JP7425565B2 - Analysis method for microorganisms in groundwater - Google Patents

Description

The present invention relates to a method for analyzing microorganisms in groundwater.

In recent years, as part of the technological development for geological disposal of high-level radioactive waste, efforts have been made to collect scientific data deep underground and to interpret the geological environment based on the collected scientific data. As one of the results, the "Scientific Characteristics Map" (Ministry of Economy, Trade and Industry, Agency for Natural Resources and Energy: July 28, 2017) has been published. However, as there is little existing knowledge about microbial data deep underground and it has been difficult to create a wide-area database, it is not currently included in the "Scientific Characteristics Map."
On the other hand, looking at international trends, it has been pointed out that it is necessary to incorporate microbial data as one of the basic data when considering geological disposal of high-level radioactive waste. Here, in order to incorporate microbial data as one of the basic data, it is essential to accumulate microbial data, and in particular, it is essential to create a wide-area database. To this end, it is necessary to efficiently collect microbial data as basic data in the same way as geological property data, hydraulic data, water quality data, etc., and it is necessary to systematize the collection of basic data.
Conventionally, microbial data is obtained by sequentially performing analysis of each analysis item such as counting the number of microorganisms, analyzing microbial community composition, analyzing microbial community structure, and analyzing microbial activity. collected. That is, as shown in FIG. 6, microbial data is collected based on multiple surveys conducted at different times. In each survey, analysis and data collection are carried out targeting one analysis item, and depending on the results of that analysis and data collection, the content of the next survey (specifically, the target of analysis and data collection) is determined. analysis items, etc.) are planned.
Here, as a method for counting the number of microorganisms, for example, the method disclosed in Patent Document 1 is known. Further, as methods for analyzing microbial communities (community composition/community structure), for example, methods disclosed in Patent Documents 2 and 3 are known. Furthermore, as a method for analyzing the activity of microorganisms, for example, the method disclosed in Patent Document 4 is known.

Japanese Patent Application Publication No. 2011-92104 Japanese Patent Application Publication No. 2006-94830 Japanese Patent Application Publication No. 2005-65605 Japanese Patent Application Publication No. 2006-238771

However, with the conventional method, the analysis period may become long.
That is, in the conventional method, groundwater is sampled each time each analysis item is analyzed. As a result, as the number of analysis items increases, the number of times water sampling is performed increases, and as a result, the analysis period may become longer. In particular, when the permeability of the ground targeted for groundwater sampling (hereinafter referred to as "targeted ground") is low, or when the depth of the target ground is deep, the period required for each water sampling may be long. Because of this, the analysis period may become even longer.
An object of the present invention is to shorten the analysis period when analyzing microorganisms in groundwater.

In order to achieve the above object, the method for analyzing microorganisms in groundwater according to the first invention includes a water sampling step of sampling groundwater, and a main analysis using the groundwater sampled in the water sampling step as a sample. The main analysis step includes a plurality of analysis items including at least one of microbial counting, microbial community analysis, and microbial activity analysis as analysis items of the main analysis, and in the water sampling step, A preliminary analysis, which is an analysis to determine the amount of groundwater sampled for each analysis item included in the main analysis, was conducted on site using the groundwater sampled in the preliminary water sampling. Based on the results , determine the amount of groundwater required for each analysis item included in the main analysis, and according to the determined amount of water , collect the groundwater required for all analysis items included in the main analysis. In carrying out the main water sampling, the preliminary water sampling and the main water sampling are performed through a series of water sampling operations, and the preliminary analysis includes microbial counting.
In the method for analyzing microorganisms in groundwater according to the first invention, preliminary analysis is performed using groundwater sampled by preliminary water sampling , and based on the results of the preliminary analysis, necessary for each analysis item included in the main analysis. The amount of groundwater to be sampled is determined, and main water sampling is carried out to sample the groundwater required for all analysis items included in the main analysis according to the determined amount of sampled water. At this time, preliminary water sampling and main water sampling will be carried out through a series of water sampling operations.
This prevents the occurrence of a shortage of samples necessary for the main analysis. Therefore, it is possible to avoid sampling water for replenishing a missing sample, and as a result, it is possible to shorten the analysis period.
Furthermore, by performing water sampling once, it is possible to sample water samples required for each analysis item included in this analysis. Therefore, even if multiple analysis items are included in this analysis, there is no need to sample water for each analysis item. Therefore, the number of times water sampling is performed is reduced, and as a result, the analysis period can be shortened.
In particular, when the permeability of the target ground is low or when the target ground is deep, the analysis period can be significantly shortened.
Furthermore, in the method for analyzing microorganisms in groundwater according to the first invention, the analysis items of the preliminary analysis include counting the number of microorganisms.
This makes it possible to appropriately determine the amount of groundwater required for this analysis. In other words, when performing various analyzes such as counting the number of microorganisms, analyzing microbial communities (community composition, community structure), and analyzing microbial activity, it is necessary to calculate the number of microorganisms present per unit volume of groundwater as a sample. , greatly influences the determination of the amount of sample (i.e., the amount of water sampled) required for the analysis. At this time, the smaller the number of microorganisms present per unit amount of water, the larger the amount of sample required. Therefore, by including microorganism count as an analysis item in the preliminary analysis, it becomes possible to appropriately determine the amount of sample required for the main analysis.
Here, the water sampling process corresponds to the second preliminary water sampling and main water sampling described later. This analysis includes counting the number of microorganisms, analyzing the microbial community (community structure, community composition, etc.), analyzing the activity of the microorganisms, analyzing the functions of the microorganisms, etc., which will be described later. This analysis process includes a microorganism counting process, a microbial community structure analysis process, a microbial community composition analysis process, a microbial activity analysis process, etc., which will be described later. The preliminary analysis corresponds to the preliminary analysis described below. The counting of the number of microorganisms corresponds to the counting of the number of microorganisms described below. Microbial community analysis corresponds to the analysis of microbial communities (community structure, community composition, etc.) described below. The microbial activity analysis corresponds to the analysis of microbial activity described below. The field refers to the water sampling site described later.

In the method for analyzing microorganisms in groundwater according to the second invention, in the method for analyzing microorganisms in groundwater according to the first invention, in the water sampling step, groundwater existing at a deep depth is sampled, A simple measurement of the metal ion concentration is carried out on the groundwater sampled by the method, and according to the result of the simple measurement, a metal ion chelating agent is added to the groundwater sampled by the preliminary water sampling. The preliminary analysis is performed using groundwater to which the agent has been added as a sample, and the preliminary water sampling, preliminary water sampling, and main water sampling are performed through a series of water sampling operations .
In the method for analyzing microorganisms in groundwater according to the second invention, a preliminary analysis is performed using groundwater to which a chelating agent has been added as a sample.
This suppresses the precipitation of metal ions dissolved in the sampled groundwater, making it possible to prevent precipitated metal ions from interfering with microscopic observation during preliminary analysis. Become.
Here, the simple measurement of metal ion concentration corresponds to the simple measurement of dissolved iron ion concentration described below. Examples of metal ions include iron ions, which will be described later. As the chelating agent, EDTA (ethylenediaminetetraacetic acid), which will be described later, is applicable.

According to the present invention, it is possible to shorten the analysis period when analyzing microorganisms in groundwater. In particular, when the permeability of the target ground is low or when the target ground is deep, the analysis period can be significantly shortened.

1 is a flowchart showing the procedure of a method for analyzing microorganisms in groundwater according to an embodiment of the present invention. It is a flowchart which shows the procedure of a metal ion precipitation suppression process. FIG. 2 is a diagram showing the correspondence between the number of microorganisms calculated by a preliminary analysis and the amount of water sampled required for each analysis item in the main analysis. 1 is a diagram showing a schematic configuration of a water sampling device 1. FIG. FIG. 2 is a diagram showing the water sampling device 1 in a state in which a pump P is installed in a pumping casing 10. FIG. 1 is a flowchart showing the procedure of a conventional method for analyzing microorganisms in groundwater.

