JPH11197691A - Method for biodegradation of organic compound and method for environmental restoration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To extend a maintenance period of the decomposing activity of a microorganism in decomposing contaminants by using the microorganism, and to efficiently and stably restore a contaminated environment, by bringing the microorganism into contact with methanol or formaldehyde after its proliferation. SOLUTION: When organic compounds (contaminants) are biodegraded by using a microorganism J1 strain (FERMBP-5102) or its mutant, an expression period of a decomposing activity is extended to improve the effectiveness of decomposing contaminants by bringing the strain into contact with methanol or formaldehyde. In this case, the concentration of methanol to be used is preferably 0.01-0.5%(V/V), and more preferably 0.05-0.2%. Moreover, the concentration of formaldehyde is preferably 0.1-5 mM, and more preferably 0.5-2 mM. Thus, the biodegradation and purification of a liquid, soil, and a gas phase, which contain contaminants such as an aromatic compound and a chlorinated ethylenic compound, can be efficiently and stably carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for biodegrading organic compounds and a method for restoring an environment containing pollutants using microorganisms.

[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, the contamination of volatile organic chlorine compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) has spread to a considerable extent in soil in the paper and pulp industry and precision machinery-related industrial areas in Japan and overseas. It has been reported that many cases were actually detected by environmental surveys. It is said that those volatile organic chlorine compounds remaining in the 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] Therefore, purification of an aqueous medium such as contaminated groundwater, soil, and the accompanying gas phase by removing and decomposing 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, in recent years, microorganisms have been reported to decompose volatile organic chlorine compounds such as TCE which are stable in the environment, and studies for practical use thereof have begun.
In biodegradation treatment using microorganisms, it is possible to decompose volatile organic chlorine compounds to harmless substances by selecting microorganisms to be used, basically no special chemicals are required,
There are advantages such as reduction of labor and cost for maintenance.

[0006] Such advantages can be applied to the treatment of contaminating with aromatic compounds, which has been a problem in the past.

[0007] Examples of isolated microorganisms having the ability to decompose volatile organochlorine compounds include TCE-degrading bacteria such as W
elcia alkenophila sero 5
(USP 4,877,736, ATCC 53570), W
elcia alkenophila sero 33
(USP 4,877,736, ATCC 53571), M
ethylcysis sp. strain M (A
gric. Biol. Chem. , 53, 2903 (1
989), Biosci. Biotech. Bioch
em. , 56,486 (1992), 56,736
(1992)), Methylosinus tric
hose OB3b (Am. Chem. So
c. Natl. Meet. Dev. Environ. M
microbiol. , 29, 365 (1989), Ap
pl. Environ. Microbiol. , 55,
3155 (1989), Appl. Biochem. B
iotechnol. , 28, 887 (1991), JP-A-02-92274, JP-A-03-29297.
No. 0), Methylomonas sp. MM2
(Appl. Environ. Microbiol.,
57, 236 (1991)), Alcaligenes.
denitrificans ssp. xylosox
idans JE75 (Arch. microbio
l. , 154, 410 (1990)), Alcalig.
enes eutrophus JMP134 (App
l. Environ. Microbiol. , 56,1
179 (1990)), Alcaligenes eu
trophus FERM P13761 (Japanese Patent Application Laid-Open
-123976), Pseudomonas a
eruginosa JI104 (Japanese Unexamined Patent Publication No. 07-236)
895), Mycobacterium vac
cae JOB5 (J. Gen. Microbio
l. , 82, 163 (1974), Appl. Envi
ron. Microbiol. , 54, 2960 (19
89), ATCC 29678), Pseudomon
as putida BH (Sewerage Association Journal, 24, 27
(1987)), Pseudomonas sp. st
line G4 (Appl. Environ. Micr
obiol. , 52, 383 (1986), 53, 9
49 (1987), 54, 951 (1988), 5
6, 1279 (1990) and 57, 1935 (199)
1), USP 4,925,802, ATCC 53617,
This bacterium was initially Pseudomonas cepacia
Pseudomonas sp.
Was changed to ), Pseudomonas mend
ocina KR-1 (Bio / Technol.,
7, 282 (1989)), Pseudomonas
putida F1 (Appl. Environ. Mi)
crobiol. , 54, 1703 (1988), 5
4,2578 (1988)), Pseudomonas
fluorescens PFL12 (Appl. En
viron. Microbiol. , 54, 2578
(1988)), Pseudomonas putid
a KWI-9 (JP-A-06-70753), P
pseudomonas cepacia KK01 (Japanese Unexamined Patent Publication No. 06-227770), Nitrosomon
as europaea (Appl. Environ.
Microbiol. , 56, 1169 (199
0)), Lactobacillus vital
is sp. nov (Int. J. Syst. Bact)
eriol. , 39, 368 (1989), ATCC
49540), Nocardia corallina
B-276 (Japanese Unexamined Patent Publication No. 08-70881, FERM)
BP-5124, ATCC 31338).

