JP7557428B2 - Method for culturing purifying bacteria and purification method - Google Patents

Description

NPMD NITE P-1471

The present invention relates to a method for culturing purification bacteria and a purification method for purifying groundwater contaminated with volatile organic chlorine compounds.

Purification technologies using microorganisms are widely used in the activated sludge and anaerobic treatment processes for wastewater treatment, etc. In recent years, bioremediation, a technology that uses microorganisms to purify soil and groundwater contaminated with hazardous chemicals, has been attracting attention as a purification method with low environmental impact and purification costs.
Chlorinated ethylenes such as tetrachloroethylene (PCE) and trichloroethylene (TCE), which are representative volatile organic compounds (VOCs), are used as inexpensive solvents with strong cleaning power against oil in a wide range of fields such as the metal industry, semiconductor industry, dry cleaning shops, etc. On the other hand, there have been many reports of soil and groundwater contamination by chlorinated ethylenes, which has become a social problem.
The bioremediation of chlorinated ethylenes utilizes anaerobic dechlorinating bacteria that render chlorinated ethylenes harmless through the reductive dechlorination reaction (chemical formula 1) of tetrachloroethylene (PCE) → trichloroethylene (TCE) → cis-1,2-dichloroethylene (cis-DCE) → chloroethylene (VCM) → ethylene.

Dehalococcoides bacteria, a type of anaerobic dechlorinating bacteria, are the only bacteria that have been reported to be capable of dechlorinating chloroethylenes to cis-DCE and beyond. In other words, in bioremediation of chlorinated ethylenes, if Dehalococcoides bacteria are not present in the soil to be treated, dechlorination can only be performed to cis-DCE. Even if Dehalococcoides bacteria are present, the amount of Dehalococcoides bacteria in the environment is very small and their proliferation rate is slow, which results in a problem of a long purification period.
In order to solve such problems, a method of artificially culturing and increasing bacteria of the genus Dehalococcoides and introducing them into the environment to be purified to promote purification (bioaugmentation) is expected. In Patent Document 1, the present inventors isolated Dehalococcoides sp. UCH007 strain (NITE P-1471, hereinafter also referred to as UCH007 strain) for the first time in Japan, and proposed a method for purifying chlorinated ethylenes using this UCH007 strain.
In addition, Dehalococcoides bacteria are said to use only hydrogen as an electron donor (Non-Patent Document 1). Therefore, Dehalococcoides bacteria have been cultivated only under a mixed gas atmosphere of nitrogen and hydrogen. However, the present inventors have proposed a cultivation method using a medium containing pyruvic acid or a salt thereof (Patent Document 2). According to this cultivation method, pyruvic acid can be used not only as a carbon source but also as an electron donor, making hydrogen gas unnecessary and allowing Dehalococcoides bacteria to be cultivated in a safe environment. Furthermore, the present inventors have proposed a method of injecting the culture solution purely cultivated by the above method into the contaminated ground by using an inert gas (nitrogen gas) and an injection pipe to supply the culture solution without contacting air (oxygen) so that the absolutely anaerobic purification bacteria are not killed (Patent Document 3).
A series of purification techniques using the UCH007 strain have been demonstrated to be effective in purifying sites contaminated with volatile organic chlorine compounds (VOCs). However, this technique is more expensive than a purification method that activates indigenous Dehalococcoides bacteria without introducing purification bacteria (biostimulation). The reason for this is the high cost of culturing the purification bacteria (see Non-Patent Document 2).

Patent No. 6103518 JP 2021-016368 A Patent Application No. 2020-073529 Patent No. 6698357 JP 2021-016369 A

Loffler, F.E., Yan, J., Ritalahti, K.M., Adrian, L., Edwards, E.A., Konstantinidis, K.T., Muller, J.A., Fullerton, H., Zinder, S.H. & Spormann, A.M. " Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi." Int. J. Syst. Evol. Microbiol.,2013,63: p.625-635. Attached document (114877) at https://www.env.go.jp/press/108518.html Journal of Environmental Biotechnology, Vol.13, No.1, p13-18, 2013

Previously, the inventors proposed a method for cultivating the necessary purification bacteria in a simple facility set up at the purification site and quickly supplying it (Patent Document 4). This technology does not use an autoclave in the cultivation process, but cultivates the purification bacteria on-site in a container filled with distilled water. However, this technology targets facultative anaerobic bacteria and cannot be applied to obligate anaerobic bacteria that die when exposed to oxygen.