Hereinafter, a method for analyzing microorganisms in groundwater according to an embodiment of the present invention will be described with reference to the drawings.

(Steps for analyzing microorganisms in groundwater)
First, the procedure of a method for analyzing microorganisms in groundwater according to an embodiment of the present invention will be explained.
FIG. 1 is a flowchart showing the procedure of a method for analyzing microorganisms in groundwater according to an embodiment of the present invention. FIG. 2 is a flowchart showing the procedure of the metal ion precipitation suppression step. FIG. 3 is a diagram showing the correspondence between the number of microorganisms calculated by the preliminary analysis and the amount of water sampled required for each analysis item in the main analysis.
The method for analyzing microorganisms in groundwater according to the present invention uses groundwater gushing out from the target ground (hereinafter referred to as "target groundwater") as a sample, and conducts an analysis regarding microorganisms (hereinafter referred to as "main analysis"), The purpose is to obtain data on microorganisms.
In particular, the method for analyzing microorganisms in groundwater according to the present invention is applicable when the sample is groundwater (target groundwater) gushing out from the ground (target ground) at a great depth (specifically, several hundred meters or more). This is the preferred method.
Here, the term "microorganisms" according to the present invention refers to minute organisms whose individual existence cannot be identified with the naked eye, and includes prokaryotes, eukaryotes (eucaria), and the like.
The term "prokaryote" refers to an organism composed of cells that do not have a nuclear envelope and have DNA exposed inside the cell, and includes bacteria (eubacteria) and archaea (archaea).
"Eukaryotes" refer to organisms that are composed of cells that have nuclear membranes inside them and clearly distinguish the nucleus and cytoplasm.
In this embodiment, an example will be described in which microorganism-related analysis is performed on prokaryotes (bacteria and archaea) to obtain data regarding the microorganisms.
In addition, the analysis items of "Analysis related to microorganisms (main analysis)" according to the present invention include counting the number of microorganisms, analysis of microbial communities (community structure, community composition, etc.), analysis of microbial activity, and analysis of microbial functions. Among analysis items such as analysis, at least one (preferably plural) analysis items are included.
In this embodiment, an example will be described in which the analysis items of the main analysis include counting the number of microorganisms, analyzing the community of microorganisms, and analyzing the activity of microorganisms.
"Number of microorganisms" refers to the number of microorganisms present per unit amount of water in a sample.
"Microbial community" refers to the contents of the microbial group present in a sample (types (classifications) of microorganisms, proportion of microorganisms belonging to each type, number of microorganisms belonging to each type, etc.).
In this embodiment, as microbial community analysis, microbial community structure analysis and microbial community composition analysis are performed.
The "microbial community structure" is the proportion (or number) of microorganisms belonging to the target classification in the microbial group present in the sample. In this embodiment, the proportion of bacteria and the proportion of archaea in the microbial group present in the sample is analyzed by analyzing the community structure of microorganisms.
"Microbial community composition" is the type of microorganisms present in the sample. In this embodiment, the types of bacteria and archaea present in the sample are analyzed by analyzing the community composition of microorganisms.

As shown in FIG. 1, the method for analyzing microorganisms in groundwater according to the present embodiment includes a water sampling preparation process, a water sampling process, a pretreatment process, and various main analysis processes. There is.
Generally, when performing microbial analysis using groundwater as a sample, it is necessary to transport the sample collected at the water sampling site to the laboratory where the analysis will be performed. In this case, the farther the distance between the water sampling site and the laboratory, the longer the time required for transportation, and as a result, the risk that the state of microorganisms present in the sample will change during transportation increases.
Therefore, in the method for analyzing microorganisms in groundwater according to the present embodiment, an on-site laboratory is temporarily set up at the water sampling site, and processes that require a quick response (specifically, the metal ion precipitation suppression process, preliminary analysis, water sampling amount determination, pretreatment process), and analyzes that require prompt implementation among the analysis items of this analysis (specifically, the microbial activity analysis process described below) will be conducted in the on-site laboratory. It is structured as follows.

(Water sampling preparation process)
In the water sampling preparation process, preparations are made for sampling the target groundwater.
Specifically, in the water sampling preparation process, first, a borehole (bore hole) is excavated to construct a well for water sampling. At this time, the borehole is drilled to a depth deeper than the depth of the target ground. Further, the borehole (borehole) may be used as a well as it is, or a well may be constructed by arranging a casing pipe within the borehole. Furthermore, when constructing a well, a fluorescent dye is added to residual water in the well (rainwater, excavated water, etc. remaining in the well).
In the water sampling preparation step, next, a water sampling device is installed in the well. Here, water sampling devices include water sampling devices that use a water sampling pump to sample water, water sampling devices that use a water sampling device to sample water, and water sampling devices that use a packer system. It is possible to use any known water sampling device, such as a water device. In particular, when sampling groundwater (target groundwater) gushing out from the ground (target ground) at a great depth (specifically, several hundred meters or more), it is preferable to use the water sampling device 1 described below. .
In the water sampling preparation process, preliminary drainage is next performed. In other words, residual water remains in the constructed well. As a result, when the installation of the water sampling device is completed, the residual water that had accumulated in the well has entered the water sampling device. Therefore, it is necessary to replace the residual water in the water sampling device with the target groundwater by preliminary drainage (drawing the target groundwater into the water sampling device).
Preliminary drainage is performed by inserting a drainage pump into the water sampling device, activating the drainage pump, and draining the remaining water in the water sampling device.
During preliminary drainage, a water quality monitoring device measures and monitors the water quality (physicochemical parameters) of the drained groundwater (residual water) over time. In this embodiment, water quality (physicochemical parameters) such as EC (electrical conductivity), temperature, water pressure, pH (hydrogen ion index), ORP (oxidation reduction potential), DO (dissolved oxygen concentration), etc. are measured and monitored. Ru. Additionally, during preliminary drainage, the fluorescent dye concentration in the drained groundwater (residual water) is measured and monitored over time. Furthermore, during preliminary drainage, the total amount of drained groundwater (residual water) is measured.
Then, (1) the water quality (physicochemical parameters) of the drained groundwater has stabilized, (2) the fluorescent dye concentration in the drained groundwater has decreased sufficiently, and (3) the system volume (specifically , the volume of the water sampling device or the volume of the well), and that a sufficient amount of groundwater has been drained. That is, if these conditions are met, it can be determined that the residual water has been replaced with target groundwater in the water sampling device.
Then, upon completion of the preliminary drainage, the target groundwater becomes ready for sampling, and the water sampling preparation process is completed.

(Water sampling process)
In the water sampling process, target groundwater will be sampled.
Specifically, in the water sampling process, first, sampling of target groundwater is started. That is, sampling of the target groundwater is started using a method suitable for the water sampling device installed in the well.
Here, the sampling of target groundwater includes the first preliminary sampling, in which target groundwater is sampled for simple measurement of dissolved iron ion concentration, which will be described later, and the target groundwater, which will be a sample for preliminary analysis, which will be described later. This includes a second preliminary water sampling and a main water sampling in which target groundwater is sampled for the main analysis (each analysis item) to be described later. Then, sampling of the target groundwater is terminated upon completion of the main sampling. At this time, sampling of the target groundwater is carried out as a series of steps from start to finish.
In other words, the target groundwater sampled during a series of processes from the start to the end of sampling target groundwater is sequentially used as a sample for simple measurement of dissolved iron ion concentration, as a sample for preliminary analysis, and then as a sample for main analysis. It is said to be a sample of Therefore, in this embodiment, the first preliminary water sampling, the second preliminary water sampling, and the main water sampling are a series of steps and are not clearly separated.
Then, the metal ion precipitation suppression process, preliminary analysis, water sampling amount determination, etc., which will be described later, are carried out in parallel with the sampling of target groundwater.
Note that the first preliminary water sampling, the second preliminary water sampling, and the main water sampling may be implemented as separate steps (temporally separated steps).

