JP2009025097A - Clarification determination method of ground water and/or soil by anaerobic microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining whether ground water and/or soil polluted by a volatile organic chloride component is purified by anaerobic microorganisms in the ground water and/or the soil. <P>SOLUTION: This method for determining whether the ground water and/or the soil polluted by the volatile organic chloride component can be purified by the anaerobic microorganisms in the ground water and/or the soil or not, includes following processes (A)-(D): (A) a process for collecting the ground water, or the ground water and the soil polluted by the volatile organic chloride component; (B) a process for cultivating the collected ground water under an anaerobic condition, or cultivating the ground water with the collected soil added thereto under an anaerobic condition; (C) a process for supplying the same ground water as the ground water used in process (B) to a cultured material in process (B), and recovering the same quantity of the cultured material as the supplied ground water; and (D) a process for analyzing the cultured material recovered in process (C), and determining whether the volatile organic chloride component is decomposed by the anaerobic microorganisms in the ground water and/or the soil or not. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for determining whether groundwater and / or soil contaminated with volatile organochlorine compounds can be purified by anaerobic microorganisms in the groundwater and / or soil.

The anaerobic microorganism purification method injects organic materials into the contaminated ground, creates an appropriate anaerobic environment in the ground, activates useful dechlorinated microorganisms, and replaces the chlorine in the VOC molecule with hydrogen. It is a purification technology (dechlorination). By artificially adjusting the redox potential in the ground, it is considered that VOC dechlorination proceeds as follows.
(i) Oxygen and nitrate ions are consumed by microorganisms by supplying organic substances, etc., and an anaerobic environment necessary for dechlorination is formed.
(ii) Microorganisms involved in dechlorination are activated by the formed anaerobic environment, and dechlorination of tetrachlorethylene (PCE) → trichloroethylene (TCE) → cis-1,2-dichloroethylene (cis-1,2-DCE) Progresses.
(iii) When the reducing environment is continued for a certain period of time, dechlorination of cis-1,2-DCE → vinyl chloride (VC) → ethylene and ethane proceeds by the dechlorinated microorganisms grown and activated in an anaerobic environment. To do.

  In general, the dechlorination of TCE to dichloroethylene (DCE) and cis-1,2-DCE to VC, ethylene, and ethane requires a reduction environment having the redox potential shown in FIG. It has been reported that complete dechlorination of VOC does not proceed unless useful dechlorinated microorganisms are present in the ground (Non-patent Document 1). Therefore, in order to apply the anaerobic microorganism purification method to a contaminated site, it is considered important to verify the purification suitability of the anaerobic microorganism purification method in advance.

  Since the involvement of bacteria belonging to the genus Dehalococcoides has been clarified in the dechlorination of cis-1,2-DCE and VC, these can be used as a method for judging the suitability for purification of anaerobic microorganism purification methods. It has been reported that it is effective to confirm useful bacteria and degradation genes by useful bacteria by DNA detection technology (Non-patent Documents 1 to 3, Patent Document 1). In addition, there are cases in which compatibility is confirmed by the combination of detection of a dehalococcides genus in groundwater by genetic analysis and a laboratory culture test (Non-Patent Documents 1, 4 and 5).

  On the other hand, there is also a method for confirming the presence of useful bacteria by conducting a culture test using soil and groundwater that are actually contaminated and confirming that VOC is dechlorinated (Patent Document 2). And 3).

  However, in the determination by the DNA detection of the decomposing microorganism and the gene involved in the decomposition, a technique for extracting the genomic DNA of the microorganism from a sample in the environment and detecting it by the PCR method is generally used in the DNA detection technique. On the other hand, environmental samples of contaminated ground may contain substances that inhibit genomic DNA acquisition and PCR in the ground environment, so if the number of bacteria belonging to the genus Dehalococcides is relatively small In some cases, accurate information cannot be obtained (Non-patent Document 6). In addition, the inhibitory effect of dechlorination by harmful substances has been reported (Non-patent Document 7), and it can be judged that dechlorination is possible only by the result of the presence of the genus Dehalococcides by genetic analysis. With the possibility of misdiagnosis of purification suitability.

A purification test to confirm that VOCs are dechlorinated by conducting a culture test using soil and groundwater obtained from actual contaminated sites is to confirm the presence of useful dechlorinated microorganisms. It is excellent in that it can be examined while confirming the conversion. However, the culture test performed by the purification suitability test method has the following problems.
(1) The culture operation is complicated and requires skill.
(2) If the artificial activation method in the contaminated ground cannot be reproduced faithfully to the indoor culture conditions, the intended result cannot be obtained. Or you may make a wrong diagnosis.
(3) Culture requires a long test period.

  To give a specific example, in the case of the culture methods that have been reported so far, the temperature setting is different from the actual ground environment, so the microflora that becomes the dominant species after cultivation is activated in the actual ground. (Patent Documents 2 and 3). In addition, the method of continuous culture using a reducing reagent such as sodium sulfide in groundwater to be dechlorinated (Patent Document 4) is different from the method of forming an anaerobic environment using organic materials, and is similar to the above. The problem remains.

