes of microorganisms after maintaining DPD2794 in a small bioreactor with an optical fiber of 38ml in volume and maintaining a constant dilution rate of 0.767 / hr divided by the operating volume of nutrient medium in the small bioreactor. When injected in a small bioreactor of mitomycin C to a concentration of 0.5 ppm, the light emitted from the DPD2794 was measured. Experimental results are shown in FIGS. 3 and 4, where BL is the luminometer of biological light emitted by DPD2794, and SBL is the light generated per DPD2794 cell divided by BL divided by OD600. The maximum SBL was 4623 and the time to reach the maximum SBL and the maximum BL was equal to 160 minutes when the time of injecting mitomycin C was 0. OD600 is the optical density at DP wavelength of 660nm of DPD2794's UV Spectrophotometer, which grows in small bioreactor, and the concentration of DPD2794 is BL and DPD2794 is biological light generated by mitomycin C. Since the degree of change over time of SBL is almost identical, it can be seen that toxicity can be detected by measuring only biological light generated by microorganisms in a small bioreactor with an optical fiber. In FIG. 4, since the microorganism's OD600 is kept constant until the injection of mitomycin C, it can be seen that the microorganism is stably grown and maintained in a small bioreactor with an optical fiber.

Example 2

Using a small bioreactor with a 38 ml operating volume Concentration in a small bioreactor of mitomycin C, a chemical that destroys the genes of microorganisms according to the dilution rate of the medium inflow rate divided by the operating volume while maintaining and maintaining the DPD2794. When the injection to 0.1ppm was measured the light generated by the DPD2794. The BL value is shown in Figure 3 at the dilution rate of 0.74 / hr.

Prior to injecting mitomycin C, the growth of microorganisms in a small bioreactor with an optical fiber is maintained stably while the light generated by the microorganisms is kept constant. From the injection of mitomycin C, the amount of light generated by the microorganism increases gradually, indicating a maximum value at 167 minutes after the injection. When the dilution rate is 0.34 / hr, 0.533 / hr, 1.06 / hr, the maximum BL values are 16000, 7700, and 441, respectively, and the detection time is 400 minutes, 335 minutes, and 210 minutes, respectively. It can be seen that it can be detected quickly.

Example 3

The biological light generated by DPD2540 when the DPD2540 is injected into the reactor so that the concentration of phenol that modifies the protein in the microorganism is 300ppm according to the dilution rate in the small bioreactor with 38m1 of optical fiber. Was measured with a luminometer. Experimental results at a dilution rate of 1.03 / hr are shown in FIG. 6 and the time of injecting phenol was set to zero.

In FIG. 6, when the 300 ppm phenol was injected into the small bioreactor equipped with the optical fiber, toxicity detection was possible at about 65 minutes. Dilution rate was 0.218 / hr, 0.498 / hr. At 1.03 / hr, the reaction detection time is 105 minutes, 90 minutes, and 61 minutes, respectively, indicating that toxic substances can be detected faster at high dilution rates.

Example 4

Example 4 shows a dilution rate of 0.829 in a second stage reactor in a toxicity detection system in which a 10 micrometer optical fiber small bioreactor with an operating volume of 10 m1 is used as a second stage reactor. When fixed in / hr and injected into the two-stage reactor so that the concentration of phenol in the two-stage reactor is 100ppm, 300ppm, 700ppm, 1400ppm, the biological light emitted by TV1061 was measured by luminometer. The results are shown in Figures 7 and 8. 7 is a result of growing and maintaining TV1061 for more than 100 hours using a toxicity detection system using a two-stage optical fiber small bioreactor.

It can be seen that the microorganisms are stably grown and maintained in the reactor because the biological light generated by the microorganisms before the toxic substance is kept constant. In addition, it can be seen that when the phenol is injected, it is recovered before the injection in a short time. 8 shows the phenomenon when 100 ppm, 300 ppm, 700 ppm, and 1400 ppm phenol were injected. In Fig. 8, the detection time of 100ppm, 300ppm and 700ppm phenol was 30 minutes, 40 minutes and 55 minutes, respectively. It can be seen that the toxicity can be detected in. In addition, as the concentration of injected phenol increases, the time taken to reach the maximum SBL at the time of phenol injection increases as the concentration of injected phenol increases with the inhibition of growth of TV1061 used for the detection of toxicity. This is because light is reduced and microbial growth gradually recovers as bacteria in the first stage reactor enter unmixed with phenol. When 1400 ppm of phenol was injected, the light generated by the toxic detection bacteria in the two-stage reactor decreased rapidly compared with the case where no phenol was injected, and then gradually recovered as the inflow of bacteria in the first stage reactor not mixed with phenol was introduced. 140 minutes after the injection can be confirmed that the recovery before phenol injection.

Even if the bacteria are genetically recombined to emit biological light according to the type of toxic substances in the bioreactor of the present invention, even if the toxic substances are detected for a long time, the microorganisms stably grow and not only minimize the consumption of nutrient medium, but also various It provides an economical way to detect toxicity.

Claims (5)

Toxicity using bioreactor consisting of nutrient medium inlet (1) and outlet (2), air inlet (3) and filter (7) to remove microorganisms from incoming air, microorganism inoculation and toxic inlet (4) In the substance detecting device, a bioreactor for stably culturing a microorganism whose gene is recombined so as to emit biological light according to the type of toxic substance and supplying the microorganism to the second stage bioreactor, the optical fiber attachment device (13) The first stage bioreactor 15, to which the optical fiber 14 is attached, and the first stage bioreactor 15 are continuously installed, where the microorganism and the sample for detection are mixed. The optical fiber 14, which transmits the change of the light emitted by the luminometer, is composed of the second stage bioreactor 16 attached by the optical fiber attachment device 13. Chakdoen small bioreactor. The small bioreactor with an optical fiber according to claim 1, wherein the first and second stage bioreactors are small bioreactors each having a volume of 57 ml or less. Toxicity using bioreactor consisting of nutrient medium inlet (1) and outlet (2), air inlet (3) and filter (7) to remove microorganisms from incoming air, microorganism inoculation and toxic inlet (4) In the material detecting apparatus, the first stage bioreactor 15 is stably incubated with the optical fiber of the microorganism in which the gene is recombined so as to emit biological light according to the type of the toxic substance, and is supplied from the bioreactor 15. The microorganism and the sample to be detected are mixed in the second stage bioreactor 16 to which the optical fiber is attached, and the microorganism detects the toxic substances by measuring the change of light emitted by the reaction with the toxic substances through the optical fiber. Toxicity detection method using a small bioreactor having an optical fiber attached. The method of claim 3, wherein the bioreactor detects toxicity by operating in a continuous or discontinuous manner. The method of claim 3, wherein the sample to be detected is wastewater, wastewater treatment plant inflow or outflow water, water, river water, water and nuclear wastewater, radioactive hazardous substances around the nuclear power plant, characterized in that the toxicity using a small bioreactor with a fiber Detection method.