It has been revealed that a purification effect can be obtained by using a bacterial liquid containing Dehalobacteria in the presence of various bacteria that are not pure cultures (Non-Patent Document 3). When culturing specific useful bacteria in a mixture of various bacteria, a large, strictly controlled culture tank (fermenter) is used. Since it is costly and difficult to install a fermenter at the purification site, the bacterial liquid is cultured in a fermenter installed in a dedicated cultivation facility, transferred to a container, and transported to the site.

In Patent Document 2 and Patent Document 5, beer barrels are used as the culture vessels. With this method, the cultured material can be transported as is to the site and then injected into the purification site using the method described in Patent Document 3. A specific operation flow diagram is shown in Figure 1. If the purification site has a temperature-controlled culture room equipped with equipment such as an autoclave, water supply system, and nitrogen cylinder, the culture can be carried out on-site, eliminating the need to transport the bacterial liquid. However, setting up a culture room incurs a certain cost. In addition, it is necessary to secure a large amount of distilled water as the culture substrate, so a water purifier or distilled water must be purchased.

When a cost analysis of the culture is performed, the cost ratio of the reducing agent (glutathione) used in Patent Document 5 is high. The reason for using a reducing agent is to remove oxygen from the medium and adjust the culture liquid to a reducing state in which anaerobic Dehalobacteria can grow. If tap water is used instead of distilled water as the culture substrate, a water purifier is not necessary, but tap water has a high oxygen concentration and oxygen is present in a nearly saturated state, so there is a problem that an anaerobic state cannot be created unless a large amount of reducing agent is added.

The present invention has been made in consideration of the above circumstances. That is, the object of the present invention is to provide a method for culturing purifying bacteria that can easily culture the purifying bacteria necessary for purification without requiring sterilization of the culture solution or the addition of a reducing agent, that is also applicable to obligate anaerobic bacteria, and that can obtain a good purification effect when the purifying bacteria are introduced into the ground, and a purification method using the purifying bacteria.

The present inventors have found that the above-mentioned problems can be solved by using locally available groundwater as a culture solution instead of distilled water as a culture substrate, and have arrived at the present invention. That is, the present invention has the following configuration.
(1) A method for culturing purification bacteria for purifying groundwater contaminated with volatile organic chlorine compounds, the purification bacteria being anaerobic purification bacteria, the purification bacteria being characterized in that trichloroethylene is added to the groundwater collected from an aquifer in which the groundwater exists to prepare a culture solution, the culture solution containing the groundwater, organic matter, and an oxidation-reduction indicator is placed in a sealable container without sterilizing the culture solution, the gas phase is replaced with nitrogen, and the culture solution is allowed to stand for a certain period of time, and after confirming that the oxidation-reduction indicator in the culture solution has changed color and the oxidation-reduction potential has decreased, the purification bacteria are added to the culture solution .
(2) A method for culturing purification bacteria for purifying groundwater contaminated with volatile organic chlorine compounds, the purification bacteria being anaerobic purification bacteria, the purification bacteria being characterized in that trichloroethylene is added to the groundwater collected from an aquifer in which the groundwater exists to prepare a culture solution, no reducing agent is added to the culture solution, the culture solution containing the groundwater, organic matter, and an oxidation-reduction indicator is placed in a sealable container, the gas phase is replaced with nitrogen, and the culture solution is allowed to stand for a certain period of time, and after confirming that the oxidation-reduction indicator in the culture solution has changed color and the oxidation-reduction potential has decreased, the purification bacteria are added to the culture solution.
(3) The method for culturing a purifying bacterium according to (1), in which no reducing agent is added to the culture solution.
(4) The method for culturing a purifying bacterium according to any one of (1) to (3), wherein the purifying bacterium is a Dehalococcoides sp. UCH007 strain (NITE P-1471) belonging to the genus Dehalococcoides.
(5) A method for purifying groundwater contaminated with volatile organic chlorine compounds using purification bacteria, the purification bacteria being anaerobic purification bacteria, the purification method comprising: a culture solution oxygen removal step of removing oxygen from a culture solution in a culture vessel; a culture step of growing the purification bacteria in the culture solution in the culture vessel; an injection pipe installation step of installing an injection pipe for injecting the culture solution into an aquifer in which the groundwater is present; an injection pipe oxygen removal step of removing oxygen inside the injection pipe; an organic solution supply step of supplying an organic solution to the aquifer and leaving it for a certain period of time to form an anaerobic ground; and a culture solution supply step of supplying the culture solution in which the purification bacteria have grown to the aquifer, the purification bacteria supplied in the culture solution supply step being cultured in the culture step using the purification bacteria culture method described in any one of (1) to (4) .