[0008] However, particularly when bacterium which decomposes chlorinated ethylene compounds such as TCE and DCE is used for actual environmental purification treatment, it is very difficult to decompose these compounds. That is, it requires a chemical substance such as an aromatic compound or methane as a producer.

For example, aromatic compounds such as phenol and toluene are very good inducers, but their toxicity is high, and their release into the environment must be carried out according to some regulations. Methane, which is a substance, is a flammable gas, and its control in the environment is extremely dangerous and complicated.

One method for solving such a problem is disclosed in JP-A-4-502277. In this method, the problem of toxicity and danger of the inducer is avoided by using tryptophan, one of aromatic amino acids, as the inducer of microbial degradation of chlorinated ethylene. Further, according to JP-A-7-171548, the above problem is avoided by using lignocellulose, which is a natural substance extracted from herbaceous plants, as an inducer.

Further, from the viewpoint of improving the degrading microorganism, a plasmid into which a DNA fragment containing a gene region coding for a degrading enzyme, oxygenase or hydroxylase, is introduced into a host bacterium, and the plasmid is introduced with a harmless inducing substance or a derivative. Attempts have been made to constitutively express a degrading activity even under conditions where no substance is present.
Examples of DNA fragment-derived strains include Pseudomonas mendocina KR-1 (JP-A-2-503866) and Pseudomonas putida KWl-9 (JP-A-6-10569).
No. 1), Pseudomonas putida BH (3rd Lecture Meeting on Research on Groundwater and Soil Pollution and Its Prevention,
213 (1994)).

Another solution is the international publication number WO92.
/ 19738. According to this report,
Mutants that degrade chlorinated ethylene compounds without using inducers such as phenol and toluene have been obtained by mutation using transposons.

Still another solution is disclosed in Japanese Unexamined Patent Publication No. 8-29.
No. 4387 and U.S. Pat. No. 5,543,317. According to this report, mutant strains (JM1 strain and G4 strain) that degrade chlorinated ethylene compounds and aromatic compounds without using inducers such as phenol and toluene.
(Strain pTOM _301c ) was obtained by treating with a mutagen, nitrosoguanidine.

[0014]

According to the studies made by the present inventors, the problem concerning the inducer when the chlorinated ethylene compound or the aromatic compound is subjected to the microbial decomposition treatment is being avoided, A major problem lies in the fact that these microorganisms lose their degrading activity in a very short time.

The causes of such a phenomenon are considered to be the autolysis of the degrading enzyme in the cells, the toxicity of the decomposed intermediate, the lack of an energy source required for the decomposition, and the like.

Accordingly, an object of the present invention is to provide a method for extending the maintenance period of the biodegradation activity when decomposing a contaminant using microorganisms, and for more efficiently and stably repairing the contaminated environment. On the point.

[0017]

An embodiment of a method for biodegrading an organic compound which can achieve the above object is described in Japanese Patent Application Laid-Open Publication No. H10-157, 1989. The microorganism J1 strain (deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry; deposit number: FERM BP- 5102) or a method for biodegrading an organic compound using a mutant strain thereof, wherein the microorganism is grown and then contacted with methanol or formaldehyde.

Further, the present invention provides a method of restoring the environment by decomposing contaminants using a microorganism J1 strain (FERM BP-5102) or a mutant thereof, wherein the microorganism is contacted with methanol or formaldehyde. I do.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION As the above mutant strain, a JMl strain (FERMBP-535), which is also deposited at the same institution, is used.
2), JMC1 strain (FERM BP-5960), JM
2N strain (FERM BP-5961), JM6U strain (F
ERM BP-5962), JM7 strain (FERM BP-
5957), JMP1000 strain (FERM P-161)
43) can be used. These are mutant strains obtained by mutating the J1 strain by mutagenesis using a mutagen and capable of decomposing organic compounds without using an inducer.

