JP7147737B2 - Method for purifying contaminated soil and groundwater - Google Patents

Description

The present invention relates to a method for remediation of soil and groundwater contaminated with chlorinated ethylene by an in situ bioaugmentation method.

As a method for promoting the purification of contaminated soil by injecting chlorinated ethylene-degrading bacteria, Patent Document 1 discloses a method in which a nutrient is injected to make groundwater under an anaerobic atmosphere, and then a culture solution containing chlorinated ethylene-degrading bacteria is added. It describes a method for performing simple and good bioaugmentation by injecting nutrients directly into groundwater and then reinjecting nutrients set to appropriate conditions (volume, concentration, pH). However, in Patent Document 1, the underground temperature is not controlled, and the temperature conditions are not necessarily optimal for the growth of chlorinated ethylene-decomposing bacteria and pollutant decomposition, so it takes a long time to decompose chlorinated ethylene. I may need it.

As a method for promoting purification by heating chlorinated ethylene-contaminated soil, Patent Document 2 discloses that the contaminated soil is energized and heated to 50 to 70° C. to elute chlorinated ethylene from the soil and remove indigenous soil. A method for degrading contaminants by co-metabolism of thermophilic bacteria (mainly Clostridium) has been described. However, thermophilic bacteria that survive in the above temperature range can mainly decompose only tetrachlorethylene (PCE) and trichlorethylene (TCE) among chlorinated ethylene, so the decomposition products dichlorethylene (DCE) and chloroethylene (CE) are remain. (It should be noted that the activity of completely decomposing DCE and CE into harmless ethylene has been reported only for Dehalococcoides bacteria, which cannot survive in this temperature range.) In the method of Patent Document 2, the subsequent gas Residual DCE and CE are collected in the suction or pumping process, and purification costs are high due to the installation of equipment.

Patent Literature 3 describes a method of injecting a microbial activator into soil and then injecting hot water to raise the underground temperature to less than 35° C., thereby promoting purification.

Patent Document 4 describes a method of heating chlorinated ethylene-contaminated soil to 20 to 35° C. to promote purification.

JP 2013-139020 A JP 2015-112556 A JP 2015-24401 A JP 2009-90183 A

N. Okutsu, W. Tamura, M. Mizumoto, T. Ueno, H. Ishida, T. Iizumi. (2012) Field demonstration of bioaugmentation in trichloroethene-contaminated groundwater. Water Practice Technol. 7: wpt2012053 G. Kitamura, Y. Yamazaki, I. Tako, I. Suzuki, T. Kobayashi and T. Shimizu. (2018) Investigation of accelerating microbial decomposition of volatile organic chlorine compounds by heating temperature control. Abstracts of the 24th Research Meeting on Groundwater/Soil Pollution and its Prevention Measures: 597-601

In the above Patent Documents 1 to 4, detailed temperature characteristics of chlorinated ethylene-degrading bacteria (Dehalococcoides bacterium) when purifying soil and groundwater contaminated with chlorinated ethylene by an in-situ bioaugmentation method. has not been clarified. Therefore, the purification treatment was not necessarily performed under optimum temperature conditions.

INDUSTRIAL APPLICABILITY The present invention is a method for purifying contaminated soil and groundwater, which can efficiently purify contaminated soil and groundwater by carrying out the method under optimal temperature conditions for cell growth and chlorinated ethylene-decomposing activity of chlorinated ethylene-degrading bacteria. intended to provide

The method for purifying contaminated soil and groundwater of the present invention is a method for purifying contaminated soil and groundwater in which chlorinated ethylene-degrading bacteria are injected into soil and groundwater contaminated with chlorinated ethylene and purified by an in-situ bioaugmentation method. , the underground temperature is maintained at the growth temperature of chlorinated ethylene-decomposing bacteria, and then the underground temperature is raised.

In one aspect of the present invention, the underground temperature is maintained at 15-30°C to grow chlorinated ethylene-degrading bacteria, and then the underground temperature is maintained at 32-34°C.

In one aspect of the present invention, the chlorinated ethylene-decomposing activity of chlorinated ethylene-decomposing bacteria is measured, and when the chlorinated ethylene-decomposing activity decreases by a predetermined level or more, the underground temperature is lowered to 25 to 30°C.