The method for cultivating and purifying purifying bacteria of the present invention can easily cultivate the purifying bacteria necessary for purification without the need for sterilization of the culture solution or the addition of a reducing agent, and can also be applied to strictly anaerobic bacteria, and can provide a good purification effect when the purifying bacteria are introduced into the ground.

FIG. 1 is a flow diagram of a conventional method for cultivating purifying bacteria. FIG. 1 is a flow chart of a method for culturing purifying bacteria according to the present invention. FIG. 1 is a graph showing the number of bacteria of the genus Dehalococcoides (UCH007 strain) after growth in groundwater medium and normal medium in Experimental Example 1. FIG. 13 is a graph showing the number of bacteria of the genus Dehalococcoides (UCH007 strain) after growth in groundwater medium and normal medium in Experimental Example 2. FIG. 13 is a graph showing VOCs decomposition activity in a groundwater medium in Experimental Example 3. FIG. 1 shows the VOCs decomposition activity in a normal medium in Experimental Example 3. FIG. 13 is a graph showing the number of bacteria of the genus Dehalococcoides (UCH007 strain) after growth in groundwater medium and normal medium in Experimental Example 4. FIG. 13 is a graph showing the VOCs concentration in groundwater on the 7th day of culture in Experimental Example 5. FIG. 13 is a graph showing the VOCs concentration in groundwater on the 16th day of culture in Experimental Example 5. FIG. 13 is a graph showing the residual rate of VOCs from the initial concentration on the 16th day of culture in Experimental Example 5. FIG. 13 shows the gene copy number of UCH007 on the 16th day of culture in Experimental Example 5. FIG. 13 shows the increase rate of UCH007 gene copy number from the start of culture to day 16 of culture in Experimental Example 5. FIG. 13 is a graph showing the concentration of vcrA in groundwater on the 16th day of culture in Experimental Example 5.

An embodiment of the present invention will be described below. However, the embodiment of the present invention is not limited to the following embodiment.
The present invention has devised a method for culturing obligate anaerobic bacteria such as Dehalococcoides bacteria on-site using a minimum amount of equipment without adding a reducing agent. Representative steps 1 to 5 in the method for culturing purifying bacteria of the present invention are shown below. In the following, an explanation will be given using obligate anaerobic purifying bacteria, Dehalococcoides bacteria, as an example of the purifying bacteria to be cultured.

[Step 1] Culture substrate (solvent for culture medium)
In order to use a liquid with as low an oxygen concentration as possible as the culture substrate, groundwater with a low oxygen concentration pumped from an aquifer at or near the site where purification is performed is used. As long as the groundwater has a low oxygen concentration, it may be either uncontaminated or contaminated with the pollutant to be purified (e.g., trichloroethylene). In addition, groundwater that has been contaminated with organic matter as a result of microbial purification or the like may be used.

The necessary chemicals are added to a sealable culture vessel that can be used to inject the bacterial liquid into the ground, and the mixture is mixed with groundwater to dissolve the chemicals, creating a culture solution. Examples of chemicals to be dissolved in groundwater include inorganic salts such as nitrogen and phosphorus (see Tables 1 to 3), reducing agents, and organic substances. Table 1 shows the composition of the basal medium, which is an inorganic nutrient source. Tables 2 and 3 show the composition of trace element solutions A and B, which are components of the basal medium. The culture vessel is described in Patent Document 3 and Patent Document 5, but a 5-gallon beer barrel (Cornelius type) can be used. A reducing agent can be added, but it is not necessary to add one. This eliminates the need to use distilled water or tap water, which not only reduces costs but also makes it possible to culture on-site at sites without water facilities.

