JP7148127B2 - Methods and kits for detecting the presence of heavy metals in seawater - Google Patents

Description

The present invention relates to methods and kits for detecting the presence of heavy metals in seawater.

In recent years, attention has been focused on the development of seabed mineral resources. There is also concern about the risk of heavy metals leaking and diffusing into the surrounding waters when extracting seabed mineral resources. Therefore, there is a demand for development of a technique for simply measuring the presence of heavy metals in seawater.

Means for measuring the abundance of heavy metals in seawater include, for example, using an inductively coupled plasma mass spectrometer. However, it is extremely difficult to meet the installation requirements for the analyzer on ships, and it is difficult to detect the presence of heavy metals in seawater in areas where seafloor mineral resources are extracted.

For example, in Patent Document 1, adherent cells are selectively adherently cultured on a cell-adhesive membrane pattern surface on a substrate, and then the adherent cells are cultured in a culture medium containing a toxicity test chemical. Next, the adherent cells are stained, and the viable and dead adherent cells are counted by observation of the substrate to determine the viability of the adherent cells, thereby quantifying the toxicity of the toxicity test chemical substance. A cytotoxicity test method is described.

Patent Document 1 also describes that flow cytometry is used for cytotoxicity testing, that only dead cells incorporate trypan blue into cells when a trypan blue solution is added to a cell solution, and that a sample consisting of a large number of cells can be analyzed using a flow cytometer. 33342 and propidium iodide staining the DNA of unfixed cells with two dyes, Hoechst 33342 and propidium iodide. It is described that the cells emit red fluorescence and are easily distinguished between the two by a flow cytometer.

JP-A-10-327854

An object of the present invention is to provide a technique for detecting the presence of heavy metals in seawater.

The present invention includes the following aspects.
[1] A method for detecting the presence of heavy metals in seawater, comprising the following steps (a) to (c): (a) collecting seawater containing microorganisms, (b) staining dead microorganisms in the seawater (c) measuring the ratio of dead microorganisms to all microorganisms in the seawater, wherein the ratio detected in step (c) is , corresponding to abundance of heavy metals in said seawater.
[2] The method according to [1], wherein the staining agent for staining dead microorganisms is Propidium Iodide.
[3] In the step (b) or (c), a staining agent that stains both dead and live microorganisms is further added to the seawater to stain all the microorganisms in the seawater, [1] Or the method described in [2].
[4] The method of [3], wherein the staining agent that stains both dead and live microorganisms is Cybergreen I or a derivative thereof.
[5] The method according to any one of [1] to [4], wherein the step (c) is performed using a flow cytometer.
[6] The method according to any one of [1] to [5], wherein in step (c), the percentage of dead microorganisms is measured for each population of microorganisms.
[7] The method according to [6], wherein the population of microorganisms is a population containing Prochlorococcus and colorless bacteria, a population containing Synechococcus, or a population containing eukaryotic algae.
[8] The method according to any one of [1] to [7], further comprising culturing the microorganisms in the seawater after step (a) and before step (b).
[9] A kit for detecting the presence of heavy metals in seawater containing a stain that stains dead microorganisms.
[10] The kit according to [9], wherein the staining agent for staining dead microorganisms is Propidium Iodide.
[11] The kit of [9] or [10], further comprising a staining agent that stains both dead and live microorganisms.
[12] The kit according to [11], wherein the staining agent that stains both dead and live microorganisms is Cybergreen I or a derivative thereof.

According to the present invention, it is possible to provide a technique for detecting the presence of heavy metals in seawater.

(a) and (b) are graphs showing the results of flow cytometry analysis in Experimental Example 1. FIG. (a) to (f) are representative histograms measured in Experimental Example 2. (a) to (f) are representative histograms measured in Experimental Example 3. (a) to (c) are representative histograms measured in Experimental Example 4. FIG. (a) to (f) are representative histograms measured in Experimental Example 5. (g) is a graph showing the ratio of PI-positive microorganisms based on the results of (a) to (f). (a) to (f) are representative histograms measured in Experimental Example 6. (g) is a graph showing the ratio of PI-positive microorganisms based on the results of (a) to (f).