The present inventor has conducted intensive research on the temperature characteristics of chlorinated ethylene-degrading bacteria (bacteria belonging to the genus Dehalococcoides) in relation to the in-situ bioaugmentation method for soil and groundwater contaminated with chlorinated ethylene. The findings 1) to 3) were obtained.

1) When the soil is heated to 15-30°C, especially 25-30°C, the growth rate of chlorinated ethylene-degrading bacteria remarkably increases. When the temperature exceeds 30°C, the cell growth rate decreases as the temperature rises.

2) After the chlorinated ethylene-degrading bacteria have grown sufficiently (as a guideline, the number of DHC bacteria is 10 ⁶ copies/mL or more, preferably 10 ⁷ copies/mL or more), the chlorinated ethylene-decomposing activity of the degrading bacteria reaches its maximum. High chlorinated ethylene cracking activity is maintained by warming to ~34°C. At a temperature of 32 to 34° C., the degrading bacteria do not proliferate vigorously, but they do not rapidly die and maintain high chlorinated ethylene decomposing activity. Also, by maintaining the soil at 32 to 34° C., the elution of chlorinated ethylene adsorbed to the soil is promoted, and efficient purification can be performed.

3) If the temperature is kept above 34°C for a long period of time, the chlorinated ethylene decomposing activity is remarkably lowered.

The present invention is based on such findings.

According to the present invention, it is possible to achieve efficient in-situ bioaugmentation by setting the temperature conditions for the entire process based on detailed temperature characteristic data of chlorinated ethylene-degrading bacteria and combining it with soil warming technology. can.

It is a graph explaining the method of the present invention. It is a graph which shows the result of an Example. It is a graph which shows the result of an Example.

In one aspect of the present invention, contaminated soil and groundwater are purified in the following steps (1) to (3).

(1) Inject chlorinated ethylene-decomposing bacteria and nutrients into contaminated soil and groundwater, then heat the ground to 15 to 30°C, especially 25 to 30°C, as shown in Fig. 1, and raise the temperature. In addition, the heating of the ground may be started prior to the injection of the chlorinated ethylene-decomposing bacteria and the nutrient. Chlorinated ethylene-degrading bacteria proliferate vigorously in the range of 15 to 30°C, especially in the range of 25 to 30°C. As the chlorinated ethylene-degrading bacteria, bacteria belonging to the genus Dehalococcoides are suitable.

(2) After the chlorinated ethylene-degrading bacteria have grown sufficiently (as a guideline, the number of DHC bacteria is 10 ⁶ copies/mL or more, preferably 10 ⁷ copies/mL or more), the underground is heated to 32-34°C and maintained. do. As shown in FIG. 1, at this temperature, degrading bacteria do not proliferate vigorously, but they do not die rapidly, and high chlorinated ethylene decomposing activity is maintained.

Also, by maintaining the soil at 32 to 34° C., the elution of chlorinated ethylene adsorbed to the soil is promoted, and efficient purification can be performed. In addition, if the purification point is maintained at a temperature exceeding 34°C for a long period of time, the proportion of hydrogen-utilizing methanogenic bacteria, which has an optimum growth temperature around 37°C, increases, and the hydrogen necessary for the chlorinated ethylene decomposition reaction is reduced. deprived of it and decomposing activity decreases.

(3) When the above step (2) is carried out, the chlorinated ethylene decomposition activity may be remarkably lowered. In this case, the underground temperature is lowered to 25 to 30° C., for example, about 30° C., and maintained at this temperature for a certain period of time (eg, 15 to 50 days) to recover the decomposition activity of the chlorinated ethylene-decomposing bacteria.

[Underground heating method]
As a method of heating the ground, (a) a method of inserting an electrode into the ground and energizing it, and heating by Joule heat generated by the electrical resistance of the soil itself; (b) heating with a heater buried in the ground. A method of warming; (c) a method of injecting heated nutrients or hot water; and the like.

As the method for heating the underground, the method (a) is preferable because the impermeable layer can be heated to 50 to 70°C. The method (a) can promote the elution of contaminants adsorbed on clay layers and silt layers, etc., into which nutrients and decomposing bacteria are difficult to penetrate, so it is possible to reduce the amount of contaminants remaining and prevent rebound after purification. .

If contaminants remain in the soil in the unsaturated zone (vent zone) above the aquifer, heat the unsaturated zone to 50-70°C and use gas suction in combination to efficiently purify it. can be done. Also in this case, pollutants eluted into the aquifer can be purified by chlorinated ethylene-degrading bacteria.

Detailed temperature characteristics of chlorinated ethylene-degrading bacteria were examined by conducting the following experimental examples.

[Experimental Example 1: Evaluation of CE decomposition activity of chlorinated ethylene-degrading bacteria under various temperature conditions]
<Culturing of chlorinated ethylene-degrading bacteria>
Dehalococcoides bacterium was used as a chlorinated ethylene-degrading bacterium.

A 10 L culture tank containing an artificial medium (described in Non-Patent Document 1, final liquid volume 6 L) was inoculated with a pre-cultured inoculum to a final concentration of 1%. Then, 50 mL each of chloroethylene (CE) and H ₂ gas were added, and culture was started under anaerobic conditions at a culture temperature of 30°C.

The culture conditions were monitored by analyzing the gas component concentrations in the culture tank headspace, and 50 mL of gas was added again when the CE and H ₂ concentrations were 5 mg/L or less and 1 mg/L or less, respectively. In order to reproduce a state in which the cells have grown sufficiently, the bacteria were cultured until the 16S rDNA reached 10 ⁷ to 10 ⁸ copies/mL.

Ethylene, methane, and H ₂ concentrations were measured by gas chromatography barrier discharge ionization detection (GC-BID). CE concentrations were determined by gas chromatography-flame ionization detection (GC-FID).

<Degradation activity evaluation test>
A decomposition activity evaluation test was started on the 96th day after inoculation in the culture tank. That is, the concentration of gas components in the headspace of the culture tank was periodically analyzed, and the generation rate of ethylene, a decomposition product of CE, was evaluated as the decomposition activity of chlorinated ethylene. The temperature of the culture tank at the beginning of the decomposition activity evaluation test was 30° C. as shown in FIG.

Prior to the decomposition activity evaluation test at each temperature, the culture tank was purged with N ₂ gas to remove CE and ethylene remaining in the culture solution. Then, N ₂ /CO ₂ gas (mixture ratio 80:20) was blown in to adjust the pH to 7.3 to 7.5, and then 50 mL each of CE and H ₂ gases were added again to start the decomposition activity evaluation test.

<Results/Discussion>
FIG. 2 and Table 1 show the measurement results of the CE decomposition rate and the methane production rate under each temperature condition.

As shown in Figure 2 and Table 1, the CE decomposition rate showed the highest value at 34°C. When the temperature exceeded 35°C, the activity decreased significantly, but a significant increase in the methane production rate was observed in inverse proportion.

The degrading microorganisms group includes archaebacteria ₍ methanogens) that utilize H2 to produce methane. Since H ₂ used for the CE decomposition reaction is also used for the methane production reaction by these bacteria, CE-degrading bacteria and methanogenic bacteria are in a competitive relationship for H ₂ . Since the optimum growth temperature for general methanogens is around 37°C, the methanogenic activity becomes dominant at 35° _C or higher in this experimental system, and the required H2 is deprived of the CE decomposition rate. This is presumed to be one of the reasons for the decline.

Incidentally, in Non-Patent Document 2, the optimum temperature for growth and decomposition activity of bacteria belonging to the genus Dehalococcoides is said to be 25 to 30°C. However, in the evaluation test of Non-Patent Document 2, the decomposition activity is evaluated immediately after inoculating a small amount of the inoculum in the medium, so the decomposition rate is greatly affected by the cell growth rate. In other words, if the optimal temperature for the enzyme group required for growth is low, the apparent optimal temperature for decomposition activity is also estimated to be low.

On the other hand, in the 10 L culture tank test this time, the test was started from the state where the bacterial cell concentration was maximized, so the effect is extremely small, and it is presumed that the true temperature characteristics of the decomposition rate can be obtained. . That is, it was suggested that the optimal temperature for growth of bacteria belonging to the genus Dehalococcoides is 25 to 30°C, while the optimal temperature for enzymes involved in CE decomposition is around 34°C.