[Step 2] Nitrogen purging: With a specified amount of groundwater in the culture vessel and the vessel sealed, the gas phase in the culture vessel is purged with nitrogen (nitrogen replacement) using a nitrogen gas generator. As the nitrogen gas generator, a PSA (Pressure Swing Adsorption) type nitrogen gas generator is preferably used.
The nitrogen purging method involves purging 500 mL of culture solution prepared in a culture vessel with nitrogen gas generated from a nitrogen gas generator at a supply rate of 0.5 L/min for 10 minutes, thereby minimizing oxygen in the culture vessel.

[Procedure 3] Addition of TCE Add a trichloroethylene (TCE) solution to the culture medium to a final concentration of 0.6 to 2.5 ml/L.

[Step 4] Creation of anaerobic conditions Static culture is performed at a temperature of 20 to 30 ° C (for information on temperature, see JP 2021-13911 A), at which the Dehalococcoides bacteria are likely to grow. Then, wait until the remaining oxygen and nitrate nitrogen in the culture vessel are consumed by bacteria other than the Dehalococcoides bacteria in the medium, creating an anaerobic state in which the Dehalococcoides bacteria in the culture vessel can grow. By not using a reducing agent and not sterilizing the culture vessel, the bacteria in the groundwater consume organic matter and create an anaerobic state. To confirm the anaerobic state, visually check that the redox indicator (such as resazurin sodium) added to the culture solution has changed color from blue-purple (pink) to transparent.

In consideration of cost and safety, stainless steel sealed containers are usually used for culturing. When a stainless steel sealed container is used, the inside cannot be seen from the outside. In the case of such a container, a transparent syringe with a needle is used. Specifically, the needle is inserted from the opening without allowing air to enter, and the culture container is tilted or turned upside down so that the inserted needle reaches the surface of the culture fluid. Then, the plunger is slightly pulled back to remove the culture fluid into the syringe so that the color of the culture fluid can be confirmed, and the color caused by the redox indicator added to the culture fluid is visually confirmed. After confirmation, the plunger is pushed back to return the culture fluid to the container, and the needle is removed from the opening while making sure that no air enters the container.

[Step 5] Inoculation and static culture of pure cultured purifying bacteriaAfter confirming that the culture solution is in a reducing state, pure cultured seed bacteria (UCH007 strain: approximately 1 x ¹⁰ cells/ml, UCH001 strain: approximately 1 x ¹⁰ cells/ml) are inoculated into the medium at a ratio of 0.1 to 1% and again cultured statically.Then, the Dehalococcoides bacteria are multiplied to a concentration of ¹⁰ cells/ml or more within 14 days of culture.

A flow diagram of the above-described culture method of the present invention is shown in FIG.
By immediately sealing groundwater collected from an aquifer where groundwater exists and anaerobicizing the groundwater using the remaining microorganisms, it is possible to grow a bacterial liquid containing Dehalococcoides bacteria without the need to add an expensive reducing agent (glutathione), which was previously required.
Furthermore, when injecting the grown purification bacteria into a contaminated site, it is necessary to purge the culture solution with nitrogen before use and to attach an activated carbon filter to the injection tube to remove VOCs in the culture solution.

The following experiments were carried out, comparing the conventional method (flow chart of the conventional method for culturing purifying bacteria, Figure 1) with the method of the present invention (flow chart of the method for culturing purifying bacteria, Figure 2). In both experiments, a culture solution containing Dehalococcoides bacteria was successfully produced.

[Experimental Example 1] Comparison of groundwater medium and normal medium
1) Objective In Experimental Example 1, we will confirm whether it is possible to cultivate purification bacteria using conventional procedures (Figure 1) using groundwater collected from the aquifer to be purified.

