JP3382393B2 - Biodegradation purification method of trichlorethylene - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biodegradation technique for trichlorethylene, and more particularly to a method for biodegrading and purifying trichlorethylene by decomposing and purifying the environment such as soil and groundwater contaminated thereby by microorganisms into a completely harmless substance.

[0002]

2. Description of the Related Art In recent years, environmental pollution due to aliphatic organochlorine compounds, which are harmful to living organisms and hardly decomposed, has become a serious problem. Especially in the soil of high-tech industries in Japan and overseas, tetrachlorethylene (PCE), trichlorethylene (TCE), dichloroethylene (DC
It is considered that the contamination by organochlorine compounds such as E) has spread to a considerable extent, and many cases actually detected by environmental surveys have been reported. It is said that these aliphatic organochlorine compounds, which remain in the soil, are dissolved in groundwater by rainwater etc. and spread throughout the surrounding area. Since such compounds are suspected to be carcinogenic and are extremely stable in the environment, pollution of groundwater used as a water source for drinking water is a major social problem. For such aliphatic organochlorine compounds, especially TCE,
Recently, aerobic degradation by microorganisms has been reported ( Metyloc containing methane monooxygenase).
ytis sp . strain M (Agric.B
iol. Chem. , 53, 2903 (1989), B
iosci. Biotech. Biochem. , 5
6,486 (1992), the same 56,736 (199)
2)), Methylosinus trichosp
orium OB3b (Am. Chem. Soc. Na
tl. Meet. Dev. Environ. Micro
biol. , 29, 365 (1989), Appl. E
nviron. Microbiol. , 55, 3155
(1989), Appl. Biochem. Biote
chnol. , 28,877 (1991)), Pseudomonas having a phenol hydroxylase.
putida BH (Sewer Association magazine, 24, 27 (1
987)), Ac having toluene monooxygenase
inetobacter sp. strain G4
(Appl. Environ. Microbiol.,
52,383 (1986) and 53,949 (198).
7), ibid. 54, 951 (1989), ibid. 56, 279.
(1990), ibid., 57, 193 (1991)), Pse.
udomonas mendocina KR-1 (B
io / Technol. , 7, 282 (1989)),
Pseudomon with toluene dioxygenase
as putida F1 (Appl. Enviro
n. Microbiol. , 54, 1703 (198
8), ibid. 54,2578 (1988)), Nitrosomonas having ammonia monooxygenase.
europaea (Appl. Environ. Mic
robiol. , 56, 1169 (1990))). All of these bacteria undergo T by an enzyme of the oxygenase system.
It is said that CE is epoxidized and then biodegraded or biodegraded. At this stage, T
Protonation of the CE epoxide results in dichloroacetic acid alongside glyoxylic acid. That is, TC by microorganisms having these oxygenase systems
In the E decomposition, dichloroacetic acid is inevitably produced at a certain ratio, but these are not decomposed by the enzyme possessed by the TCE-degrading bacterium and remain as they are.

That is, for example, when groundwater contaminated with TCE is treated in a bioreactor using these TCE-degrading bacteria, dichloroacetic acid is discharged as a result, and
Even if an attempt is made to decompose and purify TCE by these TCE-decomposing bacteria in soil contaminated with TCE, dichloroacetic acid remains as a decomposition by-product, which causes a problem of being easily dissolved in groundwater.

Dichloroacetic acid is one of the haloacetic acids, which is a disinfection by-product of tap water, along with trihalomethanes, haloacetonitriles, and haloketones, and its hepatotoxicity and mutagenicity cause a very serious problem. Therefore, it was taken up as an environmental monitoring item in 1993. These are the 23rd Japan Society for Water Environment Seminar / Analytical method accompanying revision of water quality environmental standards (Japan Society for Water Environment) Proceedings p5
5-p64 (November 1993).

As mentioned above, dichloroacetic acid is today used for TCE.
It remains as an aerobic biodegradation by-product of chlorophyll widely in the environment and is becoming a serious problem.

As a method for treating dichloroacetic acid, no practical treatment has been performed so far, and decomposition of dichloroacetic acid has only been studied at the laboratory level.

Decomposition of haloacetic acid such as dichloroacetic acid
The microorganisms thatTrichoderma,Acr
ostalagmus,Penicillium,Cl
onostacysMolds such asPseudom
onas,Arthrobacter,Rhizobi
um,Agrobacterium,Bacillu
s,Alcaligenes,Nocardia,Mi
crococcus,Achromobacter,M
oraxella(Above protein nucleic acid enzyme, 29, 1
01-110 (1984))
ing. In addition, Adachi, the unidentified fungus OS-2 strain
Decomposes acetic acid, bromoacetic acid, and iodoacetic acid to about the same degree
It has an enzyme and dichloroacetic acid is about half the activity,
Degraded (Osaka Prefectural Public Welfare Research Institute Public Health
Ed., No. 30, 89 (1992)). Also,Pseud
omonas  putida  NCIMB 12018
Extracted dehalogenase from carboxymethylcellulose
With 2 carbon atoms by immobilizing it on sucrose or thioglycolic acid
Studies have also been conducted on the decomposition of halo acid 6 (European special
Xu 179603). Other than that, halo acid dehalogenation
About the relationship between elements and genesPseudomonas  p
utida  AJ1 strain (J. Gen. Microbio
l. , 138, 675 (1992)),Pseudom
onas  cepacia  MBA4 strain (J. Bioc
hem. , 284, 87 (1992)),Pseudo
monas  sp． CBS3 strain (Biol. Chem.
Hoppe-Seyler. , 374, 489 (199
3)) is being studied.

However, all of these are evaluations of activity at the enzyme level, and the actual behavior of these microorganisms in the environment has not been studied at present.

Regarding the decomposition of the microorganism itself with dichloroacetic acid, Xanthobacter autotrophi
cus GJ10 strain (Appl. Biochem. Bi
otechnol. , 40, 158 (1993), 4
0,165 (1993)) has only been studied.

[0010]

DISCLOSURE OF THE INVENTION The problem to be solved by the present invention is such dichloroacetic acid which is a harmful substance in the microbial degradation treatment of TCE's oxygenase system, which has not been subjected to any treatment. Is generated universally, so the problem is that it must be rendered harmless.

[0011]

The above object can be achieved by the present invention described below.

Namely, the present inventors have microbial degradation process by oxygenase system of TCE, carbon dioxide by further dehalogenase activity dichloroacetic acid to result generated, water, and microorganisms decompose to the chloride ion, Toriwa
TCE that pollutes the environment such as soil and groundwater by decomposing it using microorganisms of the genus Renovobacter
I have found a way to purify the soybeans to a completely harmless state.

The method of the present invention will be described in more detail.
Microorganisms that aerobically convert TCE to epoxides of TCE, and dichloroacetic acid produced at a constant rate as a result of this reaction by dehalogenase activity into carbon dioxide,
Water and microorganisms that decompose to chlorine ions , especially
Nobakuta microorganisms of the genus at the same time or continuously, TCE
By contacting the groundwater and soil contaminated with
It decomposes and purifies TCE that contaminates groundwater and soil into completely harmless substances.

As the microorganisms used in the method of the present invention, any of the former microorganisms can be used without limitation as long as the decomposition of TCE is a decomposition route via the epoxide of TCE. All TCE-degrading bacteria can be used. Further, as such microorganisms, if they have the above-mentioned properties and abilities, naturally unidentified microorganisms, non-isolated microorganisms, symbiotic microbial groups, and newly isolated / identified microorganisms can also be used. it can. The microorganisms that have been identified and have the above-mentioned properties and capabilities include:
Bacteria belonging to the genus Pseudomonas, the genus Acinetobacter, the genus Xanthobacter, the genus Corynebacterium, etc. can be used, and particularly Pseudomonas cepacia and Pseudomonas.
Bacteria such as Putida, Pseudomonas fluorescens, Pseudomonas arginosa, etc. are effective, and for example, Pseudomonas cepacia strain KK01 (FERM BP-4235; hereinafter K) isolated from the intestine of Scorpion termites.
K01 strain) or Corynebacterium s
p. Examples include the J1 strain (FERM P-14332; hereinafter referred to as the J1 strain) and the like.

As the latter microorganism, any microorganisms capable of decomposing haloacetic acid, especially dichloroacetic acid into carbon dioxide, water, and chloride ions can be used without limitation, and the dichloroacetic acid decomposition described in the above "Prior Art" can be used. All fungi can be used. Further, as such a microorganism, similarly to the above, as long as it has the above-mentioned properties and abilities, an unidentified microorganism, an unisolated microorganism, a symbiotic microbial group,
Naturally, even newly isolated and identified microorganisms can be used.
Microorganisms that have been identified and have the above-mentioned properties and abilities include Pseudomonas and Xanthobacter .
For example, Pseudomonas Petida, Pseudomonas Deha
Roganans, Xanthobacter autotrophicus, etc.
Bacterium is effective, but in the present invention, it belongs to the genus Renovobacter.
Bacteria to be used. In particular, belongs to the genus Renovobacter
Reactor species AC strain (FE
RM P-14641; hereinafter referred to as AC strain)
It is newly isolated from the Toromu Formation and will be listed in the Examples below.
It has been found to have such excellent degrading properties.

In the present invention, the TCE decomposition processing is performed by TC
Any contaminant can be applied as long as it is a contaminant and can survive the microorganism. As can be easily understood from the examples described below, the purification method of the present invention utilizes microorganisms in an aqueous medium,
It can also be applied to various products which can be treated with an aqueous medium for a relatively short period of time, such as Seny products, paper products, leather products and the like. But here, TC in soil and groundwater, which have the highest applicability,
For the example of E, contact it with the TCE-degrading bacterium,
Further, it will be described below that the treatment can be carried out by bringing the dichloroacetic acid produced by the treatment into contact with the dichloroacetic acid decomposing bacterium. The contact between TCE-degrading bacteria and TCE can be carried out by a method such as culturing the microorganism in an aqueous medium containing TCE, or adding the aqueous medium and soil to the culture system of the microorganism. The contact between the fungus and dichloroacetic acid comprises culturing the microorganism in an aqueous medium treated with TCE by a TCE-degrading bacterium,
Alternatively, it can be carried out by a method such as adding the aqueous medium and soil to the culture system of the microorganism, and can be carried out using various methods such as a batch method, a semi-continuous method and a continuous method. The microorganism can be used in a semi-fixed state or immobilized on a suitable carrier.

The method of the present invention is applied to wastewater treatment, soil treatment, etc.
It can be applied to both closed and open systems. Further, the microorganisms may be immobilized on a carrier or the like, or various methods for promoting growth may be used in combination.

[0018]

The present invention will be described in more detail with reference to the following examples. The M9 medium used in each example has the following composition.

M9 medium composition (in 1 liter); Na ₂ HPO ₄ 6.2 g KH ₂ PO ₄ 3.0 g NaCl 0.5 g NH ₄ Cl 1.0 g Water balance (pH 7.0) TCE decomposition used in the examples Since the strains KK01 and J1 produce 2-hydroxymuconic semialdehyde having a maximum absorption at 375 nm as an intermediate product of decomposition of phenol, it is clear that they have an oxygenase, and this oxygenase decomposes TCE. Have been confirmed to be involved in.

Since the dichloroacetic acid-degrading bacteria AC strain used in the Examples decomposes dichloroacetic acid via glyoxylic acid, it is clear that the decomposition of dichloroacetic acid is the action of dehalogenase, which is a hydrolytic dehalogenase. It has become.

All TCE concentrations were measured by headspace-gas chromatography. That is, 20
Add 5 ml of M9 medium prepared to a predetermined TCE concentration to a ml vial, add 100 μl of bacterial solution, seal with a rubber stopper and an aluminum cap, and cultivate with shaking at 30 ° C. for a certain period of time, then vapor phase 0.1 ml was sampled and analyzed by gas chromatography. In the examples using soil, the TCE concentration was measured according to the method. In addition, the dichloroacetic acid concentration was measured with diluted sulfuric acid at pH 2
The resulting supernatant was extracted with diethyl ether and ethyl acetate, dried on a rotary evaporator and then redissolved in deionized water to obtain 95% 0.01N.
A high performance liquid chromatography (HPLC) method was carried out using a sulfuric acid aqueous solution / acetonitrile = 95/5 (v / v) as a solvent. It was confirmed by the GC / MS method that the peak eluting at the same retention time as that of the dichloroacetic acid sample was indeed dichloroacetic acid. Also in the examples using soil, the concentration of dichloroacetic acid was measured by directly extracting the soil with diethyl ether and ethyl acetate, and then performing the same method.

Example 1. Decomposition of TCE and dichloroacetic acid by a mixed culture system of KK01 strain and AC strain A colony of the AC strain on an agar medium is converted into a M9 medium 100 containing 0.1% yeast extract in a 200 ml Sakaguchi flask.
ml was inoculated and shake culture was carried out at 30 ° C. for 48 hours. Next, 200 ml of the KK01 colony on the agar medium
Inoculate 100 ml of M9 medium containing 0.1% of yeast extract and 200 ppm of phenol in a Sakaguchi flask.
Shaking culture was performed at 0 ° C. for 18 hours.

Next, 100 μl of the bacterial solution obtained by collecting the two strains was added to 5 ml of the same medium containing 10 ppm of TCE (concentration of each bacteria was 6 to 8 × 10 ⁸ cells / ml), and then sealed with a rubber stopper and an aluminum cap. Then, the cells were cultivated with shaking at 30 ° C., and the TCE concentration was measured every 6 hours by the method as described in the previous section of the examples. At the same time, TCE was added to 1 liter of M9 medium.
Is added to 10 ppm, and the bacteria concentration is 6-
Two strains were added so that the concentration was 8 × 10 ⁸ cells / ml, and the cells were similarly cultivated with shaking at 30 ° C., and the concentration of dichloroacetic acid was measured every 6 hours by the method shown in the preceding stage of this Example.

The results are shown in FIG. The value of the dichloroacetic acid concentration is shown as the concentration converted to the original culture solution.

It was shown that dichloroacetic acid was produced along with the decomposition of TCE (complete decomposition by 16 hours), but this was also completely decomposed by 36 hours.

Comparative Example 1. Degradation of TCE and production of dichloroacetic acid by the KK01 strain In the same manner as in Example 1, only the KK01 strain was inoculated, and the concentrations of TCE and dichloroacetic acid were measured.

The results are shown in FIG.

The decomposition rate of TCE was slightly higher than that of the mixed system with AC strain, but 700 ppb of dichloroacetic acid
It was shown to remain at the concentrations before and after.

Example 2. Degradation of TCE and dichloroacetic acid by mixed culture system of J1 strain and AC strain J1 strain and AC strain were inoculated in the same manner as in Example 1, and T
The concentrations of CE and dichloroacetic acid were measured.

The results are shown in FIG. 3 as in Example 1.

It was shown that dichloroacetic acid was produced along with the decomposition of TCE (complete decomposition by 16 hours), but this was also completely decomposed by 30 hours.

Comparative Example 2. Degradation of TCE and production of dichloroacetic acid by J1 strain In the same manner as in Example 1, but inoculating only J1 strain,
The concentrations of CE and dichloroacetic acid were measured.

The results are also shown in FIG.

The degradation rate of TCE was slightly higher than that of the mixed system with the AC strain, but dichloroacetic acid contained 350 ppb.
It was shown to remain at the concentrations before and after.

Example 3. For J1 strain and AC strain mixed culture system
Degradation of TCE and dichloroacetic acid in soil 2g of air-dried soil 5g of brown forest soil collected in Atsugi City, Kanagawa Prefecture
Put it in a 0 ml vial and the TCE concentration is 10 mg / g
  1 ml of TCE aqueous solution to become wet soil
In addition, the J1 strain and the AC strain prepared in Example 2 were used for each bacterial concentration.
6-8 x 10⁸cells / g wet soil
Inoculate, seal with an aluminum cap and let stand at 30 ° C.
After culturing, the T
The CE concentration was measured. At the same time, next, Atsugi City, Kanagawa Prefecture
Concentration of TCE of 1 in 50g of air-dried brown forest soil collected in
0 mg / g wet soil TCE water soluble
10 ml of the solution was added to the J1 strain and AC prepared in Example 2.
Each strain has a bacterial concentration of 6-8 x 10 ⁸ cells / g wet
Inoculate soybeans to give a soil of 100 ml Teflon
Static culture was carried out at 30 ° C in a bottle with an inner screw type cap.
After that, every 24 hours, 1 g of soil is collected and 5 ml of 0.0
1N sulfuric acid aqueous solution was added and stirred for 1 hour, followed by centrifugation and filtration.
After removing soil by filtration, adjust the pH to 2 or more with dilute sulfuric acid.
Introduced into the HPLC as below, dichloroacetic acid reduction daily
Measured small. The test solution is in a 100 ml Erlenmeyer flask.
Static culture was performed at 30 ° C. 1g of soil every 24 hours thereafter
Collect and add 5 ml of 0.01N sulfuric acid aqueous solution for 1 hour
After stirring and removing the soil by centrifugation and filtration,
The pH was adjusted to 2 or less with sulfuric acid and introduced into the HPLC.
The concentration of dichloroacetic acid was measured daily.

The results are shown in FIG.

The concentration of dichloroacetic acid temporarily increased as TCE was decomposed from the first day, but it was decomposed after the first day, and both TCE and dichloroacetic acid were completely decomposed on the fourth day.

Comparative Example 3. Degradation of TCE in soil and production of dichloroacetic acid by strain J1 Same method as in Example 3, but inoculating only strain J1
The concentrations of CE and dichloroacetic acid were measured.

The results are shown in FIG.

The decomposition rate of TCE is slightly higher than that of the mixed system with AC strain, but 230 μg / dichloroacetic acid
It was shown to remain at a concentration around kg wet soil.

[0041]

The biodegradation method of TCE brought about by the present invention, TCE, which has begun to be a problem at present.
Completely detoxifies the harmful substance dichloroacetic acid generated by aerobic microbial decomposition of TCE and remains in the environment
It is possible to completely decompose and purify

[Brief description of drawings]

FIG. 1 is a graph showing an implementation result of the present invention according to Example 1.

FIG. 2 is a graph showing an implementation result of Comparative Example 1 with respect to Example 1.

FIG. 3 is a graph showing an implementation result of the present invention according to Example 2.

FIG. 4 is a graph showing an implementation result of Comparative Example 2 with respect to Example 2.

FIG. 5 is a graph showing a result of carrying out the present invention according to a third embodiment.

FIG. 6 is a graph showing the execution results of Comparative Example 3 with respect to Example 3.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI // (C12N 1/20 C12R 1:38 C12R 1:38 1:01 1:01) C12R 1:15 (C12N 1/20 B09B 3/00 ZABE C12R 1:15 1:01) (56) Reference JP-A 64-34499 (JP, A) JP-A 60-214892 (JP, A) JP-A 6-296711 (JP, A) JP-A-3-292970 (JP, A) JP-A-5-23691 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C02F 3/34 A62D 3/00 B09C 1/10 WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A microorganism which introduces an oxygen atom into trichlorethylene to convert it into an epoxide, and a microorganism of the genus Renovobacter which decomposes dichloroacetic acid into chloride ion, carbon dioxide, and water by dehalogenase activity. Biodegradation and purification method for trichlorethylene. 2. The biodegradation and purification method according to claim 1, wherein the microorganism that introduces an oxygen atom into trichlorethylene and converts it into an epoxide is a microorganism that introduces an oxygen atom into trichlorethylene due to its oxygenase activity and converts into an epoxide. 3. The biodegradation and purification method according to claim 2, wherein the microorganism is a microorganism belonging to the genus Pseudomonas. 4. The method for biodegradation and purification according to claim 2, wherein the microorganism is a microorganism belonging to the genus Corynebacterium.