JPH10295366A - Novel hydrogen peroxide-resistant microorganism, method for obtaining the same, and method for repairing contaminated medium using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new microorganism JMP1000 strain useful for repair, etc., of the natural environment by allowing the microorganism to propagate in a prescribed proportion of hydrogen peroxide and further to have an ability for decomposing a chlorinated ethylene and a substituted benzene. SOLUTION: This microorganism JMP1000 strain propagates in a 1000 ppm hydrogen peroxide solution, has an ability for decomposing a chlorinated ethylene such as trichloroethylene and dichloroethylene, and a substituted benzene compound such as phenol, cresol and toluene, and is deposited as FERM P-16143. The JMP1000 strain is obtained by preparing a microorganism such as JM1 strain (FERM BP 5352) having the ability for degrading the chlorinated ethylene compound and the substituted benzene compound but having no hydrogen peroxide resistance, bringing the microorganism into contact with an inorganic salt medium containing no growth substrate of the microorganism but the hydrogen peroxide for 10-50 hr, further culturing the resultant microorganism in a medium containing both the growth substrate of the microorganism and the hydrogen peroxide, and recovering the propagated biomass.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism used for repairing a natural environment contaminated with an organic compound using a microorganism and a method for obtaining the microorganism.

[0002]

2. Description of the Related Art In recent years, environmental pollution by volatile organic chlorine compounds which are harmful to living organisms and are hardly decomposable has become a serious problem. In particular, contamination of volatile organic chlorine compounds such as chlorinated ethylene such as tetrachloroethylene (PCE), trichloroethylene (TCE), and dichloroethylene (DCE) is present in soil in the paper and pulp industry and precision machinery-related industrial areas in Japan and overseas. It is considered to be spreading in the range, and many cases actually detected by environmental surveys and the like have been reported. It is said that those volatile organic chlorine compounds remaining in soil are dissolved in groundwater by rainwater or the like and spread throughout the surrounding area. Such compounds are suspected of carcinogenicity and reproductive toxicity, and are very stable in the environment. Therefore, in particular, the pollution of groundwater used as a water source for drinking water is regarded as a major social problem.

[0003] Accordingly, purification of an aqueous medium such as contaminated groundwater, soil, and the accompanying gas phase by removal and decomposition of volatile organic chlorine compounds is an important issue from the viewpoint of environmental conservation. The technology required for purification is being developed.

For example, adsorption treatment with activated carbon, decomposition treatment with light or heat, etc. have been studied, but are not always practical in terms of cost and operability.

On the other hand, volatile organic chlorine compounds such as TCE, which are stable in the environment, have recently been reported to be decomposed by microorganisms, and research for their practical use has begun.
That is, in the biodegradation treatment using microorganisms, it is possible to decompose volatile organic chlorine compounds into harmless substances by appropriately selecting microorganisms to be used, basically no special chemicals are required, maintenance labor and There are advantages such as cost reduction.

[0006] Examples of microorganisms having the ability to decompose volatile organochlorine compounds include:
lchia alkenophila sero 5 (USP 4877736, ATCC 5357
0), Welchia alkenophila sero 33 (USP 4877736, ATCC
53571), Methylocystis sp.strain M (Agric. Biol.
Chem., 53, 2903 (1989), Biosci. Biotech. Biochem., 5
6,486 (1992), 56,736 (1992)), Methylosinus trich
osporium OB3b (Am. Chem. Soc. Natl. Meet. Dev. Env
iron. Microbiol., 29, 365 (1989), Appl. Environ. Mic
robiol., 55, 3155 (1989), Appl. Biochem. Biotechno
l., 28,877 (1991), JP-A-02-92274, JP-A-03-2
No. 92970), Methylomonas sp. MM2 (Appl. Enviro
n. Microbiol., 57,236 (1991)), Alcaligenes denitrif
icans ssp.xylosoxidans JE75 (Arch.microbiol., 15
4,410 (1990)), Alcaligenes eutrophus JMP134 (Appl.
Environ. Microbiol., 56, 1179 (1990)), Alcaligenes
eutrophus FERM-13761 (Japanese Patent Application Laid-Open No. 07-123976), Ps
eudomonas aeruginosa JI104 (JP-A-07-236895), Mycobacterium vaccae JOB5 (J. Gen. Microbio
l., 82,163 (1974), Appl. Environ. Microbiol., 54, 2
960 (1989), ATCC 29678), Pseudomonas putida BH (Journal of the Sewer Association, 24, 27 (1987)), Pseudomonas sp. Strain G
4 (Appl.Environ.Microbiol., 52,383 (1986), 53,9)
49 (1987), 54,951 (1989), 56,279 (1990), 57,193
(1991), USP 4925802, ATCC 53617, which was originally Pseu
Although classified as domonas cepacia, Pseudomonas s
p.), Pseudomonas mendocina KR-1 (Bio
/ Technol., 7,282 (1989)), Pseudomonas putida F1 (App
l. Environ. Microbiol., 54, 1703 (1988), 54, 2578 (1
988)), Pseudomonas fluorescens PFL12 (Appl. Enviro
n. Microbiol., 54, 2578 (1988)), Pseudomonas putida
KWI-9 (JP-A-06-70753), Pseudomonas cepacia
KK01 (JP-A-06-227769), Nitrosomonas euro
paea (Appl.Environ.Microbiol., 56, 1169 (1990)), La
ctobacillus vaginalis sp.nov (Int.J. Syst. Bacter
iol., 39, 368 (1989), ATCC 49540), Nocardia coralli
na B-276 (Japanese Unexamined Patent Publication No. 08-70881, FERM BP-5124, ATCC
31338).

[0007] Similarly, the purification treatment of aromatic compounds such as phenol, toluene and cresol is also shifting from a physical / chemical method to a method using microbial decomposition.

[0008] Such an environmental restoration method using microorganisms is called bioremediation, and bioaugmentation for introducing the isolated microorganisms into the environment, nutrients and oxygen. And the like can be broadly classified into biostimulation in which repair is promoted by increasing the activity of microorganisms originally present in the environment.

In any case, in bioremediation based on the aerobic decomposition of microorganisms, the supply of oxygen is indispensable.

Examples of the method for supplying oxygen include introducing air or oxygen gas to the site, adding hydrogen peroxide, and adding a peroxide salt such as calcium peroxide or magnesium peroxide.

Among them, the addition of hydrogen peroxide is a very simple and effective method which does not require a device such as gas introduction and does not lower the pH unlike a peroxide salt. Widely used. However, hydrogen peroxide itself has a strong oxidizing power and is toxic to microorganisms, and thus has a drawback that it cannot be given at a high concentration.

According to JP-A-64-34380,
In a method for microbiological degradation of chlorinated aliphatic hydrocarbons,
The initial concentration of hydrogen peroxide at the location of the microorganisms (in this case, the microorganisms originally present in the environment) (in this case, groundwater) is generally 50-200 ppm, and higher levels (up to 2000 ppm) require acclimation. It is.

According to the embodiment described in Japanese Patent Application Laid-Open No. 4-9916, a method for promoting the bio-oxidation of pollutants in a subterranean layer includes the use of microorganisms (in this case, microorganisms originally present in the environment). The initial concentration of hydrogen peroxide that does not provide a toxic environment for oxygen and has the effect of imparting oxygen to the growth is up to about 500 ppm. If it is higher than that, some complicated measures are required in how to give hydrogen peroxide. It is shown that

On the other hand, JP-A-6-226230 discloses an example in which hydrogen peroxide is used in a method for purifying contaminated soil using isolated aliphatic chlorine compound-decomposing microorganisms. According to the gazette, by introducing 10 ppm of hydrogen peroxide into the soil contaminated with trichlorethylene simultaneously with the culture solution of the decomposing microorganism, the TCE degradation rate after 3 days is doubled as compared with the introduction system of the culture solution alone. Despite the effect, 10p as an oxygen source in soil
pm of hydrogen peroxide is not sufficient.

[0015]

As described above, although hydrogen peroxide is very effective as an oxygen-imparting agent for microorganisms in bioremediation, it is not sufficient to supply oxygen due to its strong oxidizing power. Attempting to provide a concentration may cause toxicity to microorganisms.

[0016]

SUMMARY OF THE INVENTION The present invention has been made under such circumstances, and has as its object the bioremediation of natural environments such as soil and groundwater contaminated with chlorinated ethylene compounds and substituted benzene compounds. It is an object of the present invention to provide a chlorinated ethylene compound and substituted benzene compound-degrading microorganism that is resistant to hydrogen peroxide at a concentration that can sufficiently exert an oxygen-imparting effect on the microorganism in mediation.

That is, the present invention provides a microorganism JMP1000 which can grow and survive even in the presence of 1000 ppm of hydrogen peroxide and has the ability to degrade chlorinated ethylene compounds and substituted benzene compounds. The chlorinated ethylene compound in the present invention refers to trichloroethylene, dichloroethylene, and the like, and the substituted benzene compound refers to phenol, toluene, cresol (o-cresol, m-cresol, p-cresol), and the like. It is.

Another object of the present invention is to provide a novel method for obtaining the microorganism, comprising the following steps. That is, a method for obtaining a microorganism having the resolution of chlorinated ethylene compounds and substituted benzene compounds and having hydrogen peroxide resistance, having the resolution of chlorinated ethylene compounds and substituted benzene compounds and having hydrogen peroxide resistance. Preparing a microorganism that does not contain the growth substrate of the microorganism; and contacting the microorganism with an inorganic salt medium containing hydrogen peroxide for a predetermined period without containing a growth substrate of the microorganism; and contacting the microorganism with the inorganic salt medium, It is another object of the present invention to provide a method for obtaining a microorganism, comprising the steps of: culturing in a medium containing hydrogen peroxide and containing a growth substrate of the microorganism; and collecting cells grown in the medium.

That is, in the conventional method, a method of simply contacting hydrogen peroxide with a microorganism and adapting it to the condition where a high concentration of hydrogen peroxide is present has been adopted. Is a method in which a bacterium previously cultured in the presence of a predetermined growth substrate is brought into contact with an inorganic salt medium containing only hydrogen peroxide for a predetermined period (step 2), and then cultured in a medium in which the growth substrate and hydrogen peroxide coexist. (Step 3) includes steps. In particular, in step 2, contacting the bacteria with hydrogen peroxide in an inorganic salt medium containing only hydrogen peroxide, which does not contain the growth substrate of the microorganism, directly applies the strong oxidizing power of hydrogen peroxide to the microorganism. It is extremely effective in obtaining hydrogen peroxide-resistant bacteria. As a result, a hydrogen peroxide-resistant microorganism was successfully obtained more efficiently than before. In the step 2, the predetermined period for contacting the microorganism with hydrogen peroxide in an inorganic salt medium containing only hydrogen peroxide without a growth substrate is, for example, 10 to 50 hours, particularly 15 to 48 hours. When the time is set, it is preferable because hydrogen peroxide-resistant bacteria can be efficiently obtained.

[0020]

DETAILED DESCRIPTION OF THE INVENTION The novel microorganism JMP1000 strain is
A strain J1 (an international deposit number based on the Budapest Treaty: FERM BP-510) which is a strain capable of decomposing aromatic compounds and chlorinated ethylene compounds by using an inducer
2) was obtained by mutating by a mutagenesis using a mutagen, and is a JM1 strain (an international deposit number based on the Budapest Treaty: FERM BP) which is a mutant strain capable of decomposing an organic compound without using an inducer −5352)
Obtained from. The acquisition method is as follows (Example 1).

Embodiment 1 Acquisition of JMP1000 shares and
Mycological properties JM on M9 agar medium containing 1% sodium malate
2. Inoculate a colony of one strain in M9 liquid medium containing 2% sodium malate, and culture with shaking at 15 ° C. 2.2-4 days later, collect cells by centrifugation. M containing 1000-3000 ppm of hydrogen peroxide
9 resuspended in liquid medium (without addition of growth substrate of JM1)
Shake at 5 ° C. 4. After 24 hours, the bacterial suspension is diluted at a rate of 1/10 by volume,
1000 ppm hydrogen peroxide and 1% sodium malate
4. Inoculate into M9 medium containing, and shake culture at 15 ° C. When the cells have grown sufficiently (after 6 days), the culture solution is inoculated at a ratio of 1/10 by volume to an M9 medium containing 1000 ppm of hydrogen peroxide and 1% of sodium malate, and shaken at 15 ° C. Cultivate to grow. When the cells have grown sufficiently (after 3 days), the culture solution is inoculated at a ratio of 1/10 by volume into an M9 medium containing 1000 ppm of hydrogen peroxide and 1% of sodium malate, and shaken at 15 ° C. Repeat the operation of 7.6. From the culture solution obtained in this manner, colonies are formed on M9 agar medium containing 1% sodium malate, and JMP
1000 strains were isolated.

The mycological properties of the JMP1000 strain are as follows. Gram stainability and morphology: Gram-negative bacilli Growth in each medium BHIA: Good growth MacConkey: Viable Colony color: Cream Optimal temperature: 25 ° C> 30 ° C> 35 ° C Motility: negative (semi-fluid medium) TSI ( (slant / butt): alkali / alkali, H ₂ S (−) oxidase: positive (weak) catalase: positive fermentation of sugars glucose: negative sucrose: negative raffinose: negative galactose: negative maltose: negative urease: positive esculin hydrolysis ( β-glucosidase): positive Nitrate reduction: negative Indole production: negative Glucose acidification: negative Arginine dihydrolase: negative Gelatin degradation (protease): negative β-galactosidase: negative Assimilation of each compound Glucose: negative L-arabinose: negative D- Manno Source: negative D-mannitol: negative N-acetyl-D-glucosamine: negative Maltose: negative potassium gluconate: negative n-capric acid: positive adipic acid: positive dl-malic acid: positive sodium citrate: positive phenyl acetate: Negative As described above, the mycological properties of the JMP1000 strain are exactly the same as those of the parent strain JM1. However, the strain can be sufficiently grown in the presence of 1000 ppm of hydrogen peroxide, which is a condition that cannot normally grow with the JM1 strain. And can exhibit the resolution of chlorinated ethylene compounds and substituted benzene compounds. Such a characteristic of hydrogen peroxide resistance does not change even after the 5th subculture on the agar medium, and is not a mere adaptation but a mutation. The strain was deposited as a hydrogen-resistant mutant strain with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (Accession number: FERM P-16143).

The basic inorganic salt medium used for culturing the strain is not particularly limited as long as it contains components necessary for the growth of the strain, and examples thereof include M9 medium and MSB medium. A basal salt medium is used.

The composition of the M9 medium is shown below.

Na ₂ HPO ₄ : 6.2 g KH ₂ PO ₄ : 3.0 g NaCl: 0.5 g NH ₄ Cl: 1.0 g (in 1 liter of medium; pH
7.0) Culture can be performed under aerobic conditions, and may be liquid culture or solid culture. The culture temperature is preferably from 15 ° C to 30 ° C. In this embodiment, hydrogen peroxide 1000 to 30 is used.
M9 medium containing 00 ppm (without addition of growth substrate of JM1)
In this case, the contact between the JM1 strain and hydrogen peroxide was 24 hours, but the contact period is not limited to this. For example, it is preferable to set the contact period to 10 to 50 hours, particularly 15 to 48 hours, because the JM1000 strain can be obtained efficiently.

Embodiment 2 FIG . Peracid by JMP1000 strain
Decomposition of TCE and DCE in the presence of hydrogen fluoride Colonies of the JMP1000 strain on an agar medium (containing 1.0% sodium malate) were extracted from a 50% medium containing 1.0% sodium glutamate and 1000 ppm hydrogen peroxide.
200 ml of M9 medium in a 0 ml shake flask was inoculated and cultured at 15 ° C. with shaking.

As a control, a colony on an agar medium (containing 1.0% sodium malate) of the JM1 strain was used.
M in a 500 ml shake flask containing 1.0% sodium glutamate and 1000 ppm hydrogen peroxide
9 medium was inoculated into 200 ml, and cultured at 15 ° C. with shaking.

After culturing for 3 days, the JMP1000 strain and JM
The culture solution of one strain was collected by centrifugation, and resuspended in M9 medium so that the bacterial concentration was tripled.

Into a 27.5 ml vial, 9 ml of the M9 medium containing 1000 ppm of hydrogen peroxide and 9 ml of the M9 medium alone as a control were injected, and 1 ml of the bacterial suspension of the JMP1000 strain and the JM1 strain prepared as described above. Was added, and the container was completely sealed with a butyl rubber stopper with a Teflon liner and an aluminum cap.

At this time, the initial bacterial concentration was JM1 strain: 2.9.
× 10 ⁷ ; JMP1000 strain: 3.1 × 10 ⁷ (CFU
/ Ml).

TCE (trichloroethylene) and tD
Saturated vapor of CE (trans-dichloroethylene) was added with a gas tight syringe to a concentration in the liquid of 10 ppm, and the mixture was shaken at 20 ° C.

After 80 hours, the gas phase in the vial is 0.1 m
1 was collected and subjected to gas chromatography (Shimadzu Gas Chromatograph GC-14B; FID) by the headspace method.
Detector)). Table 1 shows the results. The numerical values are shown as the residual ratio (%) of each concentration based on the residual amount of a blank (no bacterial cells) of only M9 medium containing no hydrogen peroxide.

[0033]

[Table 1] According to this result, hydrogen peroxide 1 at almost the same number of bacteria
The superiority of the JMP1000 strain in TCE degradation in the presence of 000 ppm was clarified.

Embodiment 3 FIG . Peracid by JMP1000 strain
Decomposition of Phenol in the Presence of Hydrogen
Medium containing 1000 ppm of hydrogen peroxide and hydrogen peroxide, and M containing only 200 ppm of phenol as a control.
9 ml of each of the 9 culture media was injected, and 1 m of the bacterial suspension of the JMP1000 strain and the JM1 strain prepared as in Example 2
After addition of 1, it was stoppered.

After 80 hours, 0.5 ml of the bacterial solution was collected,
Spectrophotometric method using 4-aminoantipyrine (JIS
K0102-1993 28.1) to determine the amount of phenol. Table 2 shows the results. Numerical values are shown as the residual ratio (%) of each concentration based on the residual amount of a blank (no bacterial cells) of only the M9 medium without hydrogen peroxide, as in Table 1.

[0036]

[Table 2] According to this result, hydrogen peroxide 1 at almost the same number of bacteria
The superiority in the phenol degradation of the JMP1000 strain in the presence of 000 ppm was clarified.

Embodiment 4 FIG . Peracid by JMP1000 strain
Decomposition of Cresol in the Presence of Hydrogen Hydrogen In the same manner as in Example 3, the evaluation of the decomposition of o-cresol and m-cresol (200 ppm each) of the JMP1000 strain in the presence of 1000 ppm of hydrogen peroxide was performed. Each system was performed separately, and the quantification was carried out by a spectrophotometric method using p-hydrazinobenzenesulfonic acid (JI
SK 0102-1993 28.2). Table 3 shows the results. Numerical values are shown as the residual ratio (%) of each concentration based on the residual amount of a blank (no bacterial cells) of only the M9 medium without hydrogen peroxide, as in Table 1.

[0038]

[Table 3] According to this result, hydrogen peroxide 1 at almost the same number of bacteria
The superiority of the JMP1000 strain in cresol degradation in the presence of 000 ppm was clarified.

Embodiment 5 FIG . Peracid by JMP1000 strain
Decomposition of Toluene in the Presence of Hydrogen Hydrogen In the same manner as in Example 3, the evaluation of the decomposition of toluene by the JMP1000 strain in the presence of 1000 ppm of hydrogen peroxide was performed. The initial toluene concentration was 50 ppm, and the quantification was performed by sampling 0.1 ml of the gas phase in the vial and performing gas chromatography (Shimadzu Gas Chromatograph GC-14B; FID detector) by the headspace method. Table 4 shows the results. Numerical values are shown as the residual ratio (%) of each concentration based on the residual amount of a blank (no bacterial cells) of only the M9 medium without hydrogen peroxide, as in Table 1.

Table 4 shows the results. The numerical values are shown as the residual ratio (%) of each concentration based on the residual amount of a blank (no bacterial cells) of only M9 medium containing no hydrogen peroxide.

[0041]

[Table 4] According to this result, hydrogen peroxide 1 at almost the same number of bacteria
The superiority of the JMP1000 strain in the decomposition of toluene in the presence of 000 ppm was clarified.

[0042]

According to the present invention, an efficient and stable biological purification treatment of a substituted benzene compound and a chlorinated ethylene compound in the presence of hydrogen peroxide at a concentration of 1000 ppm or less can be achieved by a novel hydrogen peroxide-resistant microorganism. It becomes possible.

Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI C02F 3/34 ZAB B09B 3/00 ZABE // (C12N 1/20 C12R 1:01) (72) Inventor Tetsuya Yano 3 Shimomaruko, Ota-ku, Tokyo Chome 30-2 Canon Inc.

Claims (8)

[Claims] 1. A novel microorganism strain JMP1000 (FERM P-16143) that grows in 1000 ppm hydrogen peroxide water and has the ability to degrade chlorinated ethylene compounds and substituted benzene compounds. 2. A method for obtaining a hydrogen peroxide-resistant microorganism having a resolution of a chlorinated ethylene compound and a substituted benzene compound, comprising the steps of: A step of preparing a microorganism having no resistance; a step of contacting the microorganism with a mineral salt medium containing hydrogen peroxide without containing a growth substrate of the microorganism for a predetermined period; and a step of contacting the microorganism with the inorganic salt medium. A step of culturing in a medium containing hydrogen peroxide and containing a growth substrate of the microorganism, and collecting the cells grown in the medium. 3. The method according to claim 2, wherein the predetermined period is 10 to 50 hours. 4. The method according to claim 3, wherein the predetermined period is 15 to 48 hours. 5. The microorganism having the ability to degrade chlorinated ethylene compounds and substituted benzene compounds and having no resistance to hydrogen peroxide is JM1 strain (FERM BP-5352). A method for obtaining a microorganism according to any one of the above. 6. A step of contacting a medium containing at least one of a chlorinated ethylene compound and a substituted benzene compound as a pollutant with a microorganism capable of decomposing the pollutant in the presence of hydrogen peroxide to decompose the pollutant. In a method for repairing a contaminated medium having JMP1000,
A repair method using a strain (FERM P-16143). 7. The repair method according to claim 6, wherein the chlorinated ethylene compound is at least one compound selected from trichloroethylene and dichloroethylene. 8. The method according to claim 8, wherein the substituted benzene compound is phenol,
The repair method according to claim 6, wherein the repair method is at least one compound selected from crezone and toluene.