  Therefore, although the culture apparatus and method require a culture method that can accurately reproduce the activation method in the actual ground as much as possible, sufficient studies have not been made. Moreover, as a tendency of dechlorination after introducing organic materials, it has been confirmed that there is a stagnation period of about 2 to 12 months before dechlorination after cis-1,2-DCE proceeds. . For this reason, it is necessary to periodically collect samples to evaluate the culture state even if the culture period is prolonged, but a culture method capable of coping with long-term culture has not been established.

  The batch culture method, which has been often used for the culture of anaerobic microorganisms, is a method of culturing groundwater and organic materials collected from the ground to be purified in sealed vials. Compatibility can be evaluated by measuring, and the advantages of the batch culture method are that it can be sealed with a butyl rubber stopper, making it easy to cultivate anaerobic microorganisms, culturing on a relatively small scale, and many conditions It is mentioned that the culture method is suitable for a comparative test of the above.

However, in the VOC dechlorination test, dechlorination after cis-1,2-DCE may stagnate, and in the test for confirming dechlorination after cis-1,2-DCE, the present inventor From these studies (described later), it has been found that the following problems may occur.
(4) A large number of cultures are necessary.
(5) Culture may take longer than planned and culture results may not be obtained.
(6) When a nutrient source is insufficient, a nutrient source cannot be supplied.
(7) The redox potential is not stable.

JP 2002-345473 A JP 2006-214782 A JP 2006-26553 A JP 2006-262842 A Hendrickson, E. R., et al. 2002. Appl. Environ. Microbiol. 68: 485-495 Miller, J. A., et al. 2004, Appl. Environ. Microbiol. 70: 4880-4888 Krajmlnik-Brown R. et al. 2004, Appl. Environ. Microbiol. 70: 6347-6351 Fennell D. E, et al., 2001, Environ. Sci. Technol. 1830-1839 F. E. Loffler, et al. 2000, Appl. Environ. Microbiol. 66: 1369-1374 Nakajima Makoto et al., 2005, Research Meeting on Groundwater Soil Contamination and Prevention Measures, 242-247 Naoshi Shinjo et al., 2006, Research Meeting on Groundwater Soil Contamination and Prevention, 163-166

  In order to address the problems (1) to (7) above, the object of the present invention is to use the culture method different from the conventional ones for groundwater and / or soil contaminated with volatile organochlorine compounds. An object of the present invention is to provide a method for determining whether purification by anaerobic microorganisms in groundwater and / or soil is possible.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have conducted a semi-continuous culture method for anaerobic microorganisms contained in soil and groundwater under conditions close to the site environment of soil and groundwater contaminated with VOC. By culturing in (the following steps (B) to (C) or steps (F) to (H)), the above problems (1) to (7) can be avoided, and the actual ground environment can be avoided. The inventors have found that a more appropriate determination can be made, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for determining whether groundwater and / or soil contaminated with a volatile organochlorine compound can be purified by anaerobic microorganisms in the groundwater and / or soil, comprising the following steps ( A determination method including A) to (D).
(A) collecting groundwater contaminated with volatile organochlorine compounds, or groundwater and soil,
(B) culturing the collected groundwater under anaerobic conditions or culturing the groundwater to which the collected soil is added under anaerobic conditions;
(C) Supplying the same groundwater as the groundwater used in step (B) to the culture in step (B), recovering the same amount of culture as the supplied groundwater, and (D) recovering in step (C) Analyzing the cultured product to determine whether anaerobic microorganisms in groundwater and / or soil are decomposing volatile organochlorine compounds.

[2] The determination method according to [1], further including repeating steps (B) to (D).
[3] The determination method according to [1] or [2], further comprising culturing the groundwater in the step (B) under a temperature condition of ± 5 ° C. of the ground temperature of the collected groundwater.
[4] In any one of [1] to [3], in the step (B), the method further comprises culturing the groundwater by adding at least one of a volatile organic chlorine compound, an organic material and a nutrient salt to the groundwater. The determination method of crab.

[5] The determination method according to any one of [1] to [4], wherein the anaerobic microorganism is a bacterium belonging to the genus Dehalococcides.
[6] A method for determining whether groundwater and / or soil contaminated with a volatile organochlorine compound can be purified by anaerobic microorganisms in the groundwater and / or soil, comprising the following steps ( A determination method including E) to (I).
(E) collecting groundwater and soil contaminated with volatile organochlorine compounds,
(F) a step of culturing the groundwater to which the collected soil is added under anaerobic conditions;
(G) Separately from the step (F), a step of culturing the collected groundwater under anaerobic conditions,
(H) supplying the culture of step (F) to the culture of step (G) and recovering the same amount of culture of step (G) as the supplied culture; and (I) step (H The step of analyzing the culture collected in (1) to determine whether anaerobic microorganisms in groundwater and / or soil are decomposing volatile organochlorine compounds.