[Experimental Example 2: CE decomposition activity test at 30°C using the bacterial solution after completion of the decomposition activity evaluation test at each temperature in Experimental Example 1]
Into a vial containing 80 mL of artificial medium, 0.6 mL of CE gas and 0.2 mL of the bacterial solution after completion of each culture temperature test in Experimental Example 1 were injected to start culturing, and incubated at 30° C. for about 10 days. The cracking activity was calculated from the amount of ethylene produced later. Table 2 shows the measurement results of the CE decomposition activity (CE decomposition rate and methane production rate).

As shown in Table 2, the activity of the 34°C culture, which showed the highest decomposition activity in the above test, decreased to about 30% compared to the case of the 30°C culture.

As described above, in this experimental example, the CE decomposition rate largely depends on the growth rate of the inoculated CE-degrading bacteria. That is, since the degrading bacteria exposed to 34°C have reduced activities of the enzyme group necessary for their growth, even if they are cultured again at 30°C, their growth does not proceed immediately, and the apparent degrading activity is reduced. It was suggested that Based on these results, it is considered necessary to maintain the culture temperature at 25 to 30° C. during the growth phase of bacteria belonging to the genus Dehalococcoides.

[Experimental Example 3: CE decomposition activity test at 30°C or less]
Cultivation was initiated by injecting 0.2 mL of CE gas and 1 mL of the bacterial solution cultured at 30°C into a vial containing 100 mL of an artificial medium, followed by incubation at 15°C, 20°C, 25°C, and 30°C. FIG. 3 shows the results of measuring the amount of ethylene produced with the growth of bacteria belonging to the genus Dehalococcoides.

As shown in FIG. 3, the ethylene production rate is greatly reduced at 20° C. or lower, so it is considered necessary to maintain the temperature within a range not lower than 25° C. even during recovery of activity.

[Experimental Example 4: Dehalococcoides bacterium-derived 16S rDNA quantification]
2 mL of the culture solution after completion of the decomposition activity evaluation test at each temperature in Experimental Example 1 was sampled and allowed to stand at 4 ° C. for about 3 hours to precipitate iron powder. was centrifuged (10,000×g, 10 min, 4° C.) to extract DNA. A commercially available DNeasy Blood & Tissue Kit (QIAGEN) was used for DNA extraction. Using the extracted DNA, 16S rDNA derived from bacteria belonging to the genus Dehalococcoides was quantified according to quantitative PCR using Light Cycler (Roche) described in Non-Patent Document 1. Table 3 shows the results.

As shown in Table 3, no change was observed in the number of 16S rDNA of bacteria belonging to the genus Dehalococcoides even at temperatures above 35°C.

In addition, as shown in FIG. 2, when the bacterial solution after culturing at 38° C. is returned to 30° C., after about 1 week, it exhibits the same level of decomposition activity as during the initial 30° C. culturing. It is predicted that a certain number of bacteria belonging to the genus Dehalococcoides will survive even after culturing.

That is, it was suggested that the cause of the decrease in CE-degrading activity at 35° C. or higher is the lack of H ₂ accompanying the increase in methanogenic activity and the decrease in activity of CE-degrading enzyme in Dehalococcoides bacteria.

From these results, a certain number of bacteria of the genus Dehalococcoides survived even at a culture temperature of 34°C or higher. It is considered that high decomposition activity is exhibited again.

Claims (1)

In a method for purifying contaminated soil and groundwater, in which chlorinated ethylene-degrading bacteria are injected into soil and groundwater contaminated with chlorinated ethylene and purified by an in-situ bio-augmentation method ,
The chlorinated ethylene-degrading bacterium is a Dehalococcoides bacterium,
Maintaining the underground temperature at 15 to 30° C. to grow chlorinated ethylene-degrading bacteria, then maintaining the underground temperature at 32 to 34° C.,
The chlorinated ethylene decomposing activity of the chlorinated ethylene decomposing bacteria is measured while the underground temperature is maintained at 32 to 34 ° C. If the chlorinated ethylene decomposing activity decreases by a predetermined amount or more, the underground temperature is reduced to 25 to 30 ° C. A method for purifying contaminated soil and groundwater, characterized in that the temperature is lowered to °C.

Images