(Metal ion precipitation suppression process)
In the water sampling process, after the first preliminary sampling is completed (after the amount of target groundwater stored has reached the amount required to carry out a simple measurement of dissolved iron ion concentration), a metal ion precipitation suppression process is carried out. Implement. In the metal ion precipitation suppression step, the dissolved iron ion concentration is simply measured using the target groundwater sampled in the first preliminary water sampling as a sample. Then, depending on the simple measurement results of dissolved iron ion concentration, whether or not a metal ion chelating agent is added to the sample (preliminary analysis/main analysis sample), and metal ion to the sample (preliminary analysis/main analysis sample) are determined. Determine the amount of chelating agent added.
Generally, groundwater that exists at great depths (specifically, several hundred meters or more) is in an anaerobic environment (reducing environment), so if it is pumped to the ground and exposed to the atmosphere, , oxidation progresses rapidly, and metal ions (specifically, iron ions) dissolved in the groundwater precipitate. As a result, when performing various main analyzes using groundwater existing at great depths as a sample, microscopic observation may be hindered by the precipitated metal ions.
Therefore, it is possible to suppress the precipitation of metal ions in the sample by simply measuring the dissolved iron ion concentration in the target groundwater sample and adding a metal ion chelating agent to the sample according to the measurement result. As a result, it becomes possible to prevent the occurrence of a situation in which microscopic observation is obstructed.

As shown in FIG. 2, in the metal ion precipitation suppression step, first, a portion (5 [mL]) of the target groundwater sampled in the first preliminary water sampling is sampled (hereinafter referred to as the "first sample"). First, Ferrozine solution (0.5 [mL]) is added to the first sample and mixed in a vial. Next, a first coloring determination is performed on the first sample mixed with the Ferrozine solution.
If no coloration is confirmed as a result of the first coloration determination (no coloration), it is decided not to add the metal ion chelating agent to the sample (preliminary analysis/main analysis sample). .
In this embodiment, EDTA (ethylenediaminetetraacetic acid) is used as a chelating agent for metal ions (specifically, iron ions).
On the other hand, if coloring is confirmed as a result of the first coloring determination (in the case of coloring), a portion of the target groundwater newly sampled in the first preliminary water sampling (0.5 [mL]) As a sample (hereinafter referred to as "second sample"), the second sample is diluted 1/10 by adding distilled water (4.5 [mL]) to the second sample. Next, a Ferrozine solution (0.5 [mL]) is added to the second sample diluted 1/10 and mixed in a vial. Next, a second coloring determination is performed on the second sample mixed with the Ferrozine solution.
If no coloration is confirmed as a result of the second coloration determination (no coloration), 1/200 amount of EDTA (0 .06 [M]) (final concentration = 0.3 [mM]).
On the other hand, if light pink coloration is confirmed as a result of the second coloration determination (in the case of light pink color), 1/100 of the sample (preliminary analysis/main analysis sample) should be of EDTA (0.06 [M]) (final concentration = 0.6 [mM]).
On the other hand, if dark pink coloration is confirmed as a result of the second coloration determination (in the case of dark pink color), 1/100 of the sample (preliminary analysis/main analysis sample) should be It is decided to add EDTA (0.6 [M]) (final concentration = 6.0 [mM]).

Thereafter, the samples (preliminary analysis/main analysis samples) are processed according to the content determined in the metal ion precipitation suppression step. At this time, if the addition of EDTA is determined in the metal ion precipitation suppression process, EDTA is added to the sample (preliminary analysis/main analysis sample) according to the content determined in the metal ion precipitation suppression process. Ru. On the other hand, if it is determined in the metal ion precipitation suppression step not to add EDTA, EDTA is not added to the sample (preliminary analysis/main analysis sample).
In this embodiment, when the addition of EDTA is determined in the metal ion precipitation suppression step, EDTA is added to the sampled target groundwater in the water sampling step. That is, when it is decided to add EDTA in the metal ion precipitation suppression process, each time the sampled target groundwater reaches a predetermined amount, the metal ion precipitation suppression process determines the addition of EDTA to this predetermined amount of target groundwater. EDTA is added according to the content determined, and stored and stored as a sample (preliminary analysis/main analysis sample). On the other hand, if it is decided not to add EDTA in the metal ion precipitation suppression process, the sampled target groundwater will be stored and preserved as a sample (preliminary analysis/main analysis sample). be done.

(Preliminary analysis)
In the water sampling step, a preliminary analysis is performed after the second preliminary water sampling is completed (after the amount of stored target groundwater reaches the amount necessary for conducting the preliminary analysis). In the preliminary analysis, an analysis is performed to determine the amount of water sampled (amount of sample) required for the main analysis. In this embodiment, the number of microorganisms is counted as a preliminary analysis.
Specifically, in the preliminary analysis, first, the target groundwater sampled in the second preliminary water sampling is used as a sample (hereinafter referred to as the "preliminary analysis sample"), and the number of microorganisms (the number of microorganisms present per unit water volume) is determined. Carry out counting and evaluation. Here, when it is decided to add EDTA in the metal ion precipitation suppression step, the target groundwater to which EDTA has been added is used as a preliminary analysis sample.
As a method for counting the number of microorganisms, it is possible to use any known method such as flow cytometry, total direct count (TDC), and most probable number method (MPN). .
In this embodiment, the number of microorganisms is counted using total bacterial count (TDC) for prokaryotes (bacteria and archaea). Specifically, a preliminary analysis sample was filtered through a Nuclepore filter (pore size = 0.2 [μm]), stained with a nucleic acid stain, and 300 [cells] of the filter were stained using an epifluorescence microscope. ] For the above, count the number of prokaryotes. Then, the number of prokaryotes (N) per 1 [mL] of the preliminary analysis sample is calculated based on the following (Equation 1).
N [cells/mL] = n x (A/a) x (1/V) (Formula 1)
Here, "N" is the number of prokaryotes per 1 [mL].
"n" is the average number of prokaryotes per 100 grit.
"A" is the filtration area [μm ² ].
"a" is the area [μm ² ] of the filter per 100 grit.
"V" is the filtration amount [mL] of the preliminary analysis sample.
As the nucleic acid stain, "4,6-diamidino-2-phenylindole (DAPI)", "Acridine Orange (AO)", "SYBR Green I", etc. can be used.

Immediately after the sampled groundwater is sampled, it is exposed to an environment different from its original location, and its physicochemical properties change. Therefore, in this embodiment, preliminary analysis and determination of the amount of water sampled (amount of sample) necessary for the main analysis are performed in an on-site laboratory. This allows us to more accurately understand the state of microorganisms in situ (specifically, the number of microorganisms), and as a result, we can more appropriately determine the amount of water (sample amount) required for this analysis. It becomes possible to estimate.
Further, in this embodiment, a preliminary analysis is performed based on an unfixed sample (a sample containing living microorganisms). Thereby, preliminary analysis can be started more quickly, and as a result, the state of microorganisms in situ (specifically, the number of microorganisms) can be grasped more quickly.