[Method for detecting the presence of heavy metals in seawater]
In one embodiment, the present invention provides a method for detecting the presence of heavy metals in seawater, comprising the following steps (a) to (c): (a) collecting seawater containing microorganisms; (b) said seawater; adding a staining agent for staining dead microorganisms to stain the dead microorganisms, (c) measuring the ratio of dead microorganisms to all microorganisms in the seawater, and performing the step (c) corresponds to the abundance of heavy metals in said seawater. As will be described later in Examples, the method of the present embodiment can easily detect the presence of heavy metals in seawater.

In step (a), seawater containing microorganisms is collected. This seawater is the target seawater for detecting the presence of heavy metals, and examples thereof include seawater collected in a sea area where seabed mineral resources are extracted by seabed excavation.

Seawater contains microorganisms. In the method of this embodiment, microorganisms contained in seawater, particularly phytoplankton, are used to detect the presence of heavy metals in seawater. The seawater collected in this step is preferably applied to the following step (b) immediately after collection. Immediately after collection may be, for example, within 1 hour after collection, or within 30 minutes after collection, for example.

In step (b), a staining agent for staining dead microorganisms is added to the collected seawater to stain the dead microorganisms. The staining agent for staining dead microorganisms is not particularly limited as long as it does not stain living microorganisms. ) can be preferably used. Staining with PI results in staining of dead microorganisms but not of live microorganisms.

Subsequently, in step (c), the ratio of dead microorganisms to total microorganisms in seawater is determined. Here, total microorganisms means the sum of live and dead microorganisms. For example, dead microorganisms are stained as a result of staining microorganisms in seawater with PI. Therefore, by measuring the number of all microorganisms in seawater and the number of dead microorganisms, it is possible to calculate the ratio of dead microorganisms to all microorganisms in seawater. The ratio of dead microorganisms to all microorganisms in seawater can be calculated, for example, by the following formula (1).

Proportion of dead microorganisms to all microorganisms in seawater (%) = Total number of dead microorganisms in seawater per unit volume / (Total number of dead microorganisms in seawater per unit volume + Viable microorganisms in seawater per unit volume) Total number of microorganisms) × 100 (1)

It is convenient to perform step (c) using a flow cytometer. Also, in recent years, small flow cytometers that can be mounted on ships have become commercially available. Therefore, if such a flow cytometer is used, for example, the method of the present embodiment can be carried out on-site using seawater sampled in the sea area of the seabed mineral resource extraction site. This can significantly reduce the time from sampling seawater to detecting the presence of heavy metals in seawater.

As described below in the Examples, the ratio of dead microorganisms to total microorganisms in seawater corresponds to the abundance of heavy metals in seawater. That is, the greater the abundance of heavy metals in seawater, the greater the proportion of dead microorganisms to the total microorganisms in seawater. Therefore, the method of this embodiment can estimate the abundance of heavy metals in seawater.

In step (b) or (c), the seawater may be further added with a staining agent that stains both dead and live microorganisms to stain all microorganisms in the seawater. This facilitates the determination of total microorganisms in seawater. Moreover, as will be described later, there are microorganisms that can be identified only by staining them with a staining agent that stains both dead and live microorganisms. Therefore, by staining with such a staining agent, it is possible to increase the types of microorganisms that can be used to detect the presence of heavy metals in seawater.

Cybergreen I or a derivative thereof can be used as a staining agent that stains both dead and live microorganisms. Cybergreen I (CAS number: 163795-75-3) is a dye that specifically binds to DNA and emits fluorescence. Derivatives of Cybergreen I include, but are not limited to, Cybergreen II (CAS number: 172827-25-7).

In step (c), the percentage of dead microorganisms may be determined for each population of microorganisms. In seawater, there are mainly groups containing Prochlorococcus and colorless bacteria, groups containing Synechococcus, and groups containing eukaryotic algae. These populations are expected to have different susceptibility to heavy metals. That is, the population of microorganisms may be a population containing Prochlorococcus and colorless bacteria, a population containing Synechococcus, or a population containing eukaryotic algae. In step (c), measuring the percentage of dead microorganisms for each population of microorganisms makes it possible to estimate the abundance of heavy metals in seawater.

Alternatively, measuring the percentage of dead microorganisms by population of microorganisms has the following advantages. As will be described later in the Examples, seawater in open ocean areas (open ocean seawater) in which demand for detection of the presence of heavy metals is expected may be oligotrophic. The inventors then found that Prochlorococcus and colorless bacteria are present in open ocean seawater in amounts sufficient to detect the presence of heavy metals. On the other hand, the inventors have clarified that the abundance of Synechococcus and eukaryotic algae is low in open ocean seawater.

Therefore, in the step (c), by measuring the proportion of dead microorganisms in a population containing Prochlorococcus and colorless bacteria, the presence of heavy metals in seawater can be detected even when oligotrophic open ocean seawater is used. can do. More specifically, the percentage of dead microorganisms for a population containing Prochlorococcus and colorless bacteria can be calculated, for example, by the following formula (2).

Percentage of dead organisms for population containing Prochlorococcus and colorless bacteria (%) = total number of dead Prochlorococcus and dead colorless bacteria in seawater per unit volume/(dead in seawater per unit volume) Total number of Prochlorococcus and dead colorless bacteria + total number of live Prochlorococcus and live colorless bacteria in seawater per unit volume)×100 (2)

The method of the present embodiment may further include a step of culturing microorganisms in seawater after step (a) and before step (b).

As will be described later in Examples, the inventors have clarified that the amount of Synechococcus and eukaryotic algae present in open ocean seawater is low. Then, after step (a) and before step (b), a step of culturing microorganisms in seawater is further performed to increase the abundance of Synechococcus and eukaryotic algae, and the method of the present embodiment is performed. revealed that it is possible to

In addition, as described above, in open ocean seawater, a population containing Prochlorococcus and colorless bacteria is present in a sufficient amount for measuring the abundance of heavy metals. Prior to b), by further performing the step of culturing microorganisms in seawater, the susceptibility of the population containing Prochlorococcus and colorless bacteria to heavy metals can be improved, and the detection sensitivity of heavy metals can be improved. did.

Cultivation of microorganisms in seawater can be performed, for example, by mixing a medium with seawater and allowing the mixture to stand. The culture time may be, for example, about 4 to 48 hours. Further, the culture temperature is, for example, room temperature. Moreover, when the culture time is less than 24 hours, the light and dark conditions during culture are preferably continuous light conditions. On the other hand, when the culture time is 24 hours or more, it is preferable to culture under the conditions of a light period of 14 hours and a dark period of 10 hours. The amount of light quanta in the light period may be 40-60 μmol photons m ⁻² s ⁻¹ .

When performing the step of culturing microorganisms in seawater, the seawater collected in step (a) is not applied to step (b) immediately after collection. In this case, it is preferable to apply the method to the step of culturing the seawater collected in step (a) immediately after collection. Here, "immediately after collection" may be, for example, within 1 hour after collection, or may be, for example, within 30 minutes after collection. Further, after the step of culturing microorganisms in seawater, it is preferably applied to step (b) within, for example, one hour, and preferably applied to step (b) within 30 minutes.

[Kit for detecting abundance of heavy metals in seawater]
In one embodiment, the invention provides a kit for detecting the presence of heavy metals in seawater that includes a stain that stains dead microorganisms. With the kit of this embodiment, the above-described method for detecting the presence of heavy metals in seawater can be carried out.

In the kit of the present embodiment, the staining agent for staining dead microorganisms is not particularly limited as long as it does not stain living microorganisms. For example, Propidium Iodide can be preferably used. Propidium Iodide is the same as described above.

The kit of this embodiment may further contain a staining agent that stains both dead and live microorganisms.

A stain that stains both dead and live microorganisms may be Cybergreen I or a derivative thereof. Cybergreen I or derivatives thereof are the same as those described above.

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental example 1]
(Examination of identification conditions for microbial populations)
Various types of phytoplankton are mixed in natural seawater. It is known that natural seawater in the open ocean mainly contains three groups of algae: Prochlorococcus, Synechococcus, and eukaryotic algae. In this experimental example, flow cytometry (FCM) was used to examine conditions for distinguishing these populations.

NIES-2885 strain (National Institute for Environmental Studies) was used as Prochlorococcus. In addition, NIES-969 strain (National Institute for Environmental Studies) was used as Synechococcus. As eukaryotic algae, strain NIES-1310 (National Institute for Environmental Studies) was used. First, these microorganisms were mixed cultured. Subsequently, the culture solution of microorganisms was analyzed using a flow cytometer (model "On-chip Sort", manufactured by On-Chip Biotechnologies).

FIG. 1(a) is a graph showing a typical FCM analysis result in the case of excitation with excitation light having a wavelength of 488 nm. The horizontal axis indicates fluorescence intensity measured using a 543/22 nm bandpass filter. The notation "543/22 nm" means that the bandpass filter has a center wavelength of 543 nm and a peak transmission bandwidth of 22 nm, and so on. The vertical axis indicates the fluorescence intensity measured using a 676/37 nm bandpass filter.

As a result, it was clarified that synechococcus and eukaryotic algal populations could be distinguished from each other. However, it became clear that the population of Prochlorococcus overlapped with the noise and could not be detected. In FIG. 1(a), "Syn" indicates a Synechococcus population, "Euk" indicates a eukaryotic algal population, and "Noise" indicates noise.

Subsequently, to the medium in which Prochlorococcus, Synechococcus, and eukaryotic algae were mixed and cultured, Cybergreen I (Thermo Fisher Scientific) was added according to the instruction manual so that the final concentration was 1:1. Let stand for 15 minutes. Subsequently, analysis was performed using a flow cytometer (model “On-chip Sort”, manufactured by On-Chip Biotechnologies).

FIG. 1(b) is a graph showing a typical FCM analysis result in the case of excitation with excitation light having a wavelength of 488 nm. The horizontal axis indicates the fluorescence intensity of FL2, and the vertical axis indicates the fluorescence intensity of FL5. FL2 was detected using a 543/22 nm bandpass filter. FL5 was detected using a 676/37 nm bandpass filter.

As a result, it was revealed that the Prochlorococcus population could be separated from the noise by staining with Cyber Green I, and that the Synechococcus population, the eukaryotic algae population and the Prochlorococcus population could be distinguished from each other. In FIG. 1(b), "Syn" indicates a population of Synechococcus, "Euk" indicates a population of eukaryotic algae, "Pro" indicates a population of Prochlorococcus, and "Noise" indicates noise.

From the above results, it was clarified that staining with SYBR GREEN I enables identification of populations of Prochlorococcus, Synechococcus and eukaryotic algae. In the analysis using actual seawater, colorless bacteria are also detected in the region where Prochlorococcus is detected.

[Experimental example 2]
(Measurement of abundance of heavy metals in bay seawater)
Gulf seawater was used to determine the proportion of dead microorganisms in populations containing Prochlorococcus and colorless bacteria to detect the presence of heavy metals.

Specifically, first, the seawater in the bay immediately after sampling in the subtropical coastal waters of Japan is dispensed into containers, and zinc, lead or copper with a final concentration of 0.5 ppm, and zinc, lead or copper with a final concentration of 5 ppm. Each copper was added and allowed to stand at room temperature for 4 hours.

Subsequently, 3 μL of Propidium Iodide (Dojindo Laboratories, hereinafter sometimes referred to as “PI”) dissolved in 0.15 mM in dimethyl sulfoxide (DMSO) was added to 100 μL of each seawater, and Cyber Green I (100 times concentration stock solution) was added and left at room temperature for 15 minutes.

Subsequently, analysis was performed using a flow cytometer (model “On-chip Sort”, manufactured by On-Chip Biotechnologies). This flow cytometer can be mounted on a ship. First, it was excited with excitation light having a wavelength of 488 nm, and analysis results were obtained in which the horizontal axis indicates the fluorescence intensity of FL2 and the vertical axis indicates the fluorescence intensity of FL6. FL2 was detected using a 543/22 nm bandpass filter. FL6 was also detected using a >710 nm long-pass filter. Note that the expression “>710 nm” means that the long-pass filter transmits light with a wavelength of 710 nm or longer, and the same applies hereinafter. Subsequently, based on the analysis results obtained, the population containing Prochlorococcus and colorless bacteria was gated.

Subsequently, a histogram showing the fluorescence intensity of FL5 on the horizontal axis was obtained for the microorganisms in the set gates, and the ratio of PI-stained microorganisms was observed. Microorganisms stained with PI are dead organisms. FL5 was detected using a 676/37 nm bandpass filter. As a control, the same analysis was performed on microorganisms not exposed to heavy metals.

Figures 2(a)-(f) are representative histograms obtained. In FIGS. 2(a) to 2(f), the horizontal axis indicates the intensity of PI staining (relative value), and the vertical axis indicates the number of cells (relative value). Figure 2 (a) shows the results of microorganisms exposed to 0.5 ppm zinc, Figure 2 (b) shows the results of microorganisms exposed to 0.5 ppm lead, and Figure 2 (c) shows the results of 0.5 ppm zinc. Figure 2(d) shows the results of microorganisms exposed to copper, Figure 2(e) shows the results of microorganisms exposed to 5 ppm lead, Figure 2(f) shows the results of microorganisms exposed to 5 ppm of zinc. ) are the results for microorganisms exposed to 5 ppm copper.

As a result, the proportion of PI-stained microbes increased significantly with 0.5 ppm copper exposure, 5 ppm zinc exposure, 5 ppm lead exposure, and 5 ppm copper exposure. It became clear that This result indicates that the abundance of heavy metals in seawater can be estimated by detecting an increase in the proportion of PI-stained microorganisms.

[Experimental example 3]
(Measurement of abundance of heavy metals in open ocean seawater - eukaryotic algae)
It is envisioned that the offshore drilling area will be in the open ocean. Therefore, we measured the proportion of dead microorganisms in the microbial population in open ocean seawater, where the demand for measuring the abundance of heavy metals was assumed, and estimated the abundance of heavy metals. As open ocean seawater, freshly collected seawater from subtropical waters in Japan was used.

Preliminary studies have shown that microorganisms in open ocean seawater are often physiologically inactive compared to microorganisms in bay seawater, which are rich in nutrients, and tend to be more resistant to heavy metals than microorganisms in bay seawater. It became clear.

In this experimental example, attention was paid to eukaryotic algae in open ocean seawater, and the abundance of heavy metals was measured. As a result of preliminary investigation, it was clarified that the abundance of eukaryotic algae is small in oligotrophic open ocean seawater. Therefore, first, an ESM medium was added to open ocean seawater to a final concentration of 25%, and the mixture was allowed to stand at room temperature for 4 hours for pre-culture. The culture conditions were a continuous light period and a photon amount of 40 to 60 μmol photons m ⁻² s ⁻¹ .

Table 1 below shows the composition of the ESM medium. In Table 1, soil extracts were prepared as follows. First, 200 mL of soil (soil derived from deciduous broad-leaved trees is preferred) was added to 1000 mL of distilled water, and the mixture was autoclaved at 105° C. for 1 hour. After the sample had cooled, it was again autoclaved at 105°C for 1 hour. Subsequently, the supernatant of the sample was passed through glass fiber filter paper (model "GF/C", GE Healthcare) and Celite, and the filtrate was further passed through glass fiber filter paper (model "GF/F", GE Healthcare). passed. Subsequently, distilled water was added to the filtrate to adjust the volume to 1000 mL. Subsequently, the filtrate was transferred to a 10 mL test tube, autoclaved at 121° C. for 20 minutes, and stored in the cold.

Subsequently, the pre-cultured seawater was dispensed into a container, zinc, lead or copper with a final concentration of 1 ppm and zinc, lead or copper with a final concentration of 5 ppm were added, respectively, and allowed to stand at room temperature for 4 hours.

Subsequently, 3 μL of PI dissolved in 0.15 mM in DMSO was added to 100 μL of each seawater and allowed to stand at room temperature for 15 minutes.

Subsequently, analysis was performed using a flow cytometer (model “On-chip Sort”, manufactured by On-Chip Biotechnologies). First, it was excited with excitation light having a wavelength of 488 nm, and analysis results were obtained in which the horizontal axis indicates the fluorescence intensity of FL2 and the vertical axis indicates the fluorescence intensity of FL6. FL2 was detected using a 543/22 nm bandpass filter. FL6 was also detected using a >710 nm long-pass filter. The eukaryotic algal population was then gated based on the results of the analysis obtained.

Subsequently, a histogram showing the fluorescence intensity of FL5 on the horizontal axis was obtained for the microorganisms in the set gates, and the ratio of PI-stained microorganisms was observed. Microorganisms stained with PI are dead organisms. FL5 was detected using a 676/37 nm bandpass filter. As a control, the same analysis was performed on microorganisms not exposed to metals.

Figures 3(a)-(f) are representative histograms obtained. In FIGS. 3(a) to 3(f), the horizontal axis indicates the PI staining intensity (relative value), and the vertical axis indicates the number of cells (relative value). FIG. 3(a) shows the results of eukaryotic algae exposed to 1 ppm zinc, FIG. 3(b) shows the results of eukaryotic algae exposed to 1 ppm lead, and FIG. 3(c) shows the results of 1 ppm copper. FIG. 3(d) is the result of eukaryotic algae exposed to 5 ppm zinc, FIG. 3(e) is the result of eukaryotic algae exposed to 5 ppm lead, FIG. 3(f) shows the results of eukaryotic algae exposed to 5 ppm copper.

As a result, in eukaryotic algae exposed to 1 ppm zinc, eukaryotic algae exposed to 5 ppm zinc, and eukaryotic algae exposed to 5 ppm copper, there was a tendency for the ratio of PI-stained microorganisms to increase. . This result indicates that the abundance of heavy metals in seawater can be estimated by detecting an increase in the percentage of PI-stained eukaryotic algae. In addition, even in seawater that is oligotrophic and has a low abundance of eukaryotic algae, pre-cultivation can increase the abundance of eukaryotic algae, and furthermore, pre-cultured eukaryotic algae can be used in seawater. of heavy metals can be detected.

[Experimental example 4]
(Measurement of abundance of heavy metals in open ocean seawater - Synechococcus)
In this experimental example, attention was focused on Synechococcus in open ocean seawater, and the presence of heavy metals was detected. As open ocean seawater, freshly collected seawater from subtropical waters in Japan was used.

As a result of preliminary examination, it was clarified that Synechococcus abundance was low in oligotrophic open ocean seawater. Therefore, first, an ESM medium was added to open ocean seawater to a final concentration of 25%, and the mixture was allowed to stand at room temperature for 4 hours for pre-culture. The composition of the ESM medium and the culture conditions are as described above in Experimental Example 3.

Subsequently, the pre-cultured seawater was dispensed into a container, zinc, lead, or copper with a final concentration of 5 ppm was added thereto, and the container was allowed to stand at room temperature for 4 hours.

Subsequently, 3 μL of PI dissolved in 0.15 mM in DMSO was added to 100 μL of each seawater and allowed to stand at room temperature for 15 minutes.

Subsequently, analysis was performed using a flow cytometer (model “On-chip Sort”, manufactured by On-Chip Biotechnologies). First, it was excited with excitation light having a wavelength of 488 nm, and analysis results were obtained in which the horizontal axis indicates the fluorescence intensity of FL2 and the vertical axis indicates the fluorescence intensity of FL6. FL2 was detected using a 543/22 nm bandpass filter. FL6 was also detected using a >710 nm long-pass filter. The Synechococcus population was then gated based on the analysis results obtained.

Subsequently, a histogram showing the fluorescence intensity of FL5 on the horizontal axis was obtained for the microorganisms in the set gates, and the ratio of PI-stained microorganisms was observed. Microorganisms stained with PI are dead organisms. FL5 was detected using a 676/37 nm bandpass filter. As a control, the same analysis was performed on microorganisms not exposed to metals.

Figures 4(a)-(c) are representative histograms obtained. In FIGS. 4(a) to 4(c), the horizontal axis indicates the intensity of PI staining (relative value), and the vertical axis indicates the number of cells (relative value). Figure 4 (a) is the result of Synechococcus exposed to 5 ppm zinc, Figure 4 (b) is the result of Synechococcus exposed to 5 ppm lead, Figure 4 (c) is the result of Synechococcus exposed to 5 ppm copper This is the result.

As a result, in Synechococcus exposed to 5 ppm zinc and Synechococcus exposed to 5 ppm copper, there was a tendency that the ratio of PI-stained microorganisms increased. This result indicates that the abundance of heavy metals in seawater can be estimated by detecting an increase in the proportion of Synechococcus stained with PI. In addition, even in seawater that is oligotrophic and has a low abundance of Synechococcus, pre-culture can increase the abundance of Synechococcus. It became clear that it is possible to estimate

[Experimental example 5]
(Detection of the Presence of Heavy Metals in Open Sea Seawater - Population 1 Containing Prochlorococcus and Colorless Bacteria)
In this experimental example, the presence of heavy metals was detected by focusing on populations containing Prochlorococcus and colorless bacteria in open ocean seawater. As open ocean seawater, freshly collected seawater from subtropical waters in Japan was used.

As a result of preliminary examination, it was clarified that Prochlorococcus and colorless bacteria existed in open seawater in sufficient quantity for analysis, although open seawater is oligotrophic. Therefore, first, the presence of heavy metals was detected without performing pre-culture.

Open ocean seawater was dispensed into a container, zinc, lead or copper with a final concentration of 1 ppm and zinc, lead or copper with a final concentration of 5 ppm were added, respectively, and allowed to stand at room temperature for 4 hours.

Subsequently, 3 μL of PI dissolved at 0.15 mM in DMSO and 1 μL of SYBRGREEN I (100× stock solution) were added to 100 μL of each seawater and allowed to stand at room temperature for 15 minutes.

Subsequently, analysis was performed using a flow cytometer (model “On-chip Sort”, manufactured by On-Chip Biotechnologies). First, it was excited with excitation light having a wavelength of 488 nm, and analysis results were obtained in which the horizontal axis indicates the fluorescence intensity of FL2 and the vertical axis indicates the fluorescence intensity of FL6. FL2 was detected using a 543/22 nm bandpass filter. FL6 was also detected using a >710 nm long-pass filter. Subsequently, based on the analysis results obtained, the population containing Prochlorococcus and colorless bacteria was gated.

Subsequently, a histogram showing the fluorescence intensity of FL5 on the horizontal axis was obtained for the microorganisms in the set gates, and the ratio of PI-stained microorganisms was observed. Microorganisms stained with PI are dead organisms. FL5 was detected using a 676/37 nm bandpass filter. As a control, the same analysis was performed on microorganisms not exposed to metals.

Figures 5(a)-(f) are representative histograms obtained. FIG. 5(g) is a graph showing the proportion of PI-positive microorganisms based on the results of FIGS. 5(a)-(f). In FIGS. 5(a) to 5(f), the horizontal axis indicates the intensity of PI staining (relative value), and the vertical axis indicates the number of cells (relative value). FIG. 5(a) shows the results of microorganisms exposed to 1 ppm of zinc, FIG. 5(b) shows the results of microorganisms exposed to 1 ppm of lead, and FIG. 5(c) shows the results of microorganisms exposed to 1 ppm of copper. FIG. 5(d) is the result of microorganisms exposed to 5 ppm zinc, FIG. 5(e) is the result of microorganisms exposed to 5 ppm lead, and FIG. 5(f) is the result of 5 ppm copper. It is the result of exposed microorganisms. In FIG. 5(g), "Zn" indicates zinc, "Pb" indicates lead, and "Cu" indicates copper.

As a result, it was found that the ratio of PI-stained microorganisms was clearly increased in microorganisms exposed to 1 ppm copper and microorganisms exposed to 5 ppm copper.

This result further supports the ability to estimate the abundance of heavy metals in seawater by detecting an increase in the proportion of PI-stained microorganisms in populations containing Prochlorococcus and colorless bacteria.

In addition, even when using oligotrophic open ocean seawater, it is clear that the presence of heavy metals in seawater can be detected without pre-culture by focusing on populations containing Prochlorococcus and colorless bacteria. became.

[Experimental example 6]
(Detection of the Presence of Heavy Metals in Open Sea Seawater - Population 2 Containing Prochlorococcus and Colorless Bacteria)
In this experimental example, the presence of heavy metals was detected by focusing on populations containing Prochlorococcus and colorless bacteria in open ocean seawater. As open ocean seawater, freshly collected seawater from subtropical waters in Japan was used.

As a result of preliminary examination, it was clarified that Prochlorococcus and colorless bacteria exist in the open ocean seawater in sufficient quantity for analysis. amount was measured. First, ESM medium was added to open ocean seawater to a final concentration of 25%, and the mixture was allowed to stand at room temperature for 4 hours for pre-culture. The composition of the ESM medium and the culture conditions are as described above in Experimental Example 3.

Subsequently, the pre-cultured seawater was dispensed into a container, zinc, lead or copper with a final concentration of 1 ppm and zinc, lead or copper with a final concentration of 5 ppm were added, respectively, and allowed to stand at room temperature for 4 hours.

Subsequently, 3 μL of PI dissolved at 0.15 mM in DMSO and 1 μL of SYBRGREEN I (100× stock solution) were added to 100 μL of each seawater and allowed to stand at room temperature for 15 minutes.

Subsequently, analysis was performed using a flow cytometer (model “On-chip Sort”, manufactured by On-Chip Biotechnologies). First, it was excited with excitation light having a wavelength of 488 nm, and analysis results were obtained in which the horizontal axis indicates the fluorescence intensity of FL2 and the vertical axis indicates the fluorescence intensity of FL6. FL2 was detected using a 543/22 nm bandpass filter. FL6 was also detected using a >710 nm long-pass filter. Subsequently, based on the analysis results obtained, the population containing Prochlorococcus and colorless bacteria was gated.

Subsequently, a histogram showing the fluorescence intensity of FL5 on the horizontal axis was obtained for the microorganisms in the set gates, and the ratio of PI-stained microorganisms was observed. Microorganisms stained with PI are dead organisms. FL5 was detected using a 676/37 nm bandpass filter. As a control, the same analysis was performed on microorganisms not exposed to metals.

Figures 6(a)-(f) are representative histograms obtained. FIG. 6(g) is a graph showing the ratio of PI-positive microorganisms based on the results of FIGS. 6(a) to (f). In FIGS. 6(a) to 6(f), the horizontal axis indicates the PI staining intensity (relative value), and the vertical axis indicates the number of cells (relative value). FIG. 6(a) shows the results of microorganisms exposed to 1 ppm of zinc, FIG. 6(b) shows the results of microorganisms exposed to 1 ppm of lead, and FIG. 6(c) shows the results of microorganisms exposed to 1 ppm of copper. 6(d) is the result of microorganisms exposed to 5 ppm of zinc, FIG. 6(e) is the result of microorganisms exposed to 5 ppm of lead, and FIG. 6(f) is the result of 5 ppm of copper. It is the result of exposed microorganisms. In FIG. 6(g), "Zn" indicates zinc, "Pb" indicates lead, and "Cu" indicates copper.

As a result, it was found that the ratio of PI-stained microorganisms was clearly increased in microorganisms exposed to 1 ppm copper and microorganisms exposed to 5 ppm copper. In addition, even in microorganisms exposed to 5 ppm of zinc, there was a tendency for the ratio of PI-stained microorganisms to increase.

This result further supports the ability to estimate the abundance of heavy metals in seawater by detecting an increase in the proportion of PI-stained microorganisms in populations containing Prochlorococcus and colorless bacteria.

Moreover, even when the presence of heavy metals in seawater is detected by focusing on a population containing Prochlorococcus and colorless bacteria, pre-cultivation can increase the sensitivity to heavy metals in seawater. It became clear.

According to the present invention, it is possible to provide a technique for simply detecting the presence of heavy metals in seawater in a shipboard laboratory.

Claims (10)

A method for detecting the presence of heavy metals in seawater, comprising:
The following steps (a) to (c):
(a) collecting seawater containing microorganisms;
(b) adding a staining agent for staining dead microorganisms to the seawater to stain the dead microorganisms;
(c) measuring the percentage of dead microorganisms to total microorganisms in said seawater;
and a higher percentage detected in step (c) compared to the control indicates the presence of heavy metals in the seawater;
In step (c), the percentage of dead microorganisms is measured for each population of microorganisms;
the population of microorganisms is a population containing eukaryotic algae, and the heavy metal is 1 ppm or more of zinc or 5 ppm or more of copper;
the population of microorganisms is a population containing Synechococcus, and the heavy metal is 5 ppm or more of zinc or 5 ppm or more of copper;
A method according to claim 1, wherein said population of microorganisms is a population comprising Prochlorococcus and colorless bacteria, and said heavy metal is 5 ppm or more of zinc or 1 ppm or more of copper . 2. The method of claim 1, wherein the stain that stains dead microorganisms is Propidium Iodide. 3. The method according to claim 1 or 2, wherein in the step (b) or (c), a staining agent that stains both dead and live microorganisms is further added to the seawater to stain all the microorganisms in the seawater. described method. 4. The method of claim 3, wherein said staining agent that stains both dead and live microorganisms is SYBR Green I or a derivative thereof. The method according to any one of claims 1 to 4, wherein said step (c) is performed using a flow cytometer. The method according to any one of claims 1 to 5 , further comprising culturing the microorganisms in the seawater after step (a) and before step (b). A kit for detecting the presence of heavy metals in seawater comprising a staining agent for staining dead microorganisms and for use in the method of any one of claims 1-5 . 8. The kit of claim 7 , wherein the stain that stains dead microorganisms is Propidium Iodide. 9. A kit according to claim 7 or 8 , further comprising a staining agent that stains both dead and live microorganisms. 10. A kit according to claim 9 , wherein said staining agent that stains both dead and live microorganisms is Cybergreen I or a derivative thereof.

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