JP2000069959A - Volatile organic chlorine compound and biodegradation of aromatic compound, and restoration of environment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To conduct environmental clean-up of polluted soil, water, gas, or the like and to efficiently perform environmental restoration treatment by contacting objects to be decomposed (e.g. volatile organic chlorine compound or the like) with microorganisms under a specific condition to decompose the objects. SOLUTION: When contacting objects to be decomposed selected from volatile organic chlorine compounds and aromatic compounds (e.g. phenol, o-, m- and p-cresols, toluene, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2- dichloroethylene and 1,1-dichloroethylene) with oxygenase-developing microorganisms [e.g. J1 strain (FERM BP-5102), JM1 strain (FERM BP-5352), or the like] to decompose the objects, the present method gives the microorganisms at least one factor selected from the group consisting of a temperature shift, the feed of nutrient, oxygen supply, stirring and pH adjustment, all affecting the decomposition action of microorganisms, one or more times on the way to the decomposing process.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to trichloroethylene (TCE) and dichloroethylene (DC) using microorganisms.
Volatile organic chlorine compounds such as E), and phenol;
Biodegradation method of aromatic compounds such as toluene and cresol, in particular, purification of aqueous media such as wastewater and wastewater containing volatile organic chlorine compounds or aromatic compounds, restoration of soil contaminated by the same, and volatile organic compounds The present invention relates to an environmental restoration method useful for purifying air (gas phase) contaminated by chlorine compounds.

[0002]

2. Description of the Related Art In recent years, environmental pollution by volatile organic chlorine compounds that are harmful to living organisms and hard to decompose has become a serious problem. In particular, it is thought that the pollution of volatile organic chlorine compounds such as tetrachloroethylene (PCE), trichlorethylene (TCE) and dichloroethylene (DCE) has spread to a considerable extent in soil in industrial areas in Japan and overseas. Many cases have been reported in environmental surveys. 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. Since these compounds are suspected of being carcinogenic and stable in the environment, the pollution of groundwater used as a drinking water source has become a serious social problem.

[0003] Therefore, 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 a very important issue from the viewpoint of environmental conservation. Although technologies required for purification have been developed (for example, adsorption treatment using activated carbon, decomposition treatment using light or heat, etc.), the current technology is not always practical in terms of cost and operability. I can't say.

On the other hand, in recent years, decomposition of volatile organic chlorine compounds such as TCE which are stable in the environment by microorganisms has been reported, and the advantages are as follows: 1) Selection of microorganisms to be used in biodegradation treatment using microorganisms. And that it can decompose volatile organic chlorine compounds to harmless substances, 2) basically, no special chemicals are required, 3) labor and cost for maintenance can be reduced, and the like.

For example, TCE microorganisms include Welchia
alkenophila sero 5 (USP 4,877,736, ATCC 53570), We
lchia alkenophila sero 33 (USP 4,877,736, ATCC 535
71), Methylocystis sp. Strain M (Agric. Biol. Che
m., 53, 2903 (1989), Biosci. Biotech. Biochem., 5
6, 486 (1992), 56,736 (1992)), Methylosinus tric
hosprium OB3b (Am. Chem. Soc. Natl. Meet. Dev. Env
iron. Microbiol., 29,365 (1989), Appl. Environ. Mi
crobiol., 55, 3155 (1989)), Appl. Biochem. Biotechn.
ol., 28, 877 (1991), JP-A-02-92274, JP-A-0-92274.
3-292970), Methylomonas sp. MM2 (Appl. E
nviron. Microbiol., 57, 236 (1991)), Alcaligenes d
enitrificans ssp.xylosoxidans JE75 (Arch.Microbi
ol., 154, 410 (1990)), Alcaligenes eutrophus JMP13
4 (Appl.Environ.Microbiol., 56, 1179 (1990)), Alca
ligenes eutrophus FERM-13761 (JP-A-07-123976), Pseudomonas aeruginosa JI104 (JP-A-07-2368)
No. 95), Mycobacterium vaccae JOB5 (J. Gen. Mic
robiol., 82, 163 (1974)), Appl. Environ. Microbio
l., 54, 2960 (1989)), ATCC 29678), Pseudomonas put
ida BH (Sewer Association, 24, 27 (1987)), Pseudomona
s sp.Strain G4 (Appl.Environ.Microbiol., 52, 38
3 (1986)), 53, 949 (1987), 54, 951 (1988), 5
6, 1279 (1990), 57, 1935 (1991), USP 4,925,802,
ATCC 53617, which was originally classified as Pseudomonas cepacia, but was changed to Pseudomonas sp.), Pseud
omonas mendocina KR-1 (Bio / Technol., 7, 282 (198
9)), Pseudomonas putida F1 (Appl. Environ. Microb
iol., 54, 1703 (1988), 54, 2578 (1988)), Pseudo
monas fluorescens PFL12 (Appl. Environ. Microbio
l., 54, 2578 (1988)), Pseudomonas putida KWI-9
(JP-A-06-70753), Pseudomonas cepaciaKK01
(Japanese Unexamined Patent Publication No. Hei 06-227769), Nitrosomonas europaea
(Appl. Environ. Microbiol., 56, 1169 (1990)), La
ctobacillus vaginalis sp. Nov (Int. J.Syst. Bacte
riol., 39, 368 (1989), ATCC 49540), Nocardia cora
llina B-276 (JP 08-70881 A, FERM BP-5124, A
TCC 31338).

[0006]

However, when these microorganisms are used for actual environmental purification treatment, a problem is the continuity of the decomposition of volatile organic chlorine compounds such as TCE. For example, in an environmental purification treatment using phenol, toluene, methane, or the like as an inducer, the depletion of these inducers directly leads to the termination of the decomposition of volatile organic chlorine compounds such as TCE, so that continuous induction of the inducer is required. Supply is essential. However, on the other hand, when an inducing substance is present, the affinity of a volatile organic chlorine compound such as TCE, which is the target substance for decomposing and purifying, is considerably lower than that of the inducing substance. It is a conflicting task to achieve.
Furthermore, in an actual treatment site, it is often difficult to precisely control the concentration of the inducer, and there is an essential problem regarding the stability and reproducibility of the purification treatment.

[0007] As described above, in actual environmental purification using microorganisms, continuous expression of decomposition activity using an inducer and further increase in efficiency of decomposition purification are incompatible difficult problems. This is a major issue for the practical use of environmental purification treatment using methane.

Therefore, at present, a plasmid incorporating a DNA fragment containing a gene region encoding oxygenase or hydroxylase, which is a TCE-degrading enzyme, is introduced into a host bacterium, and the plasmid is constructed with a harmless inducer or in the absence of an inducer. Attempts have been made to express TCE-degrading activity. Such a DN
Pseudomonas mendocina KR-1 (Japanese Unexamined Patent Publication No. 2-503866), Pseudomonas putida KWI-9 (Japanese Unexamined Patent Publication No. 6-105691) and Pseudomonas putida BH (research on groundwater / soil pollution and its prevention measures) 3rd Lecture Meeting, 213
(1994)). In addition, Shields et al. Have obtained a mutant of Pseudomonas sp. G4 strain which does not require an inducer (in this case, phenol or toluene) and has TCE resolution by a technique using a transposon (Appl. Environ. Microbiol. , 58, 3977 (199
2), and WO 92/19738).

Furthermore, in Japanese Patent Application Laid-Open No. Hei 8-294487, a strain JM1 (FERM BP) capable of decomposing volatile organochlorine compounds and aromatic compounds without the need for an inducer by performing mutation with nitrosoguanidine.
−5352) has been reported.

[0010] On the other hand, during pre-culture, TC
Attempts have been made to develop E resolution and administer it to restorative sites as resting cells (Environ. Sci. Te.
chnol., 30, 1982 (1996)).

Actually, in the purification treatment that does not require these inducers, it is easier to control the purification treatment than in the case where the above-described inducers are used, and it has been reported that the purification efficiency also increases. However, with respect to the continuity of the decomposing action, the control of the growth of microorganisms is a major issue, and when quiescent cells are used, the amount of TCE decomposition or the continuation time of decomposition that can be degraded by the input quiescent cells. The problem is that it is impossible to decompose TCE beyond its capacity.

An object of the present invention is to effectively decompose and repair such aromatic compounds and / or organochlorine compounds contained as pollutants in the environment by contacting them with microorganisms. An object of the present invention is to provide a decomposition method capable of ensuring the continuity of the operation, and a method for restoring a polluted environment using the decomposition method.

[0013]

According to the decomposition method of the present invention, a microorganism having a decomposing action of a compound to be decomposed is contacted with at least one compound to be decomposed selected from a volatile organic chlorine compound and an aromatic compound. In a decomposition method having a decomposition step of decomposing the microorganism, a factor affecting the decomposition action of the microorganism is applied to the microorganism at least once during the decomposition step.

An environmental remediation method using the decomposition method of the present invention is a method for resolving a pollutant, which is at least one selected from volatile organic chlorine compounds and aromatic compounds contained in a polluted environment, by decomposing the pollutant. In an environmental remediation method having a decomposition step of decomposing the microorganism by contacting the microorganism, a factor affecting the decomposition action of the microorganism is applied to the microorganism at least once during the decomposition step. .

According to the method of the present invention, microorganisms having a decomposing action of these compounds are brought into contact with a contaminated environment such as soil contaminated with aromatic compounds and / or organochlorine compounds to decompose