(Determination of water sampling amount)
In the water sampling step, the amount of water sampled (amount of sample) required for the main analysis is determined based on the results of the preliminary analysis.
Generally, in microorganism analysis (main analysis), if the number of microorganisms to be analyzed is small, the analysis results may deviate from the state of the microorganisms in situ. Therefore, in order to derive appropriate analysis results, it is necessary to secure a certain number of microorganisms to be analyzed.
Therefore, when determining the amount of water sample (amount of sample) required for the main analysis, the larger the number of microorganisms calculated in the preliminary analysis (the number of microorganisms present per unit volume of target groundwater), the more suitable it is for the main analysis. Decide on the required amount of water sampling (amount of sample). In other words, the smaller the number of microorganisms (the number of microorganisms present per unit volume of target groundwater) calculated by the preliminary analysis, the larger the amount of water sampled (amount of sample) required for the main analysis is determined.
In particular, in this embodiment, this analysis includes multiple analysis items (specifically, counting the number of microorganisms, analyzing microbial community structure, analyzing microbial community composition, and analyzing microbial activity). It is. Then, the amount of water sampled (amount of sample) is determined individually for multiple analysis items included in this analysis. That is, for each analysis item included in this analysis, the amount of water sampled (amount of sample) required for the analysis item is determined. The total amount of water sampled (amount of sample) determined for all analysis items included in this analysis is defined as the amount of sampled water (amount of sample) required for this analysis. This makes it possible to more accurately estimate the amount of water sampled (amount of sample) required for this analysis.

Specifically, as shown in Figure 3, based on empirical rules, the number of microorganisms (N) calculated in advance through preliminary analysis and the amount of water sampled (sample amount) required for each analysis item included in the main analysis are calculated in advance. The correspondence relationship between Then, the amount of water sampled (amount of sample) required for the main analysis is determined according to the results of the preliminary analysis and the predefined correspondence relationship.
In detail, if the number of microorganisms (N) calculated by the preliminary analysis is less than 10 ⁴ [cell/mL], the amount of water sampled (sample 0.5 [L] was determined as the amount of water collected (amount of sample) required for the analysis of the microbial community structure, and 0.5 [L] was determined as the amount of water sampling (amount of sample) required for the analysis of the microbial community structure. 100 [L] is determined as the amount of water sampled (amount of sample) required for the analysis of microbial activity, and 0.2 [L] is determined as the amount of water sampled (amount of sample) required for analysis of microbial activity. As a result, the amount of water sampled (amount of sample) required for this analysis is 101.2 [L] (0.5 [L] + 0.5 [L] + 100 [L] + 0.2 [L] = 101. 2[L]) is determined.
On the other hand, if the number of microorganisms (N) calculated by the preliminary analysis is 10 ⁴ [cell/mL] or more and less than 10 ⁵ [cell/mL], counting of the number of microorganisms is one of the analysis items of the main analysis. 0.2 [L] was determined as the amount of water sampled (amount of sample) required for the analysis of the microbial community structure, and 0.2 [L] was determined as the amount of water sampled (amount of sample) required for analysis of the microbial community structure. The amount of water sampled (amount of sample) required to analyze the microbial community composition was determined to be 10 [L], and the amount of water sampled (amount of sample) required to analyze the activity of microorganisms was determined to be 0.1 [L]. [L] is determined. As a result, the amount of water sampled (amount of sample) required for this analysis is 10.5 [L] (0.2 [L] + 0.2 [L] + 10 [L] + 0.1 [L] = 10. 5[L]) is determined.
On the other hand, if the number of microorganisms (N) calculated by the preliminary analysis is 10 ⁵ [cell/mL] or more and less than 10 ⁶ [cell/mL], counting of the number of microorganisms is one of the analysis items of the main analysis. 0.2 [L] was determined as the amount of water sampled (amount of sample) required for the analysis of the microbial community structure, and 0.05 [L] was determined as the amount of water sampled (amount of sample) required for analysis of the microbial community structure. The amount of water sampled (amount of sample) required to analyze the microbial community composition was determined to be 5 [L], and the amount of water sampled (amount of sample) required to analyze the activity of microorganisms was determined to be 0.01. [L] is determined. As a result, the amount of water sampled (amount of sample) required for this analysis is 5.26 [L] (0.2 [L] + 0.05 [L] + 5 [L] + 0.01 [L] = 5. 26[L]) is determined.
On the other hand, if the number of microorganisms (N) calculated by the preliminary analysis is 10 ⁶ [cell/mL] or more, one of the analysis items of the main analysis is the amount of water sample required to count the number of microorganisms (N) of the sample. 0.1 [L] was determined as the amount of water collected (amount of sample) necessary for analyzing the microbial community structure, and 0.01 [L] was determined as the amount of water sample (amount of sample) required for analyzing the microbial community structure. 1 [L] is determined as the required amount of water sampling (amount of sample), and 0.01 [L] is determined as the amount of water sampling (amount of sample) required for analyzing the activity of microorganisms. As a result, the amount of water sampled (amount of sample) required for this analysis is 1.12 [L] (0.1 [L] + 0.01 [L] + 1 [L] + 0.01 [L] = 1. 12[L]) is determined.

In this embodiment, the end time of the water sampling process is determined based on the determined amount of water sampling (amount of sample) required for the main analysis.
In other words, the amount of target groundwater sampled in the main water sampling (the amount of target groundwater stored) has reached the amount of water sampled (amount of sample) required for the main analysis determined in the previous step. , the main water sampling (water sampling process) is completed.
In general, sampling groundwater that exists at great depths (specifically, several hundreds of meters or more) is more difficult than sampling surface water such as seawater or river water. Installation of equipment, preliminary drainage, etc. require a huge amount of time and expense. Therefore, if a shortage of samples occurs during the actual analysis, it will be necessary to sample water again, and as a result, the analysis period and analysis cost for the actual analysis will increase. Furthermore, if water is sampled individually for a plurality of analysis items included in this analysis, the number of times water sampling is performed will increase, and as a result, the analysis period and analysis cost of this analysis will increase.
Therefore, before carrying out the main analysis, the amount of water to be sampled (amount of sample) required for the main analysis is determined, and by sampling water according to the determined amount of water, the sample can be sampled during the main analysis. This makes it possible to prevent a situation where a shortage occurs. In particular, by performing water sampling at once for multiple analysis items included in this analysis, it is possible to suppress an increase in the number of times water sampling is performed.

Here, in addition to counting the number of microorganisms, the analysis items of the preliminary analysis may include analysis of the amount of mixed mineral particles.
The "amount of mixed mineral particles" is the amount of mineral particles mixed in the sample.
That is, as will be described later, in analyzing the community structure of microorganisms and analyzing the activity of microorganisms, the amount of sample to be filtered is determined according to the amount of mixed mineral particles. At this time, the greater the amount of mixed mineral particles, the greater the possibility that microscopic observation will be obstructed by the mixed mineral particles, so it is necessary to reduce the amount of sample to be filtered.
Therefore, in the preliminary analysis, the amount of mixed mineral particles may be analyzed and evaluated, and the amount of water sampled (amount of sample) required for the main analysis determined in the above process may be corrected according to the result. . At this time, as the amount of mixed mineral particles increases, the amount of water sampled (amount of sample) required for the main analysis determined in the above step is reduced.