[7] The determination method according to [6], further including repeating steps (F) to (I).
[8] The determination method according to [6] or [7], further comprising culturing the groundwater in the steps (F) and (G) under a temperature condition of ± 5 ° C. of the ground temperature of the collected groundwater. .
[9] In steps (F) and (G), the method further comprises culturing the groundwater by adding at least one of a volatile organic chlorine compound, an organic material, and a nutrient salt to the groundwater. [8] The determination method according to any of [8].
[10] The determination method according to any one of [6] to [9], wherein the anaerobic microorganism is a genus Dehalococcides.

  According to the present invention, groundwater contaminated with volatile organochlorine compounds and / or groundwater contaminated with volatile organochlorine compounds can be obtained by culturing anaerobic microorganisms under conditions close to the field environment of soil and / or soil. Alternatively, it is possible to appropriately determine whether or not the soil can be purified by the groundwater and / or anaerobic microorganisms in the soil. In addition, it is possible to determine not only the dehalococcide genus bacteria known to dechlorinate volatile organochlorine compounds at present, but also purification by unknown microorganisms.

The present invention is a method for determining whether groundwater and / or soil contaminated with volatile organochlorine compounds (VOC) can be purified by anaerobic microorganisms in the groundwater and / or soil, The following steps (A) to (D):
(A) A step of collecting groundwater contaminated with VOC, or groundwater and soil,
(B) culturing the collected groundwater under anaerobic conditions or culturing the groundwater to which the collected soil is added under anaerobic conditions;
(C) Supplying the same groundwater as the groundwater used in step (B) to the culture in step (B), recovering the same amount of culture as the supplied groundwater, and (D) recovering in step (C) Analyzing the cultured culture to determine whether anaerobic microorganisms in groundwater and / or soil are degrading VOCs.

  VOC is, for example, tetrachloroethylene (PCE) with chlorine, trichlorethylene (TCE), or cis-1,2-dichloroethylene (cis-1,2-DCE) which is a decomposition product thereof.

  An anaerobic microorganism that exists in groundwater and / or soil and decomposes VOC includes, but is not limited to, a bacterium belonging to the genus Dehalococcides, and may be an unknown microorganism that has not been identified.

  In the step (A), groundwater contaminated with VOC, or groundwater and soil are collected. At this time, it is preferable to measure the field temperature of the collected groundwater. The collected groundwater is used for culture as a medium in the step (B). When groundwater and soil are collected, the soil is added to the groundwater and used for culture.

  Hereinafter, steps (B) and (C) will be described with reference to culture device 1 (FIG. 2) preferably used in these steps, but the culture device is not limited to this.

In FIG. 2, the groundwater collected in the step (A) is put into a sterilized groundwater culture bottle 1 (capacity is preferably 0.5 to 10 L, more preferably 2 L or more, such as a screw mouth glass bottle). In order to culture under anaerobic conditions, it is preferable to put the groundwater so that the groundwater culture bottle 1 is full. The rotor 10 is inserted when stirring culture. In addition, organic materials (for example, waste bean sugar, polylactic acid, etc., the concentration is preferably 0.02 wt% to 0.1 wt%), nutrient salts (for example, KH ₂ PO ₄ , K ₂ HPO ₄ , nitrate, ammonium salt, The concentration of P, such as yeast extract, is preferably 5% or less with respect to the carbon content (C) of organic matter, VOC, etc. may be added. When soil is also collected in the step (A), the soil is further put into the groundwater culture bottle 1. The amount of soil is preferably 5 to 20% by weight of groundwater. Moreover, you may put a support | carrier in the groundwater culture bottle 1, and a silica sand, a glass bead, a polymeric material etc. are mentioned as a support | carrier, for example, It does not specifically limit. The presence of soil and carrier is considered to contribute to the formation of a micro-reducing environment and to contribute to shortening the culture period.

  The groundwater culture bottle 1 is closed with a cap capable of being sealed with the stainless tubes 2 and 3 inserted therein. In this lid, stainless tubes 2 and 3 are inserted into a butyl rubber stopper 4 and fixed with a plastic cap 5. The lid may have a structure in which a stainless tube is fixed to a plastic cap having a Teflon coating on the inside. The tip of the stainless tube 2 is installed below the center of the groundwater culture bottle 1 within a range not affected by the rotor (a position 3 cm from the bottom of the groundwater culture bottle 1 is preferable). The tip of the stainless steel tube 3 is installed at a position in contact with the upper surface of the groundwater (preferably inserted so as to protrude about 1 cm from the lower part of the lid). A stainless steel valve 6 is attached to the stainless steel tubes 2 and 3, and both valves are closed during culture.

  Filter ground water (same ground water in ground water culture bottle 1 or ground water) into sterile exchange ground water storage bottle 8 (screw cap glass bottle, etc., preferably 0.5-10L, more preferably 2L or more) Add sterilized material. This groundwater becomes the groundwater supplied in the step (C). You may further add the said organic material, nutrient salt, VOC, etc. to groundwater.

  The replacement groundwater storage bottle 8 is sealed with a butyl rubber stopper and a plastic cap in the same manner as the groundwater culture bottle 1. The stainless tube 2 and the stainless tube 13 are inserted in the butyl rubber stopper. The stainless steel tube 13 connects the replacement groundwater storage bottle 8 and the Teflon film pack 7 filled with nitrogen gas, and the valve 6 is attached to the stainless steel tube 13. The gas phase portion of the exchanged groundwater storage bottle 8 is replaced with nitrogen gas sent from the Teflon film pack 7 through the stainless steel tube 13 (nitrogen gas line). The stainless steel tube 2 is provided with a liquid feed pump 11.

  The culture in the step (B) is preferably performed at a temperature in the range of ± 5 ° C., more preferably ± 3 ° C., and even more preferably ± 1 ° C. of the ground temperature of the collected groundwater. What is necessary is just to stir at 80-100 rpm, for example using the stirrer 9 and the rotor 10, when stirring culture.

  In the step (C), a part of the groundwater cultured in the groundwater culture bottle 1 is collected. The recovered groundwater can be used as a sample for analysis. To collect, the valves 6 of the stainless tubes 2 and 3 are opened, and the groundwater in the replacement groundwater storage bottle 8 is caused to flow in the direction of the arrow of the stainless tube 2 by the liquid feed pump 11 (exchanged groundwater inflow line). Inflow. The groundwater cultured in the direction of the arrow of the stainless tube 3 flows (culture medium collection line) and can be collected (sampling) by the amount that flows. When collecting, the groundwater during cultivation can be collected without touching the air, so the anaerobic environment is not destroyed. Moreover, when a nutrient source is insufficient, an organic matter etc. can be resupplied by supplying new groundwater (medium). Further, by supplying the same amount of groundwater as the amount collected, the amount of air evacuation and pressure in the groundwater culture bottle 1 can be kept constant. Therefore, the volatilization amount of the VOC to the air can be made constant. In fact, a blank test was also conducted, and it was confirmed that there was almost no loss due to volatilization (data not shown).

  The interval and amount of collection are not particularly limited, and can be collected frequently without being limited as in a batch test. For example, measurement is performed every few days immediately after the start of the test, and in a stable state (such as the cis 1,2-DCE stagnation period), sampling may be performed about once every two weeks. If there are many analysis items to be described later, the amount of collection may be increased accordingly. When only VOC measurement is performed, for example, 1 to 5 ml may be collected, and when many TOC, ion composition, ORP, ATP, etc. are measured, for example, 10 to 25 ml (about 1% for 2L culture) is collected. Good. When the groundwater flow rate at the contaminated site is fast, the amount of groundwater collected and the number of times may be increased.

  In step (D), the groundwater collected in step (C) is analyzed to determine whether anaerobic microorganisms in the groundwater and / or soil are decomposing VOCs.

  The analysis method is not particularly limited, but a known method and a measuring device can be used. For example, measurement of VOC concentration (headspace GC-MS method, etc.), total organic carbon (TOC) measurement, redox potential (ORP) measurement, pH measurement, ion composition analysis, ATP measurement, anaerobic Design probes based on the measurement of the amount of microorganisms (MPN method, etc.), genetic analysis of anaerobic microorganisms (for example, the base sequence of bacteria belonging to the genus Dehalococcides (genbank, etc. can be searched), and probes labeled with fluorescent substances The bacteria in the analysis sample may be detected by hybridization, or a primer may be used to amplify by PCR using a nucleic acid prepared from the analysis sample as a template to detect and quantify the bacteria). A plurality of analysis methods may be combined.

The above steps (B) to (D) are repeated to obtain analysis results over time.
If VOC is decomposed over time or anaerobic microorganisms that decompose VOCs are growing, it is determined that anaerobic microorganisms in groundwater and / or soil are decomposing VOCs, and the groundwater and / or soil It is determined that purification by anaerobic microorganisms is possible.

  If it is determined that VOC is not decomposed, or anaerobic microorganisms that decompose VOC do not exist or grow, transplant anaerobic microorganisms that decompose VOC into groundwater and / or soil, or anaerobic microorganisms It is preferable to examine the development of an environment in which the bacteria grow.

Advantages of the culture apparatus 1 include the following.
(I) The structure of the culture apparatus is simple.
(II) It is possible to collect cultured groundwater in an amount necessary for water quality analysis and microbial analysis, and to collect groundwater during culture without touching the air (does not destroy the anaerobic environment).
(III) There is almost no increase in ORP even if a necessary amount of medium is collected for analysis. For example, when 20 ml is collected from a 2 L culture vessel for water quality analysis, about 1% of groundwater is exchanged and collected in the culture vessel, but the fluctuation of the ORP is slight and it is confirmed that it immediately returns to the equivalent value. is doing.
(IV) A long-term culture period from ethane to ethylene can be cultured under optimum conditions regardless of the period.
(V) Since it is easy to exchange groundwater and supply nutrients, it is also suitable for enrichment culture.
(VI) It is a culture device that can easily cope with tests that contain soil from contaminated ground.
(VII) It is a culture device that can easily handle culture with a carrier.
(VIII) It can also be used as a test method to confirm the long-term dechlorination effect by re-adding VOC as a contamination source.

The merit (VI) of the culture device 1 is that the culture method to put the soil is faithful to the phenomena that can actually occur in the ground environment by culturing under conditions closer to the ground environment compared to using only groundwater. There is a feature that can be reproduced. However, when soil is added directly to the groundwater culture bottle, the following problems may occur.
-Sorption of VOCs in cultured groundwater to soil particles-Variation in VOC measurement results due to non-uniform mixing of VOC sorptive soil particles-Other obstacles in analysis operations (such as microscopic observation)
Therefore, in the present invention, the following steps (E) to (I):
(E) Collecting groundwater and soil contaminated with VOCs,
(F) a step of culturing the groundwater to which the collected soil is added under anaerobic conditions;
(G) Separately from the step (F), a step of culturing the collected groundwater under anaerobic conditions,
(H) supplying the culture of step (F) to the culture of step (G) and recovering the same amount of culture of step (G) as the supplied culture; and (I) step (H ) And analyzing the culture collected in step) to determine whether anaerobic microorganisms in the groundwater and / or soil are degrading VOCs and / or groundwater contaminated with volatile organochlorine compounds and / or A method has been developed for determining whether soil can be purified by the groundwater and / or anaerobic microorganisms in the soil.

  In step (E), groundwater and soil contaminated with VOCs are collected. At this time, it is preferable to measure the field temperature of the collected groundwater. The collected groundwater is used for culture as a medium in the step (G). In addition, the collected soil is added to groundwater and used for culturing in step (F).

  Hereinafter, the steps (F) to (H) will be described with reference to the culture device 2 (FIG. 3) preferably used in these steps, but the culture device is not limited thereto.

  The culture apparatus 2 is an apparatus in which a soil culture bottle 12 is installed between the groundwater culture bottle 1 and the exchanged groundwater storage bottle 8. In the soil culture bottle 12, the soil collected in the step (E) is added to the groundwater and cultured (step (F)). Part of the groundwater cultured in the step (G) is collected, and the groundwater (soil supernatant) cultured in the step (F) is supplied (step (H)). By doing in this way, since the dechlorination effect of VOC by the microorganisms adhering to soil as well as the ground water existing in the contaminated ground can be confirmed, a culture test result under conditions closer to the ground environment can be obtained.

  In FIG. 3, the groundwater collected in the step (E) is put into a sterilized groundwater culture bottle 1. In order to culture under anaerobic conditions, it is preferable to put the groundwater so that the groundwater culture bottle 1 is full. When stirring culture, the rotor 10 is inserted. Moreover, you may add the organic material, nutrient salt, VOC, etc. of the said density | concentration. Furthermore, you may put a support | carrier in the groundwater culture bottle 1, As a support | carrier, silica sand, a glass bead, a polymeric material etc. are mentioned, for example, It does not specifically limit.

  The groundwater culture bottle 1 is closed with a cap capable of being sealed with the stainless tubes 2 and 3 inserted therein. The tip of the stainless tube 2 is installed below the center of the groundwater culture bottle 1 within a range not affected by the rotor (a position 3 cm from the bottom of the groundwater culture bottle 1 is preferable). The tip of the stainless steel tube 3 is installed at a position in contact with the upper surface of the groundwater (preferably inserted so as to protrude about 1 cm from the lower part of the lid). A stainless steel valve 6 is attached to the stainless steel tubes 2 and 3, and both valves are closed during culture.

  The soil in the soil culture bottle 12 sterilized is filled with ground soil contaminated with VOC (preferably soil collected from the same contaminated site as the groundwater culture bottle 1) and groundwater. The amount of soil is preferably 5 to 20% by weight of groundwater. The rotor 10 is inserted when stirring culture. Moreover, you may add the organic material of the said density | concentration (The same thing as the groundwater culture bottle 1 is preferable), nutrient salt, VOC, etc. In order to culture under anaerobic conditions, it is preferable to put the groundwater so that the soil culture bottle 12 is full. The soil culture bottle 12 is sealed with a butyl rubber stopper and a plastic cap, and stainless tubes 2 and 14 are inserted into the butyl rubber stopper.

  Ground water (same ground water as in the ground water culture bottle 1 or ground water filtered and sterilized is preferable, and organic materials, nutrient salts, VOC, etc. may be added) to the sterilized replacement ground water storage bottle 8 Insert. The replacement groundwater storage bottle 8 is sealed with a butyl rubber stopper and a plastic cap, and a stainless tube 14 with a valve 6 and a stainless tube 13 are inserted into the butyl rubber stopper. The stainless steel tube 13 connects the Teflon film pack 7 filled with nitrogen gas and the replacement groundwater storage bottle 8, and the valve 6 is attached to the stainless steel tube 13. The gas phase portion of the exchanged groundwater storage bottle 8 is replaced with nitrogen gas sent from the Teflon film pack 7 through the stainless steel tube 13 (nitrogen gas line). Further, the stainless steel tube 14 is provided with the liquid feed pump 11. During cultivation of the soil culture bottle 12, the valves 6 of the stainless tubes 2 and 14 are closed.

  The cultures in the steps (F) and (G) are preferably performed at a temperature in the range of ± 5 ° C., more preferably ± 3 ° C., and even more preferably ± 1 ° C. of the ground temperature of the collected groundwater. What is necessary is just to stir at 80-100 rpm, for example using the stirrer 9 and the rotor 10, when stirring culture.

  In the step (H), a part of groundwater cultured in the groundwater culture bottle 1 is collected. The collected groundwater can be used as a sample for analysis. To collect, the valve 6 of the stainless tubes 2, 3 and 14 is opened, and the groundwater in the replacement groundwater storage bottle 8 is caused to flow in the direction of the arrow of the stainless tube 14 by the liquid feed pump 11 (exchanged groundwater inflow line). 12, the soil supernatant in the soil culture bottle 12 flows in the direction of the arrow of the stainless tube 2 (soil liquid inflow line) and flows into the groundwater culture bottle 1. The groundwater cultured in the direction of the arrow of the stainless tube 3 flows (culture medium collection line) by the amount that flows in, and can be collected (sampling). In addition, when the soil supernatant of the soil culture bottle 12 flows into the groundwater culture bottle 1, the agitation is stopped to allow the supernatant liquid to flow after the sedimentary soil particles have settled. Min). In addition, the culture of the groundwater culture bottle 1 and the culture of the soil culture bottle 12 may be started at the same time, but the soil culture bottle 12 is conditioned in advance and the soil supernatant is used to flow into the groundwater culture bottle 1. Also good. The interval and amount of collection may be the same as in step (C).

  In step (I), the groundwater collected in step (H) is analyzed to determine whether the contaminated groundwater and / or anaerobic microorganisms in the soil are decomposing VOCs. The analysis method may be the same as in step (D).

The above steps (F) to (I) are repeated to obtain analysis results over time.
The determination as to whether purification by anaerobic microorganisms in groundwater and / or soil is possible is as described above.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In the following examples, the repetition of steps (B) to (C) or steps (F) to (H) may be referred to as a semi-continuous culture method.

[Comparative Example 1]
Evaluation of batch culture method (conventional culture method) Appropriate amounts of groundwater and organic material A (waste bean sugar) at VOC contaminated sites in 100 ml vial bottles (15 bottles) (concentration: 0.02% by weight, reference: JP 2006) -320848), sealed with a Teflon-coated butyl rubber stopper, and cultured at 20 ° C. 20 ° C is the temperature of the groundwater at the contaminated site. Vials were collected every time the culture progressed and used as analysis samples, and VOC, TOC, and ORP were measured. The VOC measurement was performed with a headspace GC-MS method (Agilent Technology), the TOC measurement with a total organic carbon meter (Shimadzu Corporation), and the ORP measurement with an oxidation-reduction potentiometer (Horiba Seisakusho).

The results are shown in FIGS.
In the batch test, 1 to 3 vials were collected for one sampling per condition and used as analysis samples. Regarding the change in VOC over time (FIG. 4), dechlorination of cis-1,2-DCE could not be confirmed even after 3 months. In addition, regarding the change with time of TOC (FIG. 5), the amount of organic matter consumed in the initial stage of cultivation was large, and 85% or more of the added amount was consumed in one month of cultivation, and dechlorination did not proceed. Regarding the time-dependent change of ORP (FIG. 6), the numerical value is not stable, and it is considered that air is mixed by long-term culture even if a teflon-processed butyl rubber stopper is used. In batch tests, there are cases where the butyl rubber stopper is inserted with a needle, or a source of nutrients is injected. In this case, the butyl rubber stopper has a hole, so that the VOC component is volatilized or mixed with air. It is considered that the correct test result cannot be obtained.

  From the above results, the batch culture method requires a large number of cultures, the culture is longer than planned, and the culture result may not be obtained, the nutrient source cannot be supplied when the nutrient source is insufficient, the oxidation It can be seen that there is a problem that the reduction potential is not stable.

[Example 1]
Evaluation of purification suitability by semi-continuous culture method Using the culture apparatus 1, put the contaminated site groundwater and the organic material A into the groundwater culture bottle 1, put the contaminated site groundwater into the replacement groundwater storage bottle 8, and replace the groundwater storage bottle 8 The air was replaced with nitrogen gas. The culture was performed at 20 ° C. while stirring with a rotor and a stirrer.

  Sampling was performed after 6 days of culture, and thereafter at a frequency of about once every two weeks. About 10 to 25 ml of groundwater in the exchanged groundwater storage bottle 8 was collected into the groundwater culture bottle 1 by pumping, and the cultured groundwater in the groundwater culture bottle 1 was extruded, and used as an analysis sample. VOC and ORP were measured in the same manner as in Comparative Example 1.

  The results are shown in FIGS. From about 100 days after the VOC culture, dechlorination of cis-1,2-DCE and increase in VC were confirmed (FIG. 7). Although the dechlorination of cis-1,2-DCE stagnated, the culture was prolonged, but a stable reduced state was maintained (FIG. 8).

[Example 2]
Comparison of culture devices 1 and 2 [Culture with culture device 1]
2 except that an appropriate amount of organic material B (polylactic acid (HRC)) was added in place of organic material A (0.05% by weight as sodium lactate, reference document: 2006-320848) in the groundwater culture bottle 1 of FIG. In the same manner as in Example 1, the cells were cultured, sampled, and VOC was measured. On the 300th day of the culture, the quantification of the genus Dehalococcides was carried out by the MPN method.

[Culture by the culture apparatus 2]
In the groundwater culture bottle 1 of FIG. 3, the same contaminated site groundwater and organic material B as the culture apparatus 1 were put. Similarly, 10 wt% of the contaminated site groundwater, the organic material B, and the contaminated site soil were added to the soil culture bottle 12 and mixed. The culture conditions were the same as in the culture apparatus 1, and both the groundwater culture bottle 1 and the soil culture bottle 12 were cultured at 20 ° C. with stirring. Sampling is performed by pumping into the groundwater culture bottle 1 using the soil supernatant of the soil culture bottle 12 (ground water cultured after 10 minutes of stirring) and extruding the cultured groundwater in the groundwater culture bottle 1. About 10 to 25 ml was taken and VOC was measured. On the 300th day of culturing, the quantification of Dehalococcides bacteria was performed by the MPN method.

  FIG. 9 shows the VOC measurement result of the culture apparatus 1, and FIG. 10 shows the VOC measurement result of the culture apparatus 2. In the case where the organic material B was used, in the culture of the culture apparatus 1, dechlorination of cis-1,2-DCE could not be confirmed even after 300 days of culture (FIG. 9). In the culture, dechlorination of cis-1,2-DCE was observed after the 100th day of culture, and VC dechlorination was confirmed (FIG. 10).

FIG. 11 shows the bacteria quantification results of the culture apparatuses 1 and 2. Proliferation of dehalococcide genus bacteria involved in dechlorination was significantly observed in the culture apparatus 2.
From the above, it can be seen that the culture apparatus 2 is particularly useful in the determination method of the present invention.

[Example 3]
Long-term dechlorination ability by supply of VOC Periodic supply of VOC as a source of contamination was performed using the culture apparatus 1 to determine whether the dechlorination ability of VOC is possible in the long term. The following groundwaters (a) to (c) were periodically supplied from the exchanged groundwater storage bottle 8 to the groundwater culture bottle 1, cultured at 20 ° C. with stirring, and VOC was measured.
(A) TCE addition (b) Organic matter addition, TCE addition (c) Organic matter + nutrient salt addition, TCE addition (nutrient salt is organic matter (C): phosphate (P): nitrate (N) = 100: 5: Phosphorus was prepared by using a phosphate buffer solution with concentrations of KH ₂ PO ₄ 21.9 g / L and K ₂ HPO ₄ 28.1 g / L with respect to the carbon content (C) of the organic matter. The organic substance was sodium lactate, and the organic substance amount was 0.02% by weight (Reference: 2006-320848).

  In the results up to the initial 56th day, no significant difference was observed between (b) and (c) (see FIGS. 12 and 13). Moreover, the usefulness of the condition of (organic matter + nutrient) of (c) was confirmed as the number of times of addition of TCE increased (when the culture period was prolonged) (FIGS. 14 to 16).

  From the above, it was found that TCE and organic matter (+ nutrient) can be repeatedly added, and the usefulness of the culture conditions can be confirmed in the long term. That is, the culture method used in the present invention can also be used as an accumulation culture method for VOC dechlorinated microorganisms.

[Example 4]
Effect of addition of phosphorus Phosphorus was supplied periodically and the effect was tested using the culture apparatus 1. The following groundwaters (d) and (e) were periodically supplied from the exchanged groundwater storage bottle 8 to the groundwater culture bottle 1 and cultured at 20 ° C. with stirring to measure VOC.
(D) Sodium lactate added (e) Sodium lactate added, phosphorus-doped _{(phosphorus, KH 2 PO 4 21.9g / L} , K 2 HPO 4 28.1g / L phosphoric acid buffer solution was prepared at a concentration of organics The amount of P was 1% with respect to the amount of carbon (C), and the amount of sodium lactate added was 0.02% by weight (Reference: JP-A-2006-320848).

  The results are shown in FIGS. Even if the amount of phosphorus added is large, dechlorination is not inhibited, but it is desirable to add as low a concentration as possible for environmental conservation, and it is preferable to set the amount to be about 1 mg / L as an effect.

  The determination method of the present invention can be applied to groundwater and / or soil purification techniques contaminated with organic matter.

It shows a redox potential suitable for dechlorination. The outline of the culture apparatus 1 used for this invention is shown. The outline of the culture apparatus 2 used for this invention is shown. The time-dependent change of VOC by a batch culture method is shown. The time-dependent change of TOC by a batch culture method is shown. The time-dependent change of ORP by a batch culture method is shown. The time-dependent change of VOC by a semi-continuous culture method is shown. The time-dependent change of ORP by a semi-continuous culture method is shown. The time-dependent change of VOC by a semi-continuous culture method (culture apparatus 1) is shown. The time-dependent change of VOC by a semi-continuous culture method (culture apparatus 2) is shown. The quantitative result of a dehalococcide genus bacterium is shown. The change of VOC until the 56th day after water culture at the time of adding VOC and organic substance to the groundwater of an exchange groundwater storage bottle is shown. The change of VOC until the 56th day after culture | cultivation at the time of adding VOC, nutrient salt, and organic substance to the groundwater of a replacement | exchange groundwater storage bottle is shown. The time-dependent change of VOC at the time of adding VOC (no organic matter addition) to the groundwater of a replacement | exchange groundwater storage bottle is shown. The time-dependent change of VOC at the time of adding organic substance and VOC to the groundwater of an exchange groundwater storage bottle is shown. The time-dependent change of VOC at the time of adding organic substance, nutrient salt, and VOC to the groundwater of a replacement | exchange groundwater storage bottle is shown. The time-dependent change of VOC at the time of adding sodium lactate to the groundwater of an exchange groundwater storage bottle is shown. The time-dependent change of VOC at the time of adding sodium lactate and phosphorus to the groundwater of an exchange groundwater storage bottle is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Groundwater culture bottle 2 Stainless steel tube 3 Stainless steel tube 4 Butyl rubber stopper 5 Plastic cap 6 Valve 7 Teflon film pack 8 Replacement groundwater storage bottle 9 Stirrer 10 Rotor 11 Liquid feed pump 12 Soil culture bottle 13 Stainless steel tube 14 Stainless steel tube

Claims (10)

A method for determining whether or not a groundwater and / or soil contaminated with a volatile organochlorine compound can be purified by anaerobic microorganisms in the groundwater and / or soil, the following steps (A) to A determination method including (D).
(A) collecting groundwater contaminated with volatile organochlorine compounds, or groundwater and soil,
(B) culturing the collected groundwater under anaerobic conditions or culturing the groundwater to which the collected soil is added under anaerobic conditions;
(C) Supplying the same groundwater as the groundwater used in step (B) to the culture in step (B), recovering the same amount of culture as the supplied groundwater, and (D) recovering in step (C) Analyzing the cultured product to determine whether anaerobic microorganisms in groundwater and / or soil are decomposing volatile organochlorine compounds.   The determination method according to claim 1, further comprising repeating steps (B) to (D).   The determination method according to claim 1 or 2, further comprising culturing the groundwater in a step (B) under a temperature condition of ± 5 ° C of an on-site temperature of the collected groundwater.   The step (B) further includes culturing the groundwater by adding at least one of a volatile organic chlorine compound, an organic material, and a nutrient salt to the groundwater. Judgment method.   The determination method according to any one of claims 1 to 4, wherein the anaerobic microorganism is a bacterium belonging to the genus Dehalococcides. A method for determining whether or not a groundwater and / or soil contaminated with a volatile organochlorine compound can be purified by anaerobic microorganisms in the groundwater and / or soil, the following steps (E) to A determination method including (I).
(E) collecting groundwater and soil contaminated with volatile organochlorine compounds,
(F) a step of culturing the groundwater to which the collected soil is added under anaerobic conditions;
(G) Separately from the step (F), a step of culturing the collected groundwater under anaerobic conditions,
(H) supplying the culture of step (F) to the culture of step (G) and recovering the same amount of culture of step (G) as the supplied culture; and (I) step (H The step of analyzing the culture collected in (1) to determine whether anaerobic microorganisms in groundwater and / or soil are decomposing volatile organochlorine compounds.   The determination method according to claim 6, further comprising repeating steps (F) to (I).   The determination method according to claim 6 or 7, further comprising culturing the groundwater in the steps (F) and (G) under a temperature condition of ± 5 ° C of an on-site temperature of the collected groundwater.   In the steps (F) and (G), the method further comprises culturing the groundwater by adding at least one of a volatile organochlorine compound, an organic material, and a nutrient salt to the groundwater. 2. The determination method according to item 1.   The determination method according to any one of claims 6 to 9, wherein the anaerobic microorganism is a bacterium belonging to the genus Dehalococcides.

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