JP6955886B2 - RHA1 strain chlorine compound resolution expression method, equipment - Google Patents

Description

The present invention relates to a technique for decomposing volatile organic chlorine compounds using microorganisms. In particular, it relates to the decomposition technology focusing on the Rhodococcus jostii RHA1 strain.

Techniques for purifying chemical pollutants diffused into the environment using microorganisms are widely used in activated sludge methods, anaerobic treatment methods, etc. in wastewater treatment.
Further, in recent years, a technique (bioremediation) for purifying soil and groundwater contaminated with harmful chemical substances by microorganisms has been attracting attention as a method having a small environmental load and purification cost.
For example, (1) a technique using anaerobic dechlorinated bacteria is disclosed in Non-Patent Document 1 and the like. In order to purify groundwater contaminated with chlorinated ethylenes such as trichlorethylene and cis-1,2-dichloroethylene, organic substances that are nutrient sources for microorganisms are injected into the aquifer from purification wells, etc., and the groundwater is anaerobic. A technique for activating anaerobic dechlorinating bacteria to purify chlorinated ethylene into harmless ethylene has been widely put into practical use.
In this technique, dechlorinating bacteria replace chlorine in chlorinated ethylene one by one with hydrogen, so that vinyl chloride monomer is always produced as an intermediate product in the purification process (Fig. 6). Vinyl chloride monomer was added to the groundwater environmental standard item in 2009 by the Ministry of the Environment. The regulated concentration of this vinyl chloride monomer is set to be one order or more smaller than that of trichlorethylene and cis-1,2-dichloroethylene, and purification by this technology produces vinyl chloride monomer that exceeds the environmental standard value in groundwater depending on the progress. As a result, secondary groundwater pollution may occur.

(2) An example of using an aerobic bacterium is disclosed in Non-Patent Document 2 and the like.
Many aerobic bacteria capable of decomposing trichlorethylene, cis-1,2-dichloroethylene, etc. have been reported.
The aerobic dechlorination reaction is mainly carried out by toluene-utilizing bacteria, phenol-utilizing bacteria, methane-utilizing bacteria and the like that grow using toluene, phenol, methane or the like as a single carbon source. The oxidase produced by these bacteria when using toluene, phenol, methane, etc. as a carbon source under aerobic conditions has a wide substrate specificity. For example, in the case of methane oxygenase, when trichlorethylene is present together with methane, the co-metabolization (cooxidation) reaction of trichlorethylene proceeds at the same time in addition to the oxidation of methane, and the dechlorination reaction of trichlorethylene is promoted. It is known (Non-Patent Document 2).
In this technology, the carbon source (electron donor) required to promote these co-metabolism reactions is harmful substances such as toluene and phenol, so the harmful substances remaining undecomposed to the underground environment. Pollution was an issue.

(3) A method for decomposing a volatile organochlorine compound using the Rhodococcus jostii RHA1 strain is disclosed in Patent Document 1 (Patent No. 2990016) and Non-Patent Document 3.
The RHA1 strain can decompose volatile organochlorine compounds such as trichlorethylene and cis-1,2-dichloroethylene by the hydroxylation reaction of biphenyldioxygenase (BphA and EtbA) (Non-Patent Document 3). In order to express these enzyme genes, it is necessary to add harmful substances such as biphenyl and ethylbenzene for induction.
When introducing the RHA1 strain into the underground environment to purify contaminated soil, sending these harmful substances to the underground environment can result in contamination by secondary injected substances. You need to be careful.

Japanese Patent No. 2990016

Chemistry and Biology, Vol. 49, No. 4, pp. 256-260, 2011 Takeda, H., Yamada, A., Miyauchi, K., Masai, E., & Fukuda, M. (2004). characterization of transcriptional regulatory genes for biphenyl degradation in Rhodococcus sp. Strain RHA1. Journal of bacteriology, 186 ( 7), 2134-2146. Kanako Chino, Kenta Yonezuka, Naoto Araki, Daisuke Kasai, & Masao Fukuda. (2014). P19-3 Rhodococcus jostii RHA1 analysis of enzyme genes involved in the degradation of cis-1,2-dichloroethylene (poster presentation). Japanese microbial ecology Proceedings of the conference, 2014, 188.

In the present invention, when the RHA1 strain is sent to the underground environment to purify the volatile organochlorine compound, the environment of the underground environment due to substances such as biphenyl and ethylbenzene that need to be added in order to develop the ability to decompose the volatile organochlorine compound. The purpose is to develop a method that does not cause pollution.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to obtain an RHA1 strain exhibiting an ability to decompose a volatile organic chlorine compound mainly by the following constitution, and to purify the volatile organic chlorine compound.
1. 1. Rhodococcus jostii RHA1 strain A gas containing a compound that expresses the ability to decompose volatile organochlorine compounds to the RHA1 strain is sent into the culture medium to obtain the RHA1 strain having volatile organochlorine compound degradability. It ’s a method of culturing,
The compound that expresses the ability to decompose volatile organic chlorine compounds is ethylbenzene or biphenyl, and after stopping the insufflation of gas containing ethylbenzene or biphenyl, air is infused to leave ethylbenzene or biphenyl in the culture solution. A method of culturing an RHA1 strain having volatile organic chlorine compound degradability characterized by removing biphenyl .
2 . 1. 1. A method for culturing an RHA1 strain having a volatile organochlorine compound resolution, which comprises performing the method for culturing the RHA1 strain described in the above in a closed system and providing a means for removing ethylbenzene or biphenyl in the exhaust system.
3 . RHA1 which is a volatile organochlorine compound equipped with air supply means A, means B for gasifying ethylbenzene or biphenyl, means for culturing RHA1 strain, and means for removing ethylbenzene or biphenyl contained in exhaust E. An apparatus for culturing a strain, the RHA1 strain culturing apparatus having a piping system D in which the insufflation means A, the means B, the means C, and the means E are connected in this order by a pipe, and a volatile organic RHA1 strain cultivated. A soil purification device comprising an injection means F for supplying soil contaminated with a chlorine compound.

1. 1. It is possible to cultivate the RHA1 strain having the ability to decompose the volatile organic chlorine compound in a state where the inducer that expresses the ability to decompose the volatile organic chlorine compound does not remain in the RHA1 strain.
2. Since the RHA1 strain that has acquired the ability to decompose volatile organochlorine compounds can be sent to the underground environment to purify volatile organochlorine compounds, substances such as biphenyl and ethylbenzene added for expression need to be injected into the soil. There is no risk of these substances polluting the underground environment.
3. 3. By adding the expression-inducing substance in a gas state, the amount of addition can be adjusted to be small, and since excess gas is adsorbed and recovered, pollution in the air can be prevented. Even if it is released, it is possible to prevent serious pollution without penetrating into the soil even if it is in a very small amount.
4. High concentrations of ethylbenzene and the like are also harmful to the RHA1 strain and interfere with culturing, so it is necessary to control the addition concentration. When a high concentration of ethylbenzene or the like is added, the RHA1 strain that comes into contact with the strain until it is uniformly dispersed may be harmed, and the cell concentration may decrease. Since the air containing vaporized ethylbenzene etc. contains a small amount of ethylbenzene and does not increase the liquid content, it is necessary to add ethylbenzene to the extent that it is necessary for expression and does not have a negative effect on the culture of the RHA1 strain. can.
The amount of compounds such as ethylbenzene required for expression is also small, and by incorporating them into the exhaust gas, the amount of ethylbenzene and the like remaining in the RHA1 strain culture solution is reduced.

A model diagram showing the pollution status of soil and groundwater by ethylene chloride. The figure which shows the culture apparatus which expresses the enzyme gene which decomposes the chlorinated ethylenes of the RHA1 strain by this invention. The graph which shows the cis-1,2-dichloroethylene decomposition rate after 7 days of the test conditions 1 to 4. The graph which shows the decomposition rate of cis-1,2-dichloroethylene after 7 days of the test condition 5-9. The graph which shows the time-dependent change of the residual ethylbenzene concentration. The figure which shows the change of the vinyl chloride monomer produced as an intermediate in a purification process in which dechlorinating bacteria replace chlorine of chlorinated ethylene one by one with hydrogen.

In the present invention, a gas containing a compound that expresses the ability of the RHA1 strain to decompose volatile organochlorine compounds is sent into the Rhodococcus jostii RHA1 strain culture medium to improve the resolution of the volatile organochlorine compounds. It is a method of culturing the possessing RHA1 strain.
The Rhodococcus jostii RHA1 strain can be obtained from the National Institute of Technology and Evaluation (NBRC 108803).
Direct addition of ethylbenzene, biphenyl, etc., which are compounds that express the ability to decompose volatile organochlorine compounds to the RHA1 strain, to the culture solution leads to contamination of the underground environment by these added compounds. In the present invention, the enzyme gene of the RHA1 strain is expressed by volatilizing these compounds and sending the vaporized gas into the culture medium to aerate it. Further, by stopping the compound gas and sending only air before the end of the culture, the compound gas dissolved in the culture solution is volatilized again and removed from the culture solution. The compound gas component dissolved in the air is provided with an adsorption means in the exhaust system to be adsorbed so as not to dissipate harmful compound gas into the environment.
This is a soil purification method that does not cause secondary pollution by injecting the RHA1 strain culture solution, which has acquired the ability to decompose volatile organochlorine compounds, into the soil to be purified.

(Regarding the ability of RHA1 strain to decompose volatile organic chlorine compounds)
The RHA1 strain utilizes the ability to decompose volatile organochlorine compounds such as trichlorethylene and cis-1,2-dichloroethylene by the hydrogenation reaction of biphenyldioxygenase.
In order to express these enzyme genes, it is necessary to add harmful substances such as ethylbenzene and biphenyl for induction. Not all of these added inducers are consumed during the expression process, but remain in the RHA1 strain culture solution, and the RHA1 strain culture solution that has acquired the ability to decompose volatile organochlorine compounds is placed in the soil. Injecting may cause secondary contamination by the inducer and cannot be used as it is.
The present invention provides an RHA1 strain culture solution that has acquired the ability to decompose volatile organochlorine compounds that does not contain harmful substances such as ethylbenzene and biphenyl.

(Inducible substance)
The inducer is ethylbenzene, biphenyl or the like. In the present invention, a gas obtained by vaporizing these inducers is used. By vaporizing and gasifying, it can be added by mixing it with air to control the concentration, and by introducing a smaller amount into the culture solution of the RHA1 strain by aeration than adding it as a liquid, the RHA1 strain is volatilized. It is possible to express a sex-organochlorine compound-degrading gene. In particular, ethylbenzene is highly volatile and easy to use.
After aeration for a predetermined time, some ethylbenzene sent into the RHA1 strain culture solution remains without being consumed by the RHA1 strain, but it can be taken out into the exhaust by aeration of air and decomposes volatile organochlorine compounds. It can be removed from the RHA1 strain culture medium that has acquired the ability.
Further, ethylbenzene taken into the exhaust gas can be removed by using an adsorbent or the like, and the exhaust gas finally released into the atmosphere does not contain harmful ethylbenzene.

(Volatile organochlorine compound)
The volatile organic chlorine compounds to be decomposed are chlorine compounds such as cis-1,2-dichloroethylene, tetratrichloroethylene, trichlorethylene, trans-1,2-dichloroethylene and 1,1-dichloroethylene.
Chlorinated ethylenes such as cis-1,2-dichloroethylene (1,2-DCE), trichlorethylene (TCE), and tetrachlorethylene (PCE) are used as cleaning agents for oil in the metal industry and the high-tech industry of semiconductors. There is. It is also used in a wide range of industrial fields such as dry cleaners. These are poorly water-soluble liquids, and when they leak to the ground from factories or the like, they do not adsorb on the way and do not decompose, but remain on the poorly permeable layer of the silt layer or clay layer.
Although it is physically poorly water-soluble, some of it dissolves in groundwater and diffuses into the soil. Therefore, the object of purification is mainly the aquifer formed above the impermeable layer, which is considerably deep.

(Soil to be washed)
As described above, the soil is purified mainly in the aquifer where ethylene chloride is diffused. The soil model is shown in FIG.
In the present invention, an injection well is provided to inject the activated RHA1 strain culture solution into the target stratum. In large-scale target areas, a culture site will be set up at the site for injection. The RHA1 strain is aerobic, so if there is little air, send air into the soil.
In addition, a mobile application machine equipped with an injection pipe and a culture solution of RHA1 strain can be used to purify a small area of a ruins such as a cleaning shop. Further, a culture device can also be mounted to make a use machine equipped with a mobile culture device.

(Device configuration)
The present invention has realized the cultivation of the RHA1 strain by developing the following culture apparatus.
Incubate the RHA1 strain that expresses the decomposition ability in the volatile organic chlorine compound equipped with the air supply means A, the means B that gasifies the compound that expresses the ability to decompose the volatile organic chlorine compound, and the RHA1 strain culture means C. It is an RHA1 strain culture device including a piping system D in which air supply means A, means B, and means C are connected by pipes in this order.
The air supply means A is an air pump or the like.
Means B for gasifying a compound is a closed container such as a tank or a tank containing a compound liquid such as ethylbenzene. Air is introduced into this container by a pipe from an air pump, and the ethylbenzene vaporized in the air is aerated. It is taken up and sent to RHA1 strain culture means C connected by a pipe.
The RHA1 strain culture means C is a closed tank or tank containing the RHA1 strain and the culture solution, and aerates air mixed with ethylbenzene gas or the like into the culture solution to introduce it. By aeration, the amount of compound such as ethylbenzene added can be controlled in a very small amount, and it is easy to diffuse it throughout. The activated RHA1 strain that has developed the ability to decompose volatile organochlorine compounds is cultured while replenishing the mixed gas for an appropriate period of time.
As the culture solution, a base material for culturing Rhodococcus josti is usually used.
While injecting the mixed gas from the culture means C, exhaust is taken. Ethylbenzene and the like contained in the exhaust gas without being purified are also harmful substances, so they are released into the atmosphere via trap means.
Furthermore, unconsumed ethylbenzene and the like may remain in the RHA1 strain culture solution, and after stopping the injection of the mixed gas, only air is introduced into the culture means C to perform a cleaning treatment to take in the remaining ethylbenzene and the like. .. Exhaust is trapped as described above.
In this way, the RHA1 strain culture apparatus can be configured as a closed system so that the activated RHA1 strain culture solution and the exhaust gas do not contain harmful substances.

Since the device itself is composed of an air pump, tank, tank, and pipe, it can be made compact, a dedicated culture device can be mounted on a mobile vehicle, and it is dispersed on a small scale such as the site of a cleaning shop. It can be applied to the purification target area.
In places such as the site of a large factory, large equipment can be installed and piped to multiple points for continuous injection.

[Embodiment 1]
FIG. 2 shows a culture apparatus 11 that expresses an enzyme gene that decomposes chlorinated ethylenes of the RHA1 strain according to the present invention.
The incubator pumps 1 and 2, the ethylbenzene volatilization tank 3, the RHA1 strain culture tank 4, the ethylbenzene adsorption tank 5, and the exhaust device 7 are each connected by a piping system 6 to form the culture device 11. The RHA1 strain culture tank 4 is filled with the RHA1 strain culture solution 9, the ethylbenzene volatilization tank 3 is filled with the ethylbenzene solution 8, and the ethylbenzene adsorption tank 5 is filled with the adsorbent 10 such as activated carbon. ing.
In this culture apparatus 11, (1) air is sent to the ethylbenzene volatilization tank 3 by an air pump 1 such as an air pump, and ethylbenzene is sent to the ethylbenzene volatilization tank 3 by an air pump 2. In the ethylbenzene volatilization tank 3, the amount of air supplied can be adjusted by the flow rate adjusting valve. (2) In the ethylbenzene volatilization tank 3, highly volatile ethylbenzene is mixed with air and sent to the culture solution 9 of the RHA1 strain culture tank 4. (3) The RHA1 strain in the culture medium is induced by the delivered ethylbenzene to express the enzyme gene. (4) The air that has passed through the RHA1 strain culture solution 9 is finally sent to the ethylbenzene adsorption tank 5 containing the adsorbent 10 such as activated carbon, where ethylbenzene is adsorbed and released into the atmosphere through the exhaust device 7.

表2 試験条件

[Test Example 1] Ethylbenzene-induced cis-1,2-dichloroethylene resolution confirmation test of RHA1 strain In this test, W inorganic salt medium (Table) was used to confirm the cis-1,2-dichloroethylene resolution of RHA1 strain induced by ethylbenzene. A decomposition test of cis-1,2-dichloroethylene was performed using 1).
(1) Pre-culture of RHA1 strain ・ The RHA1 strain was inoculated into 100 ml of LB liquid medium (1 g / l Bacto tryptone, 0.5 g / l Yeast extract, 0.5 g / l NaCl) and cultured for 24 hours.
Prepare 200 ml of W inorganic salt medium supplemented with 20 mM succinic acid, 1 g / l yeast extract, and 5 mM ammonium sulfate, add the RHA1 strain cultured in LB liquid medium for 24 hours, and add 100 rpm and 30 ° C. Was cultured for 48 hours.
(2) Activated culture After that, the air discharged from the pump is sent to the incubator via a container containing ethylbenzene and aerated for about 6 hours. One hour before the end of aeration, remove the container containing ethylbenzene and aerate.
The cells after culturing were collected by centrifugation (5,000 rpm, 10 minutes, 25 ° C.), and the cells were washed with W inorganic salt medium and used for the decomposition test.
(3) Decomposition test of cis-1,2-dichloroethylene Table 2 shows the test conditions.
In conditions 2 to 4, RHA1 strain cell concentrations were added so as to be ^{1.0 × 10 7} , 1.0 × 10 ⁸ , and 1.0 × 10 ^{9 cells / mL.}
Condition 1 was a control group to which no RHA1 strain was added.
After adding W inorganic salt medium and bacterial solution to each vial to be used, the prepared cis-1,2-dichloroethylene concentrate was added to the vial. After sealing with a butyl rubber stopper and an aluminum seal, the mixture was manually stirred to homogenize and statically cultured in a constant temperature bath at 20 ° C.
After 7 days, the vial was opened and the supernatant was collected with a pipette for analysis.
(4) Test results Fig. 3 shows the decomposition rate of cis-1,2-dichloroethylene after 7 days under each condition. The decomposition rate was calculated based on the cis-1,2-dichloroethylene concentration of condition 1 which is the control group.
From this result, it was shown that the enzyme gene was expressed by the inducing action of ethylbenzene and the cis-1,2-dichloroethylene degradation by the RHA1 strain occurred.
In addition, the amount of cis-1,2-dichloroethylene decomposition tended to increase as the number of RHA1 strains increased.
Table 1 W Inorganic salt medium composition

Table 2 Test conditions

[Test Example 2] Test for decomposition of cis-1,2-dichloroethylene by RHA1 strain using actual contaminated groundwater In this test, the resolution of cis-1,2-dichloroethylene of RHA1 strain induced by ethylbenzene is affected even in actual contaminated groundwater. It was examined whether cis-1,2-dichloroethylene was decomposed without any problem.
(1) Pre-culture of RHA1 strain
The RHA1 strain was inoculated into 100 ml of LB liquid medium (1 g / l Bacto tryptone, 0.5 g / l Yeast extract, 0.5 g / l NaCl) and cultured for 24 hours.
Prepare 200 ml of W inorganic salt medium supplemented with 20 mM succinic acid, 1 g / l yeast extract, and 5 mM ammonium sulfate, add the RHA1 strain cultured in LB liquid medium for 24 hours, and add 100 rpm and 30 ° C. Was cultured for 48 hours.
(2) Activated culture After that, the air discharged from the pump is sent to the incubator via a container containing ethylbenzene and aerated for about 6 hours.
One hour before the end of aeration, remove the container containing ethylbenzene and aerate.
The cells after culturing were collected by centrifugation (5,000 rpm, 10 minutes, 25 ° C.), and the cells were washed with W inorganic salt medium and used for the decomposition test.
(3) Decomposition test of cis-1,2-dichloroethylene Table 3 shows the test conditions.
Conditions 6 to 9 were added so that the cell concentration of the RHA1 strain was 1.0 × 10 ⁷ , 1.0 × 10 ⁸ , 5.0 × 10 ⁸ , and 1.0 × 10 ⁹ cells / mL.
Condition 5 was a control group to which RHA1 strain was not added.
After putting the actual contaminated groundwater and the bacterial solution into the vials to be used, the prepared cis-1,2-dichloroethylene concentrate was added into the vials. After sealing with a butyl rubber stopper and an aluminum seal, the mixture was manually stirred to homogenize and statically cultured in a constant temperature bath at 20 ° C. After 7 days, the vial was opened and the supernatant was collected with a pipette for analysis.
(4) Test results Fig. 4 shows the decomposition rate of cis-1,2-dichloroethylene after 7 days under each condition.
The decomposition rate was calculated based on the cis-1,2-dichloroethylene concentration of condition 5 which is the control group.
From this result, it was shown that the RHA1 strain decomposes cis-1,2-dichloroethylene even in actual contaminated groundwater.
Table 3 Test conditions

[Test Example 3] Examination of confirmation of ethylbenzene removal effect in culture solution In this test, a test was conducted to confirm whether ethylbenzene remaining in the culture solution was actually removed by the activation treatment.
(1) Pre-culture of RHA1 strain
The RHA1 strain was inoculated into 100 ml of LB liquid medium (1 g / l Bacto tryptone, 0.5 g / l Yeast extract, 0.5 g / l NaCl) and cultured for 24 hours.
Prepare 500 ml of W inorganic salt medium supplemented with 20 mM succinic acid, 1 g / l yeast extract, and 5 mM ammonium sulfate, add the RHA1 strain cultured in LB liquid medium for 24 hours, and add 100 rpm and 30 ° C. Was cultured for 48 hours.
(2) Activated culture After that, the air discharged from the pump is sent to the incubator via a container containing ethylbenzene and aerated for about 24 hours.
After that, the container containing ethylbenzene is removed, and only air is aerated to remove ethylbenzene in the culture solution. In order to confirm whether ethylbenzene has been removed, the culture broth was collected 3, 24, 48, 96, and 168 hours after the activation treatment was started (after 24 hours, aeration with air only), and a gas chromatograph was used. The ethylbenzene concentration was measured.
(3) Test Results Fig. 6 shows the change over time in the ethylbenzene concentration over time. The concentration of dissolved ethylbenzene was almost constant during the activation treatment. It was also shown that by sending only air for one day, the concentration drops to near the detection limit. At this concentration, it is unlikely that it will lead to environmental pollution even if it is introduced into the soil. It was shown that the device created this time can carry out a series of steps such as culturing the RHA1 strain, activating it, and preparing a culture solution with a low degree of contamination.

1 ... Air supply pump 2 ... Air supply pump 3 ... Ethylbenzene volatilization tank 4 ... RHA1 strain culture tank 5 ... Ethylbenzene adsorption tank 6 ... Piping system 7 ... Exhaust device 8 ...・ ・ Ethylbenzene solution 9 ・ ・ ・ RHA1 strain culture solution 10 ・ ・ ・ Adsorbent 11 ・ ・ ・ Culture device

Claims (3)

Rhodococcus jostii RHA1 strain A gas containing a compound that expresses the ability to decompose volatile organochlorine compounds to the RHA1 strain is sent into the culture medium to obtain the RHA1 strain having volatile organochlorine compound degradability. It ’s a method of culturing,
Compounds that exhibit the ability to decompose volatile organochlorine compounds are ethylbenzene or biphenyl,
After stopping the insufflation of the gas containing ethylbenzene or biphenyl, the RHA1 strain having a volatile organochlorine compound resolution characterized by insufflating air to remove the ethylbenzene or biphenyl remaining in the culture solution is cultivated. How to do it . The method for culturing the RHA1 strain according to claim 1 is carried out in a closed system, and the RHA1 strain having a volatile organochlorine compound resolution is cultivated, which comprises providing a means for removing ethylbenzene or biphenyl in the exhaust system. Method. RHA1 which is a volatile organochlorine compound equipped with air supply means A, means B for gasifying ethylbenzene or biphenyl, means for culturing RHA1 strain, and means for removing ethylbenzene or biphenyl contained in exhaust E. An apparatus for culturing a strain, the RHA1 strain culturing apparatus having a piping system D in which the insufflation means A, the means B, the means C, and the means E are connected in this order by a pipe, and a volatile organic RHA1 strain cultivated. A soil purification device comprising an injection means F for supplying soil contaminated with a chlorine compound.

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