(Pre-treatment process)
In the pretreatment process, the target groundwater (sample) collected in the main water sampling is subjected to pretreatment (preservation treatment) according to each analysis item.
In this embodiment, among the analysis items included in this analysis, counting the number of microorganisms, analyzing the community structure of microorganisms, and analyzing the community composition of microorganisms are performed in a laboratory separate from the water sampling site. . As a result, samples for each of these analysis items need to be transported from the water sampling site to the laboratory. Therefore, in this embodiment, samples for each of these analysis items are subjected to pretreatment according to the analysis item.
On the other hand, among the analysis items included in this analysis, the analysis of microbial activity will be conducted in the on-site laboratory. As a result, samples for analysis of microbial activity are not subjected to pretreatment.
Specifically, in the pretreatment process, samples for counting the number of microorganisms, samples for analyzing the community structure of microorganisms, and samples for analyzing the community composition of microorganisms are collected from the target groundwater sampled in the main water sampling. Acquire (distribute). At this time, samples for each analysis item are obtained (distributed) according to the amount of water sampled (amount of sample) required for each analysis item determined in the previous step. Then, pretreatment is performed on each of the sample for counting the number of microorganisms, the sample for analyzing the community structure of microorganisms, and the sample for analyzing the composition of microbial community.
At this time, for the sample for counting the number of microorganisms, first, neutral formalin is added to fix the sample (final concentration after fixation = 0.2 [%]). Next, the fixed sample is stored refrigerated. Here, by fixing the sample, microorganisms present in the sample are killed. This prevents the number of microorganisms in the sample from increasing, and in this analysis, it becomes possible to more accurately grasp the state of microorganisms in situ (specifically, the number of microorganisms).
On the other hand, for a sample for analysis of microbial community structure, first, paraformaldehyde is added, and the sample is fixed and membrane-treated (final concentration after fixation = 3%). Next, determine the appropriate amount of filtration by microscopic observation. At this time, the greater the amount of mixed mineral particles, the more it is necessary to reduce the amount of filtration. Then, according to the determined filtration amount, the fixed and membrane-treated sample is filtered with a Nuclepore filter (pore diameter = 0.2 [μm]). Next, the filtered sample (filter) is dehydrated and then stored frozen.
On the other hand, as for the sample for analysis of microbial community composition, after mass filtration is performed using a housing filter (particle retention capacity = 0.22 [μm]), the mass-filtered sample (filter) is stored frozen.
As mentioned above, sampled groundwater is exposed to an environment different from its original location immediately after sampling, and its physicochemical properties change. Therefore, in this embodiment, the pretreatment step is performed in an on-site laboratory. This makes it possible to more accurately grasp the state of microorganisms in situ (specifically, the number of microorganisms) in this analysis.

(Various main analysis processes)
In various main analysis steps, each analysis item included in the main analysis is analyzed. In this embodiment, as various main analysis steps, a microorganism number counting step, a microorganism community structure analysis step, a microorganism community composition analysis step, and a microorganism activity analysis step are performed.
Here, as described above, the sampled groundwater is exposed to different environments (for example, temperature, presence or absence of oxygen, pressure, etc.) compared to the original location immediately after the sampled groundwater is sampled. For example, in an underground environment at a depth of 1000 [m], considering the geothermal gradient (30 [°C/km]), it is assumed that the water temperature is 30 [°C] higher than that in the above-ground environment. As a result, when groundwater existing at a depth of 1000 [m] is pumped to the ground, the temperature environment changes by 30 [°C]. Such changes in the environment have a particularly large effect on the activity of microorganisms present in groundwater, and the activity of microorganisms present in groundwater changes significantly with the passage of time after water sampling.
Therefore, in this embodiment, among the various main analysis steps, the microorganism activity analysis step is carried out in an on-site laboratory. This makes it possible to more accurately grasp the state of microorganisms in situ (specifically, the activity of microorganisms). Here, the microbial activity analysis step is performed in parallel with the pretreatment step.
On the other hand, among the various main analysis steps, the microorganism count counting step, the microorganism community structure analysis step, and the microorganism community composition analysis step are performed in a laboratory separate from the water sampling site.

(Microorganism activity analysis process)
In the microbial activity analysis step, the activity of microorganisms present in the sample is analyzed.
Specifically, in the microbial activity analysis step, a sample for microbial activity analysis (hereinafter referred to as "activity analysis sample") is obtained from the target groundwater sampled in the main water sampling. At this time, an activity analysis sample is obtained according to the amount of water sampled (amount of sample) required for the analysis of microbial activity (analysis item) determined in the previous step. Here, in this embodiment, even if the addition of EDTA is determined in the metal ion precipitation suppression step, target groundwater to which EDTA is not added is used as the activity analysis sample.
In the microorganism activity analysis step, the microorganism activity is then analyzed and evaluated based on the obtained activity analysis sample. At this time, all known methods such as culture method, LIVE/DEAD (registered trademark) method, CTC method (CTC: 5-cyano-2,3-ditolyl tetrazolium chloride) may be used to analyze the activity of microorganisms. is possible.
In this embodiment, the activity of microorganisms is analyzed using the LIVE/DEAD method, which calculates the percentage of viable bacteria by evaluating the health of cell membranes. Specifically, first, an appropriate amount of filtration is determined by microscopic observation. At this time, the greater the amount of mixed mineral particles, the more it is necessary to reduce the amount of filtration. Then, the activity analysis sample is filtered with a Nuclepore filter (pore size = 0.2 [μm]) according to the determined filtration amount. Next, the filtered activity analysis sample was stained with SYTO9 (final concentration = 0.334 [μM]) and propidium iodide (PI) (2 [μM]) at room temperature in the dark for 15 minutes (cultured). )do. Then, using an epifluorescence microscope, the ratio of viable bacteria to the total number of microorganisms in each field (total of live bacteria and dead bacteria) was counted for 20 fields of view, and the average value was calculated as the number of microorganisms in the sample. Calculated as the ratio of viable bacteria to the total number (microbial activity).

(Microorganism count counting process)
In the microorganism counting step, the number of microorganisms present in the sample is counted.
Specifically, in the microorganism number counting step, the number of microorganisms is counted and evaluated based on the sample for counting the number of microorganisms that has been pretreated in the pretreatment step (hereinafter referred to as the "sample for counting the number of microorganisms"). Here, when it is decided to add EDTA in the metal ion precipitation suppression step, the target groundwater to which EDTA has been added is used as a sample for counting the number of microorganisms.
As a method for counting the number of microorganisms, it is possible to use any known method such as flow cytometry, total direct count (TDC), and most probable number method (MPN). .
In this embodiment, the number of microorganisms is counted using total bacterial count (TDC) for prokaryotes (bacteria and archaea). Specifically, a microbial count sample was filtered through a Nuclepore filter (pore size = 0.2 [μm]), stained with a nucleic acid stain, and an epifluorescence microscope was used to test the filter at 300 [μm]. [cells] and above, count the number of prokaryotes. Then, based on the above (Equation 1), the number of prokaryotes (N) per 1 [mL] of the preliminary analysis sample is calculated.
As the nucleic acid stain, "4,6-diamidino-2-phenylindole (DAPI)", "Acridine Orange (AO)", "SYBR Green I", etc. can be used.

(Microbial community structure analysis process)
In the microbial community structure analysis step, the microbial community structure present in the sample is analyzed.
Specifically, in the microbial community structure analysis step, the microbial community structure is determined based on the microbial community structure analysis sample (hereinafter referred to as "community structure analysis sample") that has been pretreated in the pretreatment step. Analyze and evaluate. Here, when the addition of EDTA is determined in the metal ion precipitation suppression step, the target groundwater to which EDTA has been added is used as the community structure analysis sample.
Methods for analyzing microbial community structure include the FISH method (FISH: Fluorescence in situ hybridization), the CARD-FISH method (CARD: Catalyzed reporter deposition), and the qPCR method (qPCR). All known methods such as quantitative polymerase chain reaction) It is possible to use
In this embodiment, the FISH method is used to analyze the community structure of microorganisms, targeting prokaryotes (bacteria and archaea). Since the FISH method detects cells using rRNA, cells that may have activity can be evaluated at a "single cell level." Since this method does not depend on culture operations, it is useful for analyzing prokaryotes in the natural environment.
Specifically, hybridization is performed on a community structure analysis sample, targeting bacteria and archaea, using probes targeting rRNA-specific sequences of each taxonomic group. Next, secondary staining is performed with DAPI (4',6-Diamidino-2-phenylindole, dihydrochloride). Then, using an epifluorescence microscope, the total number of cells (total number of prokaryotes) and the number of hybridized cells (number of bacteria) are counted. Furthermore, the ratio of hybridized cells to the total number of cells is calculated and used as the ratio of the targeted taxonomic group.

(Microbial community composition analysis process)
In the microbial community composition analysis step, the community composition of microorganisms present in the sample is analyzed.
Specifically, in the microbial community composition analysis step, the microbial community composition is determined based on the microbial community composition analysis sample (hereinafter referred to as "community composition analysis sample") that has been pretreated in the pretreatment step. Analyze and evaluate. Here, in this embodiment, even if the addition of EDTA is determined in the metal ion precipitation suppression step, target groundwater to which EDTA is not added is used as the community composition analysis sample.
Methods for analyzing microbial community composition include the DGGE method (DGGE: Denaturing gradient gel electrophoresis), the cloning method, and the TRFLP method (TRFLP: Terminal restriction fragment length polymer). phism), NGS method (NGS: Next generation sequencer), etc. It is possible to use a method.
In this embodiment, the microbial community composition is analyzed using the NGS method, targeting the "16S rRNA gene." Specifically, first, lysozyme, proteinase K, and SDS (sodium dodecyl sulfate) are added to a filter (a sample for community composition analysis) obtained by mass-filtering a sample, and after incubation, DNA is extracted by phenol extraction. Next, the extracted DNA is purified using PCI (Phenol chloroform isoamyl alcohol) and CAI, followed by ethanol precipitation and dissolved in TE buffer. Next, a PCR (Polymerase chain reaction) reaction is performed using primers targeting the V3-V4 region of the "16S rRNA gene" to create a library. Next, sequencing is performed using a next-generation sequencer under the condition of 2 x 300 bp. Then, after removing low-quality reads and chimeric sequences from the obtained reads, OTUs (Operational Taxonomic Units) analysis and phylogenetic inference are performed.

(Actions and effects of analysis method for microorganisms in groundwater)
In the method for analyzing microorganisms in groundwater according to the present embodiment, a preliminary analysis is performed using sampled target groundwater as a sample, and the main analysis (microorganism counting step, microorganism community structure analysis step) is performed based on the result of the preliminary analysis. , microorganism community composition analysis step, and microorganism activity analysis step) is determined, and the target groundwater is sampled according to the determined water sampling amount.
This prevents the occurrence of a shortage of samples necessary for the main analysis. Therefore, it is possible to avoid sampling water for replenishing a missing sample, and as a result, it is possible to shorten the analysis period.
Furthermore, by performing water sampling once, a sample required for this analysis can be collected. Therefore, even if multiple analysis items are included in this analysis, there is no need to sample water for each analysis item. Therefore, the number of times water sampling is performed is reduced, and as a result, the analysis period can be shortened.
In particular, when the permeability of the target ground is low or when the target ground is deep, the analysis period can be significantly shortened.
Furthermore, in the method for analyzing microorganisms in groundwater according to the present embodiment, the analysis items of the preliminary analysis include counting the number of microorganisms.
This makes it possible to appropriately determine the amount of groundwater required for this analysis. In other words, when performing various analyzes such as counting the number of microorganisms, analyzing microbial communities (community composition, community structure), and analyzing microbial activity, it is necessary to calculate the number of microorganisms present per unit volume of groundwater as a sample. , greatly influences the determination of the amount of sample (i.e., the amount of water sampled) required for the analysis. At this time, the smaller the number of microorganisms present per unit amount of water, the larger the amount of sample required. Therefore, by including the counting of the number of microorganisms in the analysis items of the preliminary analysis, it becomes possible to appropriately determine the amount of sample required for the main analysis.

Furthermore, in the method for analyzing microorganisms in groundwater according to the present embodiment, the amount of water sampled is determined for each of the plurality of analysis items included in the main analysis based on the results of the preliminary analysis.
Thereby, the amount of sample required for the main analysis can be determined in detail, and it is possible to prevent the occurrence of a shortage of samples for each analysis item.
Furthermore, in the method for analyzing microorganisms in groundwater according to the present embodiment, in the metal ion precipitation suppression step, a metal ion chelating agent is added to the sampled target groundwater according to the simple measurement result of the metal ion concentration, Preliminary analysis will be conducted using groundwater samples to which chelating agents have been added.
This suppresses the precipitation of metal ions dissolved in the sampled groundwater, making it possible to prevent precipitated metal ions from interfering with microscopic observation during preliminary analysis. becomes.

(Water sampling device 1)
Next, a water sampling device 1 suitable for the method for analyzing microorganisms in groundwater according to the present embodiment will be described.
FIG. 4 is a diagram showing a schematic configuration of the water sampling device 1. As shown in FIG.
The water sampling device 1 forms a water sampling section s in a well w, and is a device for sampling target groundwater flowing from the target ground into the water sampling section s.
As shown in FIG. 4, the water sampling device 1 includes a water sampling unit 100 inserted into a well w, and a compressor 200 disposed above ground.
The water sampling unit 100 includes a pumping casing (casing pipe) 10, a pair of packers 20a and 20b arranged on the outer peripheral surface of the pumping casing 10, a water quality monitoring device 40 arranged inside the pumping casing 10, and a pumping casing 10. It is configured to include a water sampling pipe 50 that communicates with the water sampling pipe 50 and a pressurizing head 60 that is detachable from the water pumping casing 10.

The pumping casing 10 is made of a metal material such as steel or stainless steel. The pumping casing 10 is formed into a tubular shape (pipe shape) that extends linearly as a whole. The pumping casing 10 is arranged within the well w so as to extend along the vertical direction. In the pumping casing 10, a flow path (pumping channel) is formed that extends linearly in the vertical direction from its lower end to its upper end. As a result, target groundwater that has entered from the strainer section 11, which will be described later, can be pumped toward the ground within the pumping casing 10.
The pumping casing 10 includes a strainer part (strainer pipe) 11, a pumping part (pumping pipe) 12 arranged above the strainer part 11, an in-hole valve 30 arranged between the strainer part 11 and the pumping part 12, and It is configured to include a sensor placement section (sensor placement tube) 40a.
The strainer portion 11 is formed into a tubular shape (pipe shape) that extends linearly. The lower end of the strainer section 11 is closed. A pair of packers 20a and 20b are arranged on the outer peripheral surface of the strainer section 11. A water sampling section h is configured in the area between the pair of packers 20a and 20b in the strainer section 11. The water sampling section h is configured by providing a plurality of (many) infiltration holes (not shown) in the peripheral wall of the strainer section 11. This allows groundwater gushing out from the target ground to infiltrate (draw) into the strainer section 11 via each infiltration hole.

The pumping part 12 includes a branch part (branch pipe) 12d, a small diameter part (small diameter pipe) 12a arranged above the branch part 12d, and a large diameter part (large diameter pipe) 12b arranged above the small diameter part 12a. It is composed of and.
The branch portion 12d is constituted by a so-called branch pipe (distribution pipe). Specifically, within the branching portion 12d, a main flow path extending in the vertical direction and a sub flow path branching off from the middle of the main flow path are formed. The lower end of the main flow path is connected (communicated) to a water outlet of the in-hole valve 30, which will be described later. The upper end of the main flow path is connected (communicated) with the lower end of the small diameter portion 12a. One end of the sub-channel is connected (communicated) with the main channel. Further, the other end of the sub-channel is connected (communicated) to the lower end of the water sampling pipe 50.
The small diameter portion 12a is formed into a tubular shape (pipe shape) that extends linearly. The lower end of the small diameter portion 12a is connected (communicated) with the upper end of the main flow path of the branch portion 12d. The upper end of the small diameter portion 12a is connected (communicated) to the lower end of the large diameter portion 12b via the reducer 12e.
As will be described later, the small diameter portion 12a is configured by connecting a plurality of small diameter tube bodies (pipes) according to the depth of the target ground. Each small-diameter tube body is a tube body (pipe) having a predetermined length.
The large diameter portion 12b is formed into a tubular shape (pipe shape) that extends linearly. The lower end of the large diameter portion 12b communicates with the upper end of the small diameter portion 12a via the reducer 12e. The upper end of the large diameter portion 12b extends to a predetermined height above the ground.
As described later, the large diameter portion 12b is constructed by connecting a plurality of large diameter pipes (pipes) depending on the permeability of the target ground (specifically, the depth at which the pump P described below is installed). be done. Each large-diameter tube body is a tube body (pipe) having a predetermined length.
The inner diameter of the large diameter portion 12b is configured such that the pump P can be inserted therein. On the other hand, the inner diameter of the small diameter portion 12a is smaller than the inner diameter of the large diameter portion 13b (so that the pump P cannot be inserted). This makes it possible to suppress an increase in the volume of the pumping casing 10.