The above-mentioned J1 strain and its mutant strain have all been deposited with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry (Tsukuba, Ibaraki).

For the JMl strain, the International Depository Authority mentioned above has FERM BP-5362 / based on the Budapest Treaty on the International Recognition of Deposit of Microorganisms for Patent Procedure.
Indication for identification: Corynebacterium species J
Ml strain (Corynebacterium sp. JM
It was deposited as 1). This strain was initially
Although "Corynebacterium sp. JMl strain" was indicated as belonging to the genus Corynebacterium, the identification was changed to "Not belonging to the genus Corynebacterium". JM1 strain ".

When biodegrading an organic compound (contaminant) using these microorganisms, contacting the strain with methanol or formaldehyde extends the period of expression of the decomposing activity and significantly improves the decomposing effect of the contaminant. This is preferable because it can be performed.

The concentration of methanol used is 0.01 to
0.5% (v / v) is preferable, and 0.05 to 0.2% is more preferable. If the concentration is lower than this range, the effect is small, and if the concentration is higher than this range, the microbial cells themselves are adversely affected.

The concentration of formaldehyde used is 0.1
-5 mM is preferable, and 0.5-2 mM is more preferable.
When the concentration is lower than this range, the effect is small, and when the concentration is higher than this range, a toxic effect is exerted on the microbial cells themselves.

The contact is made after the microorganism is grown on another carbon source. Resting cells that do not contain nutrients such as carbon and nitrogen sources (restingce
11) It may be contacted in a state.

In JP-A-1-160479, methanol is used as a method for restoring the activity of methane monooxygenase of methane-utilizing bacteria.
In this case, methanol is an intermediate product of an enzymatic reaction from methane to carbon dioxide via formic acid, whereas the present invention is completely different in that methanol is brought into contact with an aromatic assimilating microorganism having a completely different route. ing. Also,
Japanese Patent Application Laid-Open No. 6-245762 also uses methanol for activating methane assimilating bacteria, but the same can be said as above, and in this case, it is added as a growth substrate. Completely different.

In Japanese Patent Application Laid-Open No. 1-160479, formaldehyde is used as a method for restoring the activity of methane monooxygenase of methane-utilizing bacteria. In this case, formaldehyde is converted from methane through formic acid to form dioxide. An intermediate product of the enzymatic reaction leading to carbon,
On the other hand, the present invention is completely different in that formaldehyde is brought into contact with an aromatic assimilating microorganism having a completely different route.

Pollutants to be purified include chlorinated ethylene compounds such as trichlorethylene (TCE) and dichloroethylene (DCE), and toluene and phenol.
And aromatic compounds such as cresol,
The method of the present invention can be used to purify any of liquid, soil and air contaminated with these contaminants.

When purifying a liquid such as contaminated groundwater or wastewater, a microorganism and methanol or formaldehyde may be directly introduced into the liquid, or the liquid may be contaminated by a bioreactor using the microorganism and methanol or formaldehyde. The liquid may be treated. In this case, the microorganism may be carried on a carrier. In the case of groundwater, a carrier carrying microorganisms can be directly introduced into the groundwater stream, or the carrier can be placed in a container and introduced into the groundwater stream as a cassette-type biofilter to be treated.

When purifying the contaminated soil, a liquid containing microorganisms and methanol or formaldehyde can be mixed with the soil for pile treatment, or the microorganisms and methanol or methanol or formaldehyde can be directly discharged from a well drilled in the contaminated soil. Liquids containing formaldehyde can also be injected.
Also, when the pollutant is a volatile substance such as a chlorinated ethylene compound, a vessel containing a microorganism and a carrier containing methanol or formaldehyde is introduced into the drilled well,
The contaminated air in the soil can be sucked from the well and treated in the container.

When purifying the contaminated air, the contaminated air can be introduced into a liquid containing microorganisms and methanol or formaldehyde, or a carrier containing microorganisms and methanol or formaldehyde can be introduced. Contaminated air can also be introduced into the container for treatment.