2) Test method Groundwater medium: 500 ml of contaminated groundwater as a substrate, 0.23 g of sodium pyruvate, 1.25 g of sodium bicarbonate, 0.5 g of glutathione (reducing agent), and 0.5 ml of 0.1% sodium resazurin were added to a 720 ml glass vial. The glass vial was sealed with a butyl rubber stopper and sterilized by purging with nitrogen gas for 15 minutes (121°C, 20 minutes). After cooling, 0.5 ml of vitamin solution and 2.0 ml of TCE solution (final concentration 1.2 mg/L) were added, and 2 ml of coculture culture solution of UCH007 and UCH001 strains (UCH007: 1.05 x 10 ⁸ cells/ml, UCH001: 1.0 x 10 ⁹ cells/ml) was inoculated. The composition of the vitamin solution is shown in Table 4.
Conventional medium: A conventional medium was prepared by changing the water substrate. 500 ml of distilled water as the substrate, 0.23 g of sodium pyruvate, 0.5 g of glutathione (reducing agent), and 0.5 ml of 0.1% resazurin sodium were added to a glass vial in addition to inorganic nutrient sources (Table 1). After 15 minutes of nitrogen purging, the medium was sterilized and cooled, and 0.5 ml of vitamin solution and 2.0 ml of TCE solution (final concentration 1.2 mg/L) were added to adjust the medium, and 2 ml of coculture culture solution of UCH007 and UCH001 strains (UCH007: 1.05 x 10 ⁸ cells/ml, UCH001: 1.0 x 10 ⁹ cells/ml) was inoculated.

3) Test results Both the groundwater medium and the regular medium were statically cultured in a thermostatic chamber at 22°C, and on the 14th day of culture, Dehalococcoides genus bacteria (UCH007 strain) had proliferated to over ¹⁰⁷ cells/ml (Figure 3). Experimental Example 1 confirmed that by using groundwater collected from the aquifer to be purified, it was possible to cultivate purification bacteria equivalent to or better than those in the regular medium.

[Experimental Example 2] Test to culture groundwater using a reducing agent without sterilizing it
1) Purpose of the test To confirm whether it is possible to culture purifying bacteria without the sterilization process, in addition to the method of Example 1.

2) Test method Groundwater medium: 40 ml of groundwater as substrate, 40 mg of sodium pyruvate, 0.1 g of sodium bicarbonate, 40 mg of glutathione (reducing agent), 40 μL of vitamin solution, inorganic nutrient source (Table 1), and 40 μL of 0.1% resazurin sodium were placed in a 60 ml glass vial. The vial was sealed with a butyl rubber stopper and purged with nitrogen gas for 5 minutes, after which 0.2 ml of TCE solution (final concentration 1.2 mg/L) was added, and 0.4 ml of coculture culture solution of UCH007 and UCH001 strains (UCH007: 7.7 x ¹⁰⁷ cells/ml, UCH001: 1.0 x ¹⁰⁹ cells/ml) was inoculated.
Normal medium: As in the case of the groundwater medium, 40 ml of distilled water as a substrate, 40 mg of sodium pyruvate, 0.1 g of sodium bicarbonate, 40 mg of glutathione (reducing agent), inorganic nutrient source (Table 1), and 40 μL of 0.1% resazurin sodium were added to a glass vial. The vial was sealed with a butyl rubber stopper, purged with nitrogen gas for 5 minutes, and then sterilized (121°C, 20 minutes). After cooling, 40 μL of vitamin solution and 0.2 ml of TCE solution (final concentration 1.2 mg/L) were added, and 0.4 ml of coculture culture solution of UCH007 and UCH001 strains (UCH007: 7.7 x ¹⁰⁷ cells/ml, UCH001: 1.0 x ¹⁰⁹ cells/ml) was inoculated.

3) Test results Both the groundwater medium and the regular medium were cultured statically in an incubator at 25°C, and on the 14th day of culture, it was confirmed that Dehalococcoides bacteria (UCH007 strain) had proliferated at more than 10 ⁸ cells/ml in both media (Figure 4).

[Experimental Example 3] Test to confirm the decomposition activity of volatile organic chlorine compounds (VOCs) in the medium not subjected to sterilization treatment obtained in Experimental Example 2
1) Test Objectives In Experimental Example 2, the purifying bacteria grew in the unsterilized groundwater medium in the same way as in the sterilized normal medium. Unsterilized groundwater contains many bacteria other than the purifying bacteria. In order to confirm that a purification effect equivalent to that of a normal medium in which pure culture is performed using a non-sterile bacterial solution (a state in which other bacteria are present), the cultured bacterial solution is introduced into actual contaminated groundwater to confirm the purification effect.

