KR20180059227A - Method for analyzing living organism in water to be treated - Google Patents

Abstract

The present invention relates to a method for analyzing a survival status of organisms present in treated water such as ballast water. In particular, the present invention provides the method for analyzing the survival status of the organisms present in the treated water, including the steps of: establishing respective extinction references for zooplankton, phytoplankton, and bacteria from sterilized water; radiating a single wavelength, which is selected within a range between 250 nm and 700 nm, to treated measurement target water and measuring an intensity of emitted light by using a fluorescence analysis device; calculating respective concentrations of proteins, fulvic acids, and humic acids from the measured intensity of the light; and determining presence or absence of at least one of the zooplankton, the phytoplankton, and the bacteria by comparing the concentrations of the proteins, the fulvic acids, and the humic acids with the extinction references.

Description

METHOD FOR ANALYZING LIVING ORGANISM IN WATER TO BE TREATED [0002]

The present invention relates to a method for analyzing the viability of an organism present in treated water. More specifically, the present invention relates to a method for analyzing whether or not an organism existing in a treatment water such as ship ballast water exists.

Ballast water is seawater that is filled in a tank installed in the vessel to maintain the center of gravity of the ship when the vessel is operated. When the cargo is shipped, discard the seawater that is being loaded, and when the cargo is released, put the seawater again and grab the center of gravity of the ship. As the international trade volume increases, the number of vessels is increasing and the size of vessels is increasing. The ship will operate in several countries, where the ballast water filled in one country will be discharged when entering ports of other countries. The discharged ballast water contains harmful plankton and bacteria, which causes migration and inflow of foreign marine species due to discharging of ballast water, resulting in disturbance of indigenous marine ecosystem. . To prevent this, the United Nations International Maritime Organization (IMO) adopted the International Convention for the Control of Ballast Water in 2004 in order to prevent marine pollution. According to this, new ships and existing vessels are obliged to install a ship ballast water treatment facility approved by IMO from 2014 to 2020. Currently, more than 30 countries have ratified the Convention, but the shipowners' associations argue that it is not possible to ratify the convention without standardization of analysis methods for organisms in ballast water.

The Biodegradation Criteria for Marine Ballast Water (IMO D-2) measures biological populations using a variety of standard measurement methods. For example, APHA Standard method 9215, APHA Standard method 9222 B (measurement of total coliforms by membrane filtration), APHA Standard method 10200 C (enrichment technique), EPA 445.0: 1997 (phytoplankton (Determination of chlorophyll-a of marine phytoplankton using fluorescence in the identification of life and death), and UNESCO 4: 2003 (cell count). The above methods have a problem in that a large number of labor and time are required for the analysis because the number of cells must be directly counted.

As a method of analyzing the organisms present in the ship ballast water, a precise analysis method has been used in which a certain amount of ballast water samples are taken from the pipe through which the ballast water is discharged, and the collected water is concentrated on the net and then inspected using a microscope. This precise analysis method requires a considerable amount of time and manpower for the inspection, and there is a problem that the inspection result on the ballast water discharged in real time can not be obtained.

As another method, Korean Patent Registration No. 10-1551816 discloses a method of analyzing the equilibrium of a living organism in a ballast water by analyzing ballast water collected from a collecting device by using a fluorescence analysis method.

In addition, although many devices for analyzing the indicator have been already known, only one living organism is measured according to the measurement method, and thus the viability of the whole microorganism can not be discriminated and the accuracy is lowered.

In general, ballast water is removed by sterilization before it is loaded on board. As a sterilization treatment method, there is known a method of decomposing organic matter through chemical treatment such as electrolytic treatment, ozone treatment or chemical treatment, and a method of photodecomposing organic matter through physical treatment such as UV irradiation or UV / TiO ₂ treatment . The sterilized ship ballast water is stored in the ballast tanks of vessels for a long period of voyage, and the organisms that have not been removed during the sterilization process will continue to multiply in the tank while continuing protein synthesis. However, there is no known method for precisely measuring in real time the presence of organisms present in the equilibrium at the discharge stage.

It is an object of the present invention to provide a method for analyzing the viability of a living organism in a ship ballast water at the discharge stage. It is another object of the present invention to provide a method for determining the presence or absence of living organisms in water purification plants, tap water, sterilized water used in laboratories, etc., in addition to the equilibrium water of ships.

The above-mentioned problem is solved by a method for producing a microorganism, comprising the steps of: setting a mortality criterion for zooplankton, phytoplankton, and bacteria from sterilized water; Irradiating a single wavelength selected from the range of 250 nm to 700 nm to the treatment water to be measured and measuring intensity of emitted light using a fluorescence analyzer; Calculating concentrations of protein, fulvic acid, and humic acid from the measured light intensities, respectively; And determining the presence or absence of at least one of zooplankton, phytoplankton, and bacteria by comparing the concentration of the protein, fulvic acid, and humic acid with the killing criterion to determine the viability of an organism present in the treated water .

Preferably, the criterion of killing of the zooplankton is that 10 animals of zooplankton are added to 1 m ³ of sterilized water and kept for 24 hours and then irradiated with a single wavelength selected from 250 nm to 450 nm and measured with a fluorescence analyzer The concentration of protein, fulvic acid and humic acid may be divided by 24.

Preferably, the criterion for killing the phytoplankton is 100 pellets of phytoplankton in 10 ml of sterilized water, maintained for 24 hours, irradiated with a single wavelength selected from 250 nm to 450 nm, and measured with a fluorescence analyzer The concentration of protein, fulvic acid and humic acid may be divided by 24.

Preferably, the bacteria are killed by adding 100 pellets of phytoplankton to 10 ml of the sterilized water, keeping the pellet plankton for 24 hours, irradiating a single wavelength selected from 250 nm to 450 nm, , The concentration of fulvic acid and humic acid, respectively, divided by 240.

Preferably, when the concentration of the protein in the treated water is higher than the concentration of fulvic acid or humic acid in the treated water, it is determined whether or not the zooplankton is killed compared to the criterion of death of the zooplankton.

Preferably, when the concentration of the protein in the treated water is lower than the concentration of fulvic acid or humic acid in the treated water, it is determined whether or not the phytoplankton is killed compared to the criterion of death of the phytoplankton.

Also preferably, when the concentration of the protein, fulvic acid and humic acid in the treated water is lower than that of the phytoplankton, it is determined whether the bacteria are killed or not, in comparison with the criterion of killing the bacteria.

Preferably, the fluorescence analysis apparatus may include a chamber for containing a sample, a light source for irradiating the chamber with light, a filter for changing the light emitted from the light source into a single wavelength, and a sensor for measuring intensity of light emitted from the sample have.

Preferably, the method further comprises the step of calculating the concentration of chlorophyll-a by measuring the light intensity after irradiating the treated water with light of 620 to 700 nm.

Also preferably, the treated water is a ship ballast water.

According to the method of the present invention, it is possible to simultaneously and accurately analyze the survival of phytoplankton, zooplankton, and bacteria in the treatment water such as ship equilibrium.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart showing a method for analyzing organisms present in a treatment water such as ship ballast water according to the present invention. FIG.
FIG. 2 shows a result of a 3D fluorescence spectrum analysis on ship ballast water according to an embodiment of the present invention.
3 schematically shows the configuration of the analysis process and the fluorescence analysis apparatus according to the present invention.

Unless defined otherwise, all technical terms used in the present invention have the following definitions and are consistent with the meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.

The term "about" is used herein to refer to a reference quantity, a level, a value, a number, a frequency, a percent, a dimension, a size, a quantity, a weight, or a length of 30, 25, 20, 25, 10, 9, 8, 7, Level, value, number, frequency, percent, dimension, size, quantity, weight or length of a variable, such as 4, 3, 2 or 1%.

Throughout this specification, the words "comprises" and "comprising ", unless the context requires otherwise, include the steps or components, or groups of steps or elements, Steps, or groups of elements are not excluded.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1 is a flowchart showing a method for analyzing living organisms present in treated water according to the present invention. The treated water may be one species selected from the group consisting of ship equilibrium water, water treatment plant treated water, tap water, and sterilized water in a laboratory, and more preferably, equilibrium water of ships.

(S11) setting the death criterion of zooplankton and phytoplankton, respectively; (S12) of collecting the treatment water to be measured, irradiating a single wavelength selected in the range of 250 nm to 700 nm, and measuring the intensity of light emitted using the fluorescence analyzer; Calculating a concentration of protein, fulvic acid, and humic acid from the intensity of the measured light (S13); And determining the presence of zooplankton, phytoplankton, and bacteria by comparing the concentration of the protein, fulvic acid and humic acid with the killing criterion (S14).

According to an embodiment of the present invention, a step (S15) of calculating the concentration of chlorophyll-a by measuring light absorbance using a fluorescence analyzer after irradiating light of 620 to 700 nm to the ballast water to be measured after step S14, . ≪ / RTI > This makes it possible to more accurately determine whether phytoplankton is killed.

Each step will be described in detail below.

First, the step (S11) of setting the mortality criterion of the zooplankton and the phytoplankton, respectively. The IMO D-2 standard established by IMO for animal and phytoplankton is below 10 cells / ml. Table 1 below shows IMO D-2 standard.

Target creature  standard Plankton,> 50㎛ <10 viable cells / m ³ Plankton, 10 ~ 50㎛  <10 viable cells / ml Toxicogenic Vibrio Cholerae
(01 and 0139)  <1 cfu / 100ml Escherichira coli  <250 cfu / 100 ml intestinal Enterococci  <100 cfu / 100 ml

The mortality criterion defined in the present invention is set out in a different manner, although it starts from the IMO D-2 standard. Zooplankton, phytoplankton and bacterium are different from each other, and since the zooplankton is relatively large, the amount of the zooplankton is high, and the mortality criterion may be high.

Criteria for the death of zooplankton shall be determined in the following manner.

First, the treated water to be treated is sterilized and sterilized Prepare 1 m ³ . The sterilization method may be one of the methods selected from the group consisting of electrolytic treatment, ozone treatment, chemical treatment, UV irradiation, and UV / TiO ₂ treatment. Through the sterilization process, all the plankton and bacteria in the treated water are killed, and the protein, fulvic acid and humic acid are all sterilized. Ten animals of animal plaques are added to the sterilized water and maintained for 24 hours, followed by stirring and collecting 5 ml. The collected sterilized water is filtered with a filter having a size of 10 μm or less, and then irradiated with a single wavelength of 275 nm, 330 nm, and 370 nm, respectively, using a fluorescence analyzer to measure the intensity of the emitted wavelength. The concentrations of protein (275 nm), fulvic acid (330 nm), and humic acid (370 nm) are calculated and divided by 24 to set the criterion for killing of zooplankton. Preferably, the death criterion can be determined by repeating this step several times and calculating the average.

Criteria for killing phytoplankton are determined by the following method.

The treated water to be treated is sterilized, and 10 ml of sterilized water is prepared. 10 x 10 phytoplankton are introduced (10 on the basis of IMO D-2 / ml). The sterilization treatment is the same as the above-mentioned method. After the addition, the mixture is kept for 24 hours, stirred, and 5 ml is collected. The collected treatment water is irradiated with a single wavelength of 275 nm, 330 nm, and 370 nm, respectively, using a fluorescence analyzer to measure the intensity of the emitted wavelength. The concentrations of protein, fulvic acid, and humic acid were then calculated This is divided by 24 and set as the criterion for the death of phytoplankton. Preferably, the death criterion can be determined by repeating this step several times and calculating the average.

In the above, holding for 24 hours after the introduction of plankton imparts the time for the biosynthesis of plankton. Also, the concentration of protein, fulvic acid, and humic acid measured after fluorescence analysis is divided by 24 and determined as the death criterion. This is based on the assumption that the measurement takes about one hour after the sampling of the treated water and the measurement using the fluorescence analyzer, and the average concentration is discharged for one hour. Plankton can be biosynthesized for as long as 1 hour or less, so it is maintained for 24 hours and divided by 24, and average value is calculated.

The criteria for the death of bacteria are as follows.

The treated water to be treated is sterilized, and 10 ml of sterilized water is prepared. 10 x 10 phytoplankton are introduced (10 on the basis of IMO D-2 / ml). The sterilization treatment is the same as the above-mentioned method. After the addition, the mixture is kept for 24 hours, stirred, and 5 ml is collected. The collected treatment water is irradiated with a single wavelength of 275 nm, 330 nm, and 370 nm, respectively, using a fluorescence analyzer to measure the intensity of the emitted wavelength. The concentrations of protein, fulvic acid, and humic acid were then calculated This is divided by 240 and set as the criterion for bacterial killing. Preferably, the death criterion can be determined by repeating this step several times and calculating the average.

The fluorescence analysis apparatus used in the present invention comprises a chamber for storing a sample, a light source for irradiating light to the chamber, a filter for converting light emitted from the light source into a single wavelength, and a sensor for measuring the intensity of light emitted from the sample . Place the sample in the chamber and fix the excitation and emission wavelength at 250 ~ 600 nm and scan for about 40 minutes.

Next, the process water to be measured is sampled and irradiated with a single wavelength selected in the range of 250 nm to 700 nm, and the intensity emitted by each wavelength is measured using the fluorescence analyzer (S12). Preferably 250 to 450 nm, and more preferably 275 nm, 330 nm, and 370 nm.

Treatment water such as ship equilibrium water can be decomposed by chemical treatment such as electrolysis, ozone treatment or chemical treatment in ship ballast water treatment equipment (BWTS), physical treatment such as UV irradiation or UV / TiO ₂ treatment The organic matter is photo-degraded and sterilized. Since the biodegradable organisms such as zooplankton and phytoplankton are biosynthesized, many metabolites such as protein, fulvic acid and humic acid are dissolved in ballast tanks. According to one embodiment of the present invention, the step of storing sterilized ship ballast water in a ballast tank and collecting the ballast water before release to the sea is used to measure the intensity of light emitted by each wavelength using a fluorescence analyzer . The sample collected using the fluorescence analyzer can be irradiated with a wavelength of 250 to 425 nm at intervals of 5 nm, and the intensity of light emitted from the sample can be measured by wavelength.

Preferably, when the sample is irradiated with light having wavelengths of 275 nm, 330 nm, and 370 nm, wavelengths corresponding to protein, fulvic acid, and humic acid are emitted, respectively. Investigate one wavelength at a time and measure the emitted wavelength. According to one embodiment of the present invention, when the sample is irradiated with light of 275 nm and the intensity of the emitted 250 to 600 nm wavelength is analyzed by a sensor of the fluorescence analyzer (about 5 nm step) The amount of protein in the strength can be measured. In the same manner, the amount of humic acid can be measured by irradiating the sample with light of 330 nm and irradiating the amount of fulvic acid with light of 370 nm. The intensity of the emitted wavelength is proportional to the amount of living organisms in the sample taken.

The amount of metabolites such as protein, fulvic acid, and humic acid present in the treated water can be calculated using the light intensity measured in this step (S13). The amount of metabolites can be calculated by measuring distilled water as a sample and calculating the amount of protein, fulvic acid, and humic acid by converting the difference of the light intensity measured for the treated water to the intensity of the emitted light .

The treated water collected in this step, preferably the ballast water, can be measured after being filtered with a filter having a size of about 10 μm or less, and in the case of measuring phytoplankton, it can be measured with a filter having a size of about 50 μm or less. Filters can be selectively applied. For example, the concentration of protein, fulvic acid, and humic acid can be measured by applying a 50 μm filter to measure chlorophyll-a, discarding treated water, and applying a 10 μm filter.

Next, the step of determining the presence of zooplankton, phytoplankton, and bacteria by comparing the concentrations of the protein, fulvic acid, and humic acid with the killing criteria of the zooplankton, phytoplankton, and bacteria (S14). As described above, the criterion of killing of zooplankton, phytoplankton, and bacteria may be flexible depending on the sterilization condition of the treated water to be measured.

It is judged that the amount of zooplankton is dominant when the concentration of the protein calculated from the sample is higher than that of fulvic acid and humic acid, respectively. In this case, the concentration measured by the fluorescence analyzer is compared with the killing criterion of the zooplankton, and it is judged that the zooplankton is killed if it is low. If the measured concentration is higher than the mortality criterion, it is judged that the zooplankton survived. In this case, it is necessary to ask the specialist agency for precise inspection to determine whether or not the treated water is discharged.

In addition, it is judged that the amount of phytoplankton is dominant when the concentration of the protein calculated from the sample is lower than that of fulvic acid and humic acid, respectively. In this case, the concentration measured by the fluorescence analyzer is compared with the killing criterion of the phytoplankton, and it is judged that the phytoplankton is killed when the concentration is lower than the killing criterion. If the measured concentration is higher than the mortality criterion, it is judged that the phytoplankton survived. In this case, it is necessary to ask the specialist agency for precise inspection to determine whether or not the treated water is discharged.

FIG. 2 shows the results of 3D fluorescence spectroscopic analysis of the protein, fulvic acid, and humic acid measured by the above method. Referring to FIG. 2, the fluorescence spectrum of the treated water is measured on the basis of the predetermined death criterion, and it is judged whether or not the biological killing is based on the death criterion.

When the protein concentration is higher than the concentration of fulvic acid or humic acid after irradiation with a single wavelength selected from the range of 250 nm to 700 nm with respect to the treated water to be measured and the fluorescence analysis is carried out, And if the concentration of the protein, fulvic acid, and humic acid in the measured ship equilibrium is lower than the criterion of death of the zooplankton, it is judged to be biodegradation.

When the concentration of the protein is lower than the concentration of fulvic acid or humic acid, fluorescence analysis is carried out on the ballast water to be measured, and it is judged that the phytoplankton is the criterion of death of the phytoplankton, and the measured protein of the ship equilibrium, If the concentration of humic acid is lower than the criterion of phytoplankton death, it is judged to be biodegradation.

If the concentration of protein, fulvic acid, and humic acid is lower than 1/10 of the killing rate of the phytoplankton, it can be judged that no phytoplankton is present. Therefore, it is analyzed on the basis of bacteria.

According to an embodiment of the present invention, the amount of chlorophyll-a may be calculated by measuring the light intensity after irradiating light of 620 nm to 700 nm further after S14 (S15). For the measurement of the absorption intensity, the fluorescence analysis apparatus described above may be used. In the case of calculating the amount of chlorophyll-a, the treated water can be measured after filtration with a 50 탆 filter. This can be used as an aid to improve the accuracy of phytoplankton survival.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

Setting the kill criteria for zooplankton, phytoplankton, and bacteria from the sterilized water, respectively;
Irradiating a single wavelength selected from the range of 250 nm to 700 nm to the treatment water to be measured and measuring intensity of emitted light using a fluorescence analyzer;
Calculating concentrations of protein, fulvic acid, and humic acid from the measured light intensities, respectively; And
Determining the presence or absence of at least one of zooplankton, phytoplankton, and bacteria by comparing the concentration of the protein, fulvic acid, and humic acid to the kill criterion to determine the viability of an organism present in the treated water. [Claim 2] The method according to claim 1, wherein the killing of the zooplankton is carried out by adding 10 zooplankton to 1 m &lt; ³ &gt; of sterilized water and keeping it for 24 hours, irradiating a single wavelength selected from 250 nm to 450 nm, Wherein the concentration of the protein, fructic acid and humic acid, respectively, is divided by 24. [2] The method according to claim 1, wherein the phytosphictal phytoplankton death criterion is determined by measuring a single wavelength selected from the range of 250 nm to 450 nm after irradiating 100 ml of phytoplankton into 10 ml of the sterilized water, holding it for 24 hours, Wherein the concentration of protein, fulvic acid and humic acid is divided by 24, respectively. [3] The method according to claim 1, wherein the bacterial death criterion is such that 100 dogs of phytoplankton are added to 10 ml of sterilized water, maintained for 24 hours, irradiated with a single wavelength selected from 250 nm to 450 nm, , The concentration of fulvic acid and the concentration of humic acid is divided by 240, respectively. The method according to claim 1, wherein the concentration of the protein in the treated water is higher than the concentration of fulvic acid or humic acid in the treated water, thereby determining whether or not the zooplankton is killed in comparison with the criterion of death of the zooplankton. The method according to claim 1, wherein the concentration of the protein in the treated water is lower than the concentration of fulvic acid or humic acid in the treated water to determine whether or not the phytoplankton is killed compared to the criterion of death of the phytoplankton. The method according to claim 1, wherein the concentration of the protein, fulvic acid and humic acid in the treated water is lower than the mortality criterion of phytoplankton to determine whether the bacteria are killed or not in comparison with the bacterial death criterion. [2] The fluorescence analysis apparatus of claim 1, wherein the fluorescence analysis apparatus includes a chamber for containing a sample, a light source for irradiating the chamber with light, a filter for changing the light emitted from the light source into a single wavelength, and a sensor for measuring intensity of light emitted from the sample &Lt; / RTI &gt; 2. The analysis method according to claim 1, further comprising the step of calculating the concentration of chlorophyll-a by measuring light absorption intensity after irradiating the treated water with light of 620 to 700 nm. The analytical method according to any one of claims 1 to 9, wherein the treated water is a ship ballast water.

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