In any case, it is also possible to supply methanol in a gaseous state over a wide range.

The contact between the strain and the substance to be decomposed can be carried out by any method as long as the strain can exhibit the degrading activity, and can be carried out using various methods such as a batch method, a semi-continuous method, and a continuous method. it can.

The strain can be used in a semi-fixed state or immobilized on a suitable carrier.

The method of the present invention is applicable to both closed and open wastewater treatment, soil treatment and air treatment. Various methods for promoting growth may be used in combination.

[0036] The medium used for culturing microorganisms basically needs to contain a carbon source, a nitrogen source, a phosphorus source, inorganic salts, and the like necessary for growth.

The basic inorganic salt medium used for culturing microorganisms is not particularly limited as long as it contains components necessary for the growth of the strain. For example, a basic salt medium such as M9 medium or MSB medium can be used. A medium is used.

The composition of the M9 medium (pH 1 in 1 liter of 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 Culture can be carried out under aerobic conditions, and liquid culture or solid culture may be used. . The culture temperature is preferably 15 to 30 ° C.

[0040]

The present invention will be described in more detail with reference to the following examples.

Example 1 Effect of Methanol on Degradation of TCE (Liquid System) by JM1 Strain A colony of strain JM1 on M9 agar medium (containing 1% sodium glutamate) was transformed with M9 containing 0.5% sodium glutamate. Inoculate 200 ml of medium and 500 ml
Shaking culture was performed at 15 ° C. in a shake flask.

After 45 hours, which is the late stage of logarithmic growth, the cells were collected by centrifugation, and suspended again in an equal volume of M9 medium containing no carbon source to prepare a bacterial suspension.

Next, three 27.5 ml vials (vials (a) to (c)) were prepared, and the vial (a) was prepared.
Each of (b) and (b) was charged with 10 ml of the above bacterial suspension, and methanol was added to vial (a) to a final concentration of 2 mM. Vial bottle (b)
Was added with the same amount of sterilized water as methanol added to the vial (a). The vial (c) is blank
10 ml of M9 medium without cells and a vial (a)
The same amount of sterilized water as the methanol added to was added.

Next, each of the vials (a) to (c) was charged with TC so that the initial TCE concentration was about 5 ppm.
A saturated aqueous solution of E was added, and the mixture was completely sealed with a butyl rubber stopper with a Teflon liner and an aluminum cap, and shaken at 15 ° C.
24 hours after the start of shaking, the gas phase in each vial was 0.1
The TCE concentration was measured by gas chromatography (trade name: GC-14B, equipped with FID detector; manufactured by Shimadzu Corporation) by the headspace method (TCE concentration at the end of the first day). .

Next, the vials (a) to (c) whose TCE concentrations were measured were left at 15 ° C. for 1 hour with the stoppers open to volatilize the remaining TCE, and then re-initialized. Add a TCE saturated aqueous solution to each of the vials (a) to (c) so that the concentration becomes about 5 ppm,
After completely sealed with a butyl rubber stopper with a Teflon liner and an aluminum cap, the mixture was shaken at 15 ° C., and 24 hours after the start of shaking, the TCE concentration in the gas phase of each vial was measured in the same manner as described above (at the end of the second day). TCE concentration).

The above operation was repeated for each of the vials (a) to (c), and the TCE concentration in the gas phase in each vial was measured (TCE at the end of the third day). concentration).

Table 1 shows the results of this example. From Table 1,
It can be seen that the vial (a) system continued to degrade TCE on the third day.

[0048]

[Table 1]

Example 2 Effect of Methanol on Decomposition (Liquid System) of DCE by JMl Strain cis-1,2-dichloroethylene (c
is-1,2-DCE), trans-1,2-dichloroethylene (trans-1,2-DCE), and 1,
1-dichloroethylene (1,1-DCE)
Except for ppm, the effect of the present invention was evaluated in the same manner as in Example 1.

Table 2 shows the results. From these results, the effect of the present invention was shown in the decomposition treatment of liquid DCE using the JM1 strain.

[0051]

[Table 2]

Example 3 Effect of Methanol on Decomposition (Liquid System) of Toluene by JM1 Strain Example 1 except that the substance to be decomposed was 20 ppm of toluene.
The effect of the present invention was evaluated in the same manner as described above. Table 3 shows the results. From the results, the effect of the present invention was shown in the decomposition treatment of liquid toluene using the JM1 strain.