2) Test method In this test, 40 ml of actual contaminated groundwater was placed in a 60 ml glass culture vessel and anaerobicized by adding organic materials beforehand. The quality of the anaerobic groundwater is shown in Table 5. A groundwater medium in which UCH007 strain had been grown and a normal medium were added to this anaerobic groundwater, and the decomposition activity of VOCs was compared. The UCH007 strain in both the groundwater medium and the normal medium when added to the contaminated groundwater was 1 x 10 ⁸ cells/ml, and 0.4 ml (1%) was added to the contaminated groundwater. Cultivation was performed as a static culture at 25°C.

3) Test results The test results are shown in Figures 5 and 6. Whether using the UCH007 strain grown in non-sterile groundwater (Figure 5) or the UCH007 strain grown in normal medium (pure culture) (Figure 6), the TCE concentration had dropped significantly by the 14th day, meaning there was no difference in the purification effect on actual contaminated groundwater. This confirmed that purification was possible even when using a bacterial solution containing bacteria grown other than the purification bacteria.

[Experimental Example 4] Test to culture groundwater without sterilization or reducing agents
1) Purpose of the test In Experimental Example 2 (cultivating groundwater under non-sterile conditions), we will confirm whether purification bacteria can be cultivated without using an expensive reducing agent (glutathione).

2) Test method Groundwater medium: 100 ml of groundwater as a substrate, 100 mg of sodium pyruvate, 100 μL of vitamin solution, inorganic nutrient source (Table 1), and 100 μL of 0.1% resazurin sodium were placed in a 120 ml glass vial. The vial was sealed with a butyl rubber stopper and purged with nitrogen gas for 10 minutes, after which 0.5 ml of TCE solution (final concentration 1.4 mg/L) was added and 1.0 ml of coculture culture solution of UCH007 and UCH001 strains (UCH007: 7.7 x ¹⁰⁷ cells/ml, UCH001: 1.0 x ¹⁰⁹ cells/ml) was inoculated.
Normal medium: As with the above groundwater medium, 80 ml of distilled water as a substrate, 80 mg of sodium pyruvate, 0.2 g of sodium bicarbonate, 80 mg of glutathione (reducing agent), inorganic nutrient source (Table 1), and 80 μL of 0.1% resazurin sodium were added to a glass vial. The vial was sealed with a butyl rubber stopper, purged with nitrogen gas for 10 minutes, and then sterilized (121°C, 20 minutes). After cooling, 80 μL of vitamin solution and 0.3 ml of TCE solution (final concentration 0.52 mg/L) were added, and 1.0 ml of coculture culture solution of UCH007 and UCH001 strains (UCH007: 7.7×10 ⁷ cells/ml, UCH001: 1.0×10 ⁹ cells/ml) was inoculated.

3) Test results: Both groundwater medium and normal medium were cultured in a thermostatic chamber at 25℃, and on the 14th day of culture, it was confirmed that UCH007 had proliferated to more than ¹⁰⁷ cells/ml in both media (Figure 7). Therefore, it was confirmed that the necessary amount of purification bacteria can be cultured under non-sterile conditions without using a reducing agent.

From the above results, it was found that the method for culturing purifying bacteria of the present invention uses groundwater as a culture substrate, and as a result, compared to conventional culture methods, sterilization is not required and it is possible to culture without using a reducing agent.
The method for culturing purifying bacteria of the present invention is expected to have the following effects.
(1) Since groundwater is used, there is no need to use distilled water (cost reduction; cultivation is possible on-site even if the purification site does not have a running water supply).
(2) Cultivation is possible if you have a container, seed culture, and a room where the temperature can be controlled.
(3) No reducing agent is required (cheap).
(4) It can be cultivated locally (no transportation costs).
(5) It is easy to manage (it is not a pure culture).
(6) High safety. Only groundwater (bacteria) from the site where the purification is carried out and bacteria (isolated strains) that have been confirmed to be safe are used for cultivation.
(7) The cultivation period is short (reduced number of containers).

[Experimental Example 5] Effect of concentration of VOCs added to groundwater on the proliferation of purification bacteria
1) Purpose of the test To investigate the optimal concentration range for VOCs (TCE) to be added to groundwater.

2) Test method A culture solution containing TCE, groundwater, sodium pyruvate, inorganic nutrient sources (Table 1), vitamins, sodium bicarbonate, and 0.1% resazurin sodium was prepared according to the composition shown in Table 6.
The 10 types of culture medium listed in Table 6 were added to 10 glass vials with a total volume of 120 ml. The gas phase was purged with nitrogen for 5 minutes, and TCE solution of each condition was added and cultured at 25°C. After one week of culture, 0.2 ml of coculture medium of UCH007 strain (UCH007: 4.7 x ¹⁰⁷ cells/ml, UCH001: 6.0 x ¹⁰⁸ cells/ml) was inoculated into each vial, and the subsequent growth of UCH007 strain was confirmed.

3) Test results Figure 8 shows the VOCs concentration on the 7th day after the start of cultivation. As the concentration of added TCE increased, the concentration became lower than the initial concentration, and under almost all conditions TCE was dechlorinated to cis-1,2-DCE. Cultivation was continued thereafter, and the VOCs concentration on the 16th day after the start of cultivation is shown in Figure 9. It was confirmed that the dechlorination of VOCs had progressed further, except when the initial TCE concentration was 3.0 mg/L.

Furthermore, the residual rate of VOCs from the initial concentration on the 16th day from the start of culture is shown in Figure 10. The gene copy number of UCH007 on the 16th day from the start of culture is shown in Figure 11. The rate of increase in the gene copy number of UCH007 from the start of culture to the 16th day of culture is shown in Figure 12.
From the above investigation, the following became clear:
(1) It is possible to grow UCH007 predominantly in groundwater by culturing it at an initial VOCs concentration (additive concentration) of 3.0 mg/L or less.
(2) From the results shown in Figures 10 and 12, the final concentration of VOCs added to groundwater is preferably 0.6 to 2.5 mg/L, and more preferably 1.0 to 2.5 mg/L.
Figure 13 shows the results of measuring the concentration of vcrA (the functional gene for dechlorination) in groundwater on the 16th day after the start of cultivation. From these results, the reason why the dechlorination rate progressed even under conditions where the TCE addition rate was low and the UCH007 strain did not increase significantly is thought to be because dechlorinating bacteria other than the UCH007 strain were present in the original groundwater.

Next, a method for purifying groundwater contaminated with volatile organic chlorine compounds using the culture solution of the purifying bacteria obtained by the above-mentioned method for culturing the purifying bacteria will be described.
The purification method of this embodiment includes a culture solution oxygen removal process for removing oxygen from the culture solution in a culture vessel; a cultivation process for growing purification bacteria in the culture solution in the culture vessel; an injection pipe installation process for installing an injection pipe for injecting the culture solution into an aquifer where groundwater exists; an injection pipe oxygen removal process for removing oxygen inside the injection pipe; an organic solution supply process for forming an anaerobic ground by supplying an organic solution to the aquifer and leaving it for a certain period of time; and a culture solution supply process for supplying the culture solution in which purification bacteria have grown to the aquifer.

The culture solution oxygen removal step of removing oxygen from the culture solution corresponds to the above-mentioned step of tightly sealing the culture vessel and purging with nitrogen gas.
The culture process for growing the purifying bacteria in the culture solution in the culture vessel corresponds to the process of adding a TCE solution to the culture solution using the above-mentioned groundwater, inoculating the purifying bacteria, and allowing it to undergo static culture in an incubator.

In the injection pipe installation process, an injection pipe is installed in the ground to inject the culture solution in which the purification bacteria have been grown into the groundwater aquifer. The injection pipe is installed to a length equivalent to the depth of the aquifer so that it reaches the aquifer where the contaminated groundwater exists. There are no limitations on the method of installing the injection pipe underground, and for example, the injection pipe may be inserted into a borehole formed by drilling the ground.

In the injection pipe oxygen removal process, oxygen is removed from inside the injection pipe installed in the ground. Specifically, oxygen is removed from inside the injection pipe by supplying nitrogen gas from a nitrogen gas generator to the bottom of the injection pipe. It is preferable to supply nitrogen gas at least three times the volume inside the injection pipe.

In the organic solution supply process, an anaerobic ground is formed by supplying an organic solution to the aquifer and leaving it for a certain period of time. By supplying organic matter to the ground, aerobic bacteria present in the ground consume oxygen and decompose the organic matter, creating an anaerobic environment in the ground. The leaving period is about 1 to 2 months and is determined by monitoring the sulfate ion concentration. Once a decrease in the sulfate ion concentration is confirmed, proceed to the next step. There are no particular restrictions on the organic matter (e.g., sodium lactate, etc.). The organic matter concentration in the organic solution is preferably within the range of 500 mg/L to 50 g/L. When the organic solution is injected into the injection tube, nitrogen gas pressure from a nitrogen gas generator is used.

In the culture solution supplying process, in which a culture solution in which purifying bacteria have been grown is supplied to the aquifer, the culture solution of the purifying bacteria cultivated in the above-mentioned cultivation process is supplied to the ground (aquifer). The purifying bacteria supplied in the culture solution supplying process are those cultivated in the above-mentioned cultivation process using the above-mentioned cultivation method for the purifying bacteria. When the culture solution is injected into the injection pipe, the pressure of nitrogen gas from a nitrogen gas generator is used.

According to the purification method of this embodiment, when a culture solution containing organic matter is supplied into the ground, aerobic bacteria present in the ground consume oxygen while decomposing the organic matter in the culture solution, forming an anaerobic environment in the ground. When an anaerobic environment is formed in the ground, the anaerobic bacteria become activated and decompose the organic matter, supplying hydrogen (electron donor). The anaerobic purification bacteria then use this hydrogen to proceed with purification (dechlorination).

Claims (5)

A method for culturing purification bacteria for purifying groundwater contaminated with volatile organic chlorine compounds, comprising:
The purification bacteria are anaerobic purification bacteria,
The groundwater is collected from an aquifer in which the groundwater exists, and trichloroethylene is added to the groundwater to be used as a culture solution ;
The culture medium is not sterilized,
The method for cultivating purifying bacteria is characterized in that a culture solution containing the groundwater, organic matter, and an oxidation-reduction indicator is placed in a sealable container, the gas phase is replaced with nitrogen, and the solution is allowed to stand for a certain period of time. After confirming that the oxidation-reduction indicator in the culture solution has changed color and the oxidation-reduction potential has decreased, the purifying bacteria are added to the culture solution . A method for culturing purification bacteria for purifying groundwater contaminated with volatile organic chlorine compounds, comprising:
The purification bacteria are anaerobic purification bacteria,
The groundwater is collected from an aquifer in which the groundwater exists, and trichloroethylene is added to the groundwater to be used as a culture solution;
No reducing agent is added to the culture medium,
The method for cultivating purifying bacteria is characterized in that a culture solution containing the groundwater, organic matter, and an oxidation-reduction indicator is placed in a sealable container, the gas phase is replaced with nitrogen, and the solution is allowed to stand for a certain period of time. After confirming that the oxidation-reduction indicator in the culture solution has changed color and the oxidation-reduction potential has decreased, the purifying bacteria are added to the culture solution. The method for culturing purifying bacteria according to claim 1 , wherein no reducing agent is added to the culture solution. The method for culturing a purifying bacterium according to any one of claims 1 to 3 , wherein the purifying bacterium is a Dehalococcoides sp. UCH007 strain (NITE P-1471) belonging to the genus Dehalococcoides. A method for purifying groundwater contaminated with volatile organic chlorine compounds using purification bacteria, comprising:
The purification bacteria are anaerobic purification bacteria,
A culture solution oxygen removal step of removing oxygen from the culture solution in the culture vessel;
A culture step of growing the purifying bacteria in the culture solution in the culture vessel;
An injection pipe installation step of installing an injection pipe for injecting the culture solution into an aquifer where the groundwater exists;
an oxygen removal process in the injection tube for removing oxygen from inside the injection tube;
an organic solution supplying step of supplying an organic solution to the aquifer and leaving it for a certain period of time to form an anaerobic ground;
A culture solution supplying step of supplying the culture solution in which the purification bacteria have been grown to the aquifer,
The purification method, characterized in that the purification bacteria supplied in the culture solution supplying step are cultured in the culturing step using the purification bacteria culture method described in any one of claims 1 to 4 .

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