The sensor arrangement portion 40a is formed in a tubular shape (pipe shape) that extends linearly. The lower end of the sensor arrangement section 40a is connected (communicated) with the upper end of the strainer section 11. The upper end of the sensor arrangement portion 40a is connected (communicated) to a water inlet of the in-hole valve 30, which will be described later. A water quality monitoring device 40 is arranged within the sensor arrangement section 40a. This allows the water quality monitoring device 40 to measure the quality of water pumped into the sensor arrangement section 40a.
The in-hole valve 30 includes a valve box (valve chamber) 12c, and a valve body 30a and a valve seat (not shown) arranged in the valve box 12c.
A water inlet (not shown) is provided on the bottom surface of the valve box 12c, and a water outlet (not shown) is provided on the top surface of the valve box 12c. The upper end of the sensor arrangement part 40a is connected (communicated) to the water inlet, and the lower end of the main flow path of the branch part 12d is connected (communicated) to the water outlet.
The valve body 30a of the in-hole valve 30 opens and closes the water inlet. When the water inlet is open, target groundwater can be pumped (infiltrated) from the strainer section 11 into the pumping section 12 via the sensor arrangement section 40a and the in-hole valve 30. On the other hand, when the water inlet is closed, backflow (infiltration) of target groundwater from the pumping section 12 to the strainer section 11 is prevented.

The water quality monitoring device 40 measures the quality of groundwater pumped from the strainer unit 11 and transmits the measurement results by wire or wirelessly to a computer (not shown) located on the ground. Here, the electronic computer is placed in an observation room installed on the ground.
In this embodiment, the water quality monitoring device 40 can measure electrical conductivity (EC), temperature, water pressure, pH (hydrogen ion index), ORP (oxidation-reduction potential), DO (dissolved oxygen concentration), etc. It has become.
The water sampling pipe 50 is composed of a pipe made of a metal material such as steel or stainless steel, or a tube made of a material such as nylon or Teflon (registered trademark) that does not affect the quality of groundwater. ing. The water sampling pipe 50 may be configured in a series, or may be configured by connecting a plurality of pipe bodies (or tubes) depending on the depth.
The lower end of the water sampling pipe 50 is connected (communicated) to the sub-channel of the branch portion 12d. Further, the upper end of the water sampling pipe 50 extends to the ground, and the target groundwater discharged from the other end of the water sampling pipe 50 can be received by a water sampling container (not shown). Furthermore, a valve b1 that opens and closes the flow path of the water sampling pipe 50 is arranged at the upper end of the water sampling pipe 50.

The pressure head 60 is configured to be removable from the upper end of the large diameter portion 12b. When the pressurizing head 60 is attached to the upper end of the large diameter portion 12b, the upper end of the pumping casing 10 (large diameter portion 12b) is sealed. The pressure head 60 is provided with a pressure port (not shown).
The compressor 200 supplies (pressurizes) compressed gas to the pressurizing port of the pressurizing head 60 via the air tube t. As a result, when compressed gas is supplied from the compressor 200 to the pressurizing port of the pressurizing head 60 with the pressurizing head 60 attached to the upper end of the pumping casing 10 (large diameter portion 12b), the pumping casing 10 (large diameter portion 12a) is pressurized.

(Target groundwater sampling method using water sampling device 1)
Next, a method for sampling target groundwater using the water sampling device 1 will be explained.
FIG. 5 is a diagram showing the water sampling device 1 in a state where the pump P is installed inside the pumping casing 10.
When sampling target groundwater using the water sampling device 1, the water sampling unit 100 is first installed (configured) in the well w in the water sampling preparation process described above.
To do this, first, the water sampling unit 100 with the small diameter part 12a and the large diameter part 12b removed is inserted into the well w. At this time, the valve body 30a (water inlet) of the in-hole valve 30 is open, and the target groundwater can be pumped from the strainer section 11 to the pumping section 12. Furthermore, the valve b1 of the water sampling pipe 50 is open.
Next, a plurality of small-diameter pipe bodies are connected to the upper end of the main channel of the branching part 12d according to the depth forming the predetermined water sampling section s (hereinafter referred to as "water sampling depth"). By adding the plurality of large diameter parts 12b, a small diameter part 12a is formed, and by adding a plurality of large diameter parts 12b to the upper end of the small diameter part 12a, a large diameter part 12b is formed. At this time, the upper end of the small diameter portion 12a and the lower end of the large diameter portion 12b are connected via the reducer 12e. This places the strainer section 11 at the water sampling depth.
Next, each packer 20a, 20b is expanded to form a water sampling section s.
With the above steps, the installation of the water sampling unit 100 in the well w is completed. When the installation of the water sampling unit 100 is completed, the upper end of the water pumping casing 10 (large diameter portion 12b) is placed at a predetermined height from the ground. At this time, the pressurizing head 60 is not attached to the upper end of the pumping casing 10 (large diameter section 12b). When the installation of the water sampling unit 100 is completed, the water quality monitoring device 40 starts monitoring the quality of the groundwater pumped from the strainer section 11.

Next, perform preliminary drainage.
To do this, as shown in FIG. 5, first, the pump P is inserted from the upper end of the pumping casing 10 (large diameter section 12b), and the pump P is installed at the lower end of the large diameter section 12b. Next, the pump P is operated to start draining (preliminary draining) of the residual water pumped to the lower end of the large diameter portion 12b. At this time, the valve body 30a (water inlet) of the in-hole valve 30 is open. Further, the valve b1 of the water sampling pipe 50 is open.
Here, the residual water drained (suctioned) by the pump P is guided to the ground via the water pump T1. A branch pipe T2 is connected to the upper end of the lift pipe T1. In the branch pipe T2, a main flow path r1 and two sub flow paths (specifically, a water quality measurement path r2 and a sampling path r3) that branch from the middle of the main flow path r1 are formed. .
One end of the main flow path r1 is connected (communicated) with the upper end of the water pump T1. Moreover, the other end of the main flow path r1 is connected (communicated) with a drainage port (sewer etc.). Furthermore, a flow meter F and a flow control valve b2 are arranged at the other end of the main flow path r1.
One end of the water quality measurement path r2 is connected (communicated) with the main flow path r1. Further, the other end of the water quality measurement path r2 is connected (communicated) to a drainage outlet (sewer etc.). Further, a second water quality monitoring device 80 is arranged in the drainage water quality measuring path r2. The second water quality monitoring device 80 measures the quality of residual water (residual water drained by pump P) passing through water quality measurement path r2. In this embodiment, the second water quality monitoring device 80 measures CTD (electrical conductivity, temperature, water depth), pH (hydrogen ion index), ORP (redox potential), DO (dissolved oxygen amount), etc. .
One end of the sampling channel r3 is connected (communicated) with the main channel r1. The target groundwater discharged from the other end of the sampling channel r3 can be received by a water sampling container (not shown). Furthermore, a valve b3 that opens and closes the sampling channel r3 is arranged at the other end of the sampling channel r3.
When performing preliminary drainage, the valve b3 of the sampling channel r3 is closed, and the flow rate control valve b2 of the main channel r1 is opened. As a result, the residual water drained by the pump P flows into the main flow path r1 via the pumping pipe T1. The residual water that has flowed into the main flow path r1 passes through the flow meter F and the flow control valve b2, and is discharged to the drain port. At this time, a portion of the residual water that has flowed into the main flow path r1 flows into the water quality measurement path r2. Then, the residual water that has flowed into the water quality measuring path r2 is discharged to the drain after the water quality is measured by the second water quality monitoring device 80.

As preliminary drainage progresses, target groundwater that has gushed out from the target ground to the water sampling section s is drawn into the strainer section 11 via the water sampling section h. Then, the target groundwater drawn into the strainer section 11 is pulled upward within the pumping casing 10. That is, the target groundwater drawn into the strainer section 11 is pulled up to the lower end of the water pumping section 12 via the sensor arrangement section 40a (water quality monitoring device 40) and the in-hole valve 30, and then further pumped into the water pumping section 12. can be pulled upwards.
At this time, if the permeability of the target ground is low, the water level of the residual water in the large diameter section 12b becomes lower than the depth where the suction port of the pump P is installed during preliminary drainage, and the pump Drainage due to P may become impossible. In this case, it is necessary to temporarily stop the operation of the pump P and wait for the level of the residual water to recover before restarting the operation of the pump P. At this time, the lower the permeability of the target ground, the smaller the amount of target groundwater gushing out per unit time, and therefore the longer the time required to restore the water level of the residual water.
While the preliminary drainage is in progress, the water quality monitoring device 40 monitors changes over time in the water quality (the quality of groundwater pumped to the lower end of the small diameter section 12a), and also monitors the second water quality. Changes over time in the water quality measured by the monitoring device 80 (the quality of the residual water drained by the pump P) are monitored. Further, while preliminary drainage is in progress, the measurement results of the water quality monitoring device 40 and the measurement results of the second water quality monitoring device 80 are compared.

The preliminary drainage is completed on the condition that the residual water is replaced with target groundwater at least at the lower end of the pumping section 12.
Specifically, when the water quality measured by the water quality monitoring device 40 remains stable for a predetermined period of time, the preliminary drainage is terminated. That is, when this condition is satisfied, it can be determined that the residual water has been replaced with target groundwater at least in the lower end of the pumping section 12 within the pumping casing 10. Here, the term "stable state" refers to a state in which there is little change in water quality over time.
After the preliminary drainage is completed, the pump P is recovered from the inside of the pumping casing 10 (the lower end of the large diameter portion 12b), and thereafter, target consideration water is sampled in the water sampling process described above.

In the water sampling process, target groundwater is sampled through the water sampling pipe 50 by pressurizing the upper end of the pumping casing 10 .
To do this, first, the valve body 30a (water inlet) of the in-hole valve 30 is closed. This prevents the target groundwater from flowing backward from the pumping section 12 to the strainer section 11. Note that the valve b1 of the water sampling pipe 50 is open. This makes it possible to sample the target groundwater from the upper end of the water sampling pipe 50.
Next, the pressurizing head 60 is attached to the upper end of the pumping casing 10 (large diameter portion 12b). Thereby, the upper end side of the pumping casing 10 is sealed.
Next, the compressor 200 is operated to start sampling the target groundwater pumped to the lower end of the pumping section 12 (main water sampling). That is, when the compressor 200 operates, gas is pressurized from the compressor 200 into the upper end of the pumping casing 10 through the pressurizing port of the pressurizing head 60. As a result, the water surface of the residual water in the pumping casing 10 is pressurized downward. At this time, the valve body 30a of the in-hole valve 30 is closed, which prevents the target groundwater from flowing back from the pumping section 12 to the strainer section 11, so that the water is pumped to the lower end of the pumping section 12. Target groundwater is pushed out to the ground via the water sampling pipe 50. This makes it possible to store (receive) target groundwater discharged from the upper end of the water sampling pipe 50 in the water sampling container.
At this time, there is a possibility that residual water may have entered the water sampling pipe 50. Therefore, at the start of the main sampling, after discarding at least an amount of water equivalent to the volume of the water sampling pipe 50 from the upper end of the water sampling pipe 50, the water is stored in the water sampling container (receiving water). Start. This makes it possible to prevent turbidity of residual water in sampled target groundwater.
Furthermore, during water sampling, if the amount of water sampled due to pressurization increases, there is a risk that residual water pumped into the water pumping section 12 may be sampled. Therefore, during sampling, every time a predetermined amount of target groundwater is sampled, the operation of the compressor 200 is temporarily stopped (main water sampling is temporarily stopped), and the valve body 30a of the in-hole valve 30 is opened. After waiting for recovery of the level of residual water in the pumping casing 10 (large diameter section 12b) (drawing of target groundwater to the lower end of the pumping section 12), the valve element 30a of the in-hole valve 30 is closed, and the compressor It is necessary to restart the operation of 200 (restart main water sampling).
As described above, it becomes possible to perform target groundwater sampling (first preliminary water sampling, second preliminary water sampling, and main water sampling).

(Functions and effects of water sampling device 1)
Next, the functions and effects of the water sampling device 1 will be explained.
The water sampling device 1 includes an in-hole valve 30 that prevents backflow of groundwater from the pumping section 12 to the strainer section 11, a water sampling pipe 50 that guides groundwater from the lower end of the pumping section 12 to the ground, and an upper end of the pumping casing 10. and a compressor 200 that supplies compressed gas to.
This prevents underground water from flowing backward from the pumping section 12 to the strainer section 11, and by supplying compressed gas to the upper end of the pumping casing 10, water is pumped to the lower end of the pumping section 12. The target groundwater is pushed out by gas pressure and guided to the ground via the water sampling pipe 50.
Therefore, even if the entire residual water in the pumping casing 10 is not replaced with the target groundwater, if the residual water at the lower end of the pumping part 12 is replaced with the target groundwater, the target groundwater can be sampled above ground. Therefore, there is no need to replace the entire residual water in the pumping casing 10 with target groundwater, and it becomes possible to shorten the time required to sample target groundwater.
In particular, even if the permeability of the target ground is low or the depth of the target ground is deep, it is sufficient to replace the residual water at the lower end of the pumping section 12 with the target groundwater when sampling the target groundwater. It becomes possible to significantly shorten the time required to sample target groundwater.

1 Water sampling device 10 Pumping casing 11 Strainer part 12 Pumping part 12a Small diameter part 12b Large diameter part 12c Valve box 20a, 20b Packer 40 Water quality monitoring device 50 Water sampling pipe 60 Pressurizing head 30 In-hole valve 100 Water sampling unit 200 Compressor

Claims (2)

A water sampling process of sampling groundwater;
A main analysis step of performing the main analysis using the groundwater sampled in the water sampling step as a sample,
The analysis items of the main analysis include a plurality of analysis items including at least one of microbial counting, microbial community analysis, and microbial activity analysis,
In the water sampling process, a preliminary analysis is conducted on-site to determine the amount of groundwater required for each analysis item included in the main analysis, using the groundwater sampled through preliminary water sampling as a sample . Based on the results of the preliminary analysis, determine the amount of groundwater required for each analysis item included in the main analysis, and determine the amount of groundwater required for all analysis items included in the main analysis according to the determined water sampling amount. In carrying out the main water sampling to sample groundwater, the preliminary water sampling and the main water sampling are carried out through a series of water sampling operations,
A method for analyzing microorganisms in groundwater, characterized in that the preliminary analysis includes counting the number of microorganisms. In the water sampling step, groundwater existing at great depth is sampled, and a simple measurement of metal ion concentration is performed on the groundwater sampled by preliminary water sampling , and the preliminary water sampling is performed according to the result of the simple measurement. A metal ion chelating agent is added to the groundwater sampled by , and the preliminary analysis is performed using the groundwater to which the chelating agent has been added as a sample. The method for analyzing microorganisms in groundwater according to claim 1 , wherein preliminary water sampling and the main water sampling are performed .

Images