[0053]

[Table 3]

Example 4 Effect of Methanol on Decomposition (Liquid System) of Phenol and Cresol by JM1 Strain (I) A bacterial suspension was prepared in the same manner as in Example 1. Next, three 50 ml sterile tubes (sterile tube (a)
To (c)), 10 ml of the above bacterial suspension was added to each of the sterile tubes (a) and (b), and methanol was further added to the sterile tube (a) to a final concentration of 2 mM. . The sterilized tube (b) was used as a control sample by adding sterilized water in the same amount as methanol added to the sterilized tube (a). Further, to the sterilization tube (c), 10 ml of M9 medium containing no cells and sterilized water in the same amount as methanol added to the sterilization tube (a) were added.

Next, a phenol saturated aqueous solution was added to each of the sterile tubes (a) to (c) so that the initial phenol concentration was about 50 ppm, and the mixture was shaken at 15 ° C. Twenty-four hours after the start of shaking, 0.1 ml of the solution was collected from each sterilized tube, and was subjected to an absorption spectrophotometric method using 4-aminoantipyrine (JIS K0102-1993 2).
The phenol concentration was measured according to 8.1) (phenol concentration at the end of the first day).

Three 50 ml sterile tubes (sterile tubes (d) to (f)) were newly prepared, and 10 ml of the above bacterial suspension was added to each of the sterile tubes (d) and (e). To (d), methanol was added to a final concentration of 2 mM. The sterilized tube (e) was used as a control sample by adding the same amount of sterilized water as methanol added to the sterilized tube (d). Further, the sterile tube (f) contains 10 ml of M9 medium containing no cells,
The same amount of sterilized water as the methanol added to the sterile tube (d) was added.

These sterilized tubes (d) to (f) were
After standing for 4 hours, a phenol saturated aqueous solution was added to each of the sterilized tubes (d) to (f) so that the initial phenol concentration was about 50 ppm, and the mixture was shaken at 15 ° C. Twenty-four hours after the start of shaking, the phenol concentration in each of the sterile tubes (d) to (f) was measured in the same manner as described above (phenol concentration at the end of the second day).

Further, three 50 ml sterile tubes (sterile tubes (g) to (i)) were newly prepared, and 10 ml of the above bacterial suspension was added to each of the sterile tubes (g) and (h).
Was added to the sterilized tube (g), and methanol was added to a final concentration of 2 mM. The sterilized tube (h) was used as a control sample by adding the same amount of sterilized water as methanol added to the sterilized tube (g). Further, the sterile tube (i) contains 10 ml of M9 medium containing no cells,
The same amount of sterilized water as the methanol added to the sterile tube (g) was added.

These sterilized tubes (g) to (i) were
After allowing to stand for 8 hours, a saturated aqueous phenol solution was added to each of the sterilized tubes (g) to (i) so that the initial phenol concentration became about 50 ppm, and the mixture was shaken at 15 ° C. Twenty-four hours after the start of shaking, the phenol concentration in each of the sterilized tubes (g) to (i) was measured in the same manner as described above (the phenol concentration at the end of the third day). Table 4 shows the results.

(II) o-cresol and m-cresol were used in place of phenol, and the initial concentration was 30 ppm.
The o-cresol concentration and the m-cresol concentration in each sterilized tube were measured in the same manner as in the above (I) except that the above conditions were used. Table 4 shows the results.

From the results in Table 4, it can be seen that methanol has an effect of extending the maintenance period of the activity in the degradation of phenol or cresol using the JM1 strain.

[0062]

[Table 4]

Example 5 Effect of Methanol on Degradation of TCE by Jl Strain (Liquid System) Agar M9 medium of Jl strain (1% sodium glutamate,
And 2 mM phenol)).
200 ml of M9 medium containing sodium glutamate and 2 mM phenol was inoculated, and shake-cultured at 15 ° C. in a 500 ml shake flask.

At the 50th hour, which is the latter half of logarithmic growth (at this time, the culture solution has a yellow color derived from 2-hydroxymuconic acid semialdehyde, which is an intermediate product of phenol degradation)
Were collected by centrifugation and resuspended in an equal volume of M9 medium without a carbon source to prepare a bacterial suspension.

A TCE degradation experiment was performed by the J1 strain in the same manner as in Example 1 except that the bacterial suspension containing the J1 strain was used instead of the bacterial suspension containing the JM1 strain in Example 1. .

The results are as shown in Table 5, and
An effect of extending the TCE decomposition activity of the J1 strain by methanol was observed.

[0067]

[Table 5]

Example 6 Effect of Methanol on Degradation of TCE by Other Strains (Liquid System) As other degrading bacteria, strains JMC1, JM2N and JM were used.
The effects of the present invention were evaluated for 6U strain, JM7 strain, and JMP1000 strain in the same manner as in Example 1. Table 6 shows the results. From these results, the JMCl strain, JM2N strain, JM6U
Liquid TC using strains JM7 and JMP1000
The effect of the present invention was shown in the decomposition treatment of E.

[0069]

[Table 6]

<Example 7> TCE in soil by JM1 strain
Effect of Methanol on Decomposition Treatment of Bacteria A bacterial suspension containing the JM1 strain was prepared in the same manner as in Example 1.

Next, three vials of 68 ml capacity (vial bottles (a) to (c)) were prepared, and 50 g of Sahara through sand (13% water content) was added to each of the vials (a) and (b).
And 5 ml of the bacterial suspension, methanol was added to the vial (a) to a final concentration of 2 mM, and the vial (a) was added to the vial (b) as a control.
The same amount of sterilized water as the methanol added to was added. further,
The vial (c) is a blank containing no cells.
10 ml of 9 medium and sterilized water in the same amount as methanol added to the vial (a) were added.

Next, a TCE saturated aqueous solution was added to each of the vials (a) to (c) so that the initial TCE concentration was about 5 ppm (per the total water content in the vials), and then the Teflon liner was added. Completely sealed with a butyl rubber stopper and an aluminum cap, and allowed to stand in an environment of 15 ° C. After 24 hours, 0.1 ml of a gas phase in each vial was collected, and subjected to gas chromatography (trade name: The concentration of TCE was measured using GC-14B with FID detector (manufactured by Shimadzu Corporation) (TCE concentration at the end of the first day).

Next, these vials (a) to (c)
And left at 15 ° C. for 1 hour to volatilize the residual TCE in the vial.
A saturated aqueous solution of TCE was added so as to give ppm, and the mixture was completely sealed in the same manner and shaken at 15 ° C. Twenty-four hours after the start of shaking, the TCE concentration in the gas phase of each vial was measured in the same manner as described above (the TCE concentration at the end of the second day).

The above operation was repeated for each of the vials (a) to (c) to measure the TCE concentration in the gas phase in each vial (TCE at the end of the third day). concentration).

The results are as shown in Table 7, and the effect of extending the activity of decomposing TCE in the soil of the JM1 strain by methanol was observed.

[0076]

[Table 7]

Example 8 Effect of Methanol on Decomposition Treatment of TCE in Gas Phase by Bioreactor Using JMl Strain A bacterial suspension of JM1 strain was prepared in the same manner as in Example 1.

Next, a porous cellulose carrier (average pore size of about 500 μm, diameter of about 5 mm) was sterilized in an autoclave and cooled to room temperature or lower to obtain a diameter (inner diameter) of 3c.
m, packed in a 10 cm long column.

At a temperature of 15 ° C., a suspension obtained by adding methanol to the bacterial suspension prepared as described above to a concentration of 2 mM was added until the solution was slightly eluted from the lower end of the column. Air containing TCE (approximately 200 ppm in gas concentration) at a flow rate of 0.2
flow continuously through the column at ml / min, after 1 day, 2 days,
After 3 days, the TCE gas concentration was measured.

The amount of TCE was determined by quantifying TCE in the air flowing out from the lower end of the column by gas chromatography (PID detector). As controls, the same experimental system without methanol and JM1
The quantification of TCE in the system without adding the strain was also performed.

Table 8 shows the results. From this result, JM
The effect of the present invention was demonstrated in the decomposition treatment of gas phase TCE by a bioreactor using one strain.

[0082]

[Table 8]

Example 9 Effect of Formaldehyde on Decomposition of TCE by JM1 (Liquid System) Decomposition of TCE was carried out in the same manner as in Example 1 except that formaldehyde was used in place of methanol used in Examples. .

The results are as shown in Table 9, and the effect of extending formaldehyde on the TCE degradation activity of the JM1 strain was confirmed.

[0085]

[Table 9]

Example 10 Effect of Formaldehyde on Decomposition (Liquid System) of DCE by JM1 Strain cis-1,2-dichloroethylene (c
is-1,2-DCE), trans-1,2-dichloroethylene (trans-1.2-DCE), and 1.
1-dichloroethylene (1,1-DCE)
Except for ppm, the effect of the present invention was evaluated in the same manner as in Example 9.

Table 10 shows the results. From this result, JMl
The effect of the present invention was shown in the decomposition treatment of DCE in a liquid system using the strain.

[0088]

[Table 10]

Example 11 Effect of Formaldehyde on Decomposition of Toluene (Liquid System) by JMl Strain Example 9 except that the substance to be decomposed was 20 ppm of toluene.
The effect of the present invention was evaluated in the same manner as described above. Table 11 shows the results.
Shown in From these results, the effect of the present invention was shown in the decomposition treatment of liquid toluene using the JMl strain.

[0090]

[Table 11]

Example 12 Effect of Formaldehyde on Decomposition (Liquid System) of Phenol and Cresol by JMl Strain Phenol and cresol were prepared in the same manner as in Example 4 except that formaldehyde was used in place of methanol used in Example 4. Was decomposed.

The results are as shown in Table 12, and
Formaldehyde exhibited an effect of extending the phenol-degrading activity and cresol-degrading activity of the JM1 strain.

[0093]

[Table 12]

Example 13 Effect of Formaldehyde on Decomposition of TCE by J1 Strain (Liquid System) Decomposition of TCE was carried out in the same manner as in Example 5 except that formaldehyde was used instead of methanol in Example 5.

The results are as shown in Table 13, and
An effect of extending the TCE decomposition activity of the J1 strain by formaldehyde was observed.

[0096]

[Table 13]

<Example 14> TCE using other strains
Of Formaldehyde on Decomposition (Liquid System) of Others As other decomposing bacteria, JMCl strain, JM2N strain, JM
The effect of the present invention was evaluated in the same manner as in Example 9 for the 6U strain, the JM7 strain, and the JMP1000 strain. Table 14 shows the results.
Shown in From these results, the JMCl strain, JM2N strain, JM6
Liquid T using U strain, JM7 strain and JMP1000 strain
The effect of the present invention was shown in the decomposition treatment of CE.

[0098]

[Table 14]

<Example 15> TC in soil by JM1 strain
Effect of Formaldehyde on Decomposition Treatment of E TCE was decomposed in the same manner as in Example 7 except that formaldehyde was used instead of methanol in Example 7.

The results are as shown in Table 15, and
Formaldehyde had an effect of extending the activity of decomposing TCE in soil of the JM1 strain.

[0101]

[Table 15]

<Example 16> Effect of formaldehyde on decomposition treatment of TCE in gas phase by bioreactor using JMl strain Same as Example 8 except that formaldehyde was used in place of methanol used in Example 8 To decompose TCE.

The results are as shown in Table 16, and
An effect of extending formaldehyde decomposition activity by formaldehyde was observed.

[0104]

[Table 16]

[0105]

As is apparent from the above description, according to the present invention, efficient and stable biodegradation purification of liquid, soil and gas phase containing pollutants such as aromatic compounds and chlorinated ethylene compounds. Processing becomes possible.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 1/20 B01D 53/34 134E B09B 3/00 ZABE

Claims (31)

[Claims] 1. The microorganism J1 strain (FERM BP-510)
2) Or a method of biodegrading an organic compound using a mutant thereof, wherein the microorganism is grown and then contacted with methanol. 2. The microorganism J1 strain (FERM BP-510)
2) Or a method of biodegrading an organic compound using a mutant thereof, wherein the microorganism is grown and then contacted with formaldehyde. 3. The mutant strain is a JMl strain (FERM BP).
-5352), JMC1 strain (FERM BP-596)
0), JM2N strain (FERM BP-5961), JM
6U strain (FERM BP-5962), JM7 strain (FE
RM BP-5957), JMP1000 strain (FERM
The biodegradation method according to claim 1 or 2, which is P-16143). 4. The biodegradation method according to claim 1, wherein the organic compound is a chlorinated aliphatic compound. 5. The biodegradation method according to claim 4, wherein the chlorinated aliphatic compound is at least one of trichloroethylene and dichloroethylene. 6. The biodegradation method according to claim 1, wherein the organic compound is an aromatic compound. 7. The biodegradation method according to claim 6, wherein said aromatic compound is at least one selected from toluene, phenol and cresol. 8. The method according to claim 8, wherein the contaminant is a microorganism Jl strain (FERM B).
P-5102) or a method for restoring the environment by using a mutant thereof to decompose the microorganism, wherein the microorganism is contacted with methanol. 9. The method according to claim 1, wherein the contaminant is a microorganism Jl strain (FERM B)
P-5102) or a method for restoring the environment by using a mutant thereof to restore the environment, comprising contacting the microorganism with formaldehyde. 10. The mutant strain is a JMl strain (FERM B).
P-5352), JMC1 strain (FERM BP-596)
0), JM2N strain (FERM BP-5961), JM
6U strain (FERM BP-5962), JM7 strain (FE
RM BP-5957), JMP1000 strain (FERM
P-16143), The environmental restoration method of Claim 8 or 9. 11. The method according to claim 8, 9 or 10, wherein the contaminant is a chlorinated aliphatic compound. 12. The method according to claim 11, wherein the chlorinated aliphatic compound is at least one of trichloroethylene and dichloroethylene. 13. The method according to claim 8, 9 or 10, wherein the contaminant is an aromatic compound. 14. The method according to claim 13, wherein the aromatic compound is at least one selected from toluene, phenol and cresol. 15. The method according to claim 8, wherein the environment is a liquid. 16. The environmental restoration method according to claim 15, wherein the contaminant is decomposed by bringing the liquid into contact with a carrier supporting the microorganism. 17. The method according to claim 16, wherein the liquid is introduced from one end of a container having a carrier carrying the microorganism, and discharged from the other end to the outside of the container. 18. The method according to claim 8, wherein the environment is soil. 19. The environmental restoration method according to claim 18, further comprising a step of introducing a liquid containing the microorganism into the soil. 20. The environmental restoration method according to claim 19, wherein the introduction of the microorganism into the soil is performed from an injection well provided in the soil. 21. The method according to claim 18, wherein the soil is introduced into a solution containing the microorganism. 22. The environmental restoration method according to claim 18, wherein the soil containing the microorganism is mixed with the soil. 23. The environmental restoration method according to claim 18, wherein said soil is brought into contact with a carrier supporting said microorganism. 24. The method according to claim 8, wherein the environment is air. 25. The environmental restoration method according to claim 24, wherein the air is introduced into a solution containing the microorganism. 26. The environmental remediation method according to claim 24, wherein the pollutant is decomposed by bringing the air into contact with a carrier carrying the microorganism. 27. The environmental restoration method according to claim 26, wherein said air is introduced from one end of a container having a carrier carrying said microorganism, and discharged from the other end to the outside of said container. 28. A microorganism Jl strain (FERM BP-51)
02) or a method of biodegrading an organic compound using a mutant strain thereof, comprising a step of growing the microorganism and then contacting it with methanol, and a step of contacting the microorganism contacted with methanol with the organic compound. A method for biodegrading an organic compound, comprising: 29. An environment contaminated with contaminants,
In a method of repairing the strain using strain I (FERM BP-5102) or a mutant thereof, the step of growing the microorganism and then contacting with methanol, and the step of contacting the microorganism contacted with methanol with the contaminant are performed. An environmental restoration method comprising: 30. A microorganism Jl strain (FERM BP-51)
02) or a method of biodegrading an organic compound using a mutant thereof, comprising a step of growing the microorganism and then contacting it with formaldehyde, and a step of contacting the microorganism contacted with formaldehyde with the organic compound. A method for biodegrading an organic compound, comprising: 31. An environment contaminated with contaminants is treated with microorganisms J.
In a method of repairing the strain using the L. strain (FERM BP-5102) or a mutant thereof, the step of growing the microorganism and then contacting the microorganism with formaldehyde, and the step of contacting the microorganism contacted with formaldehyde with the contaminant are performed. An environmental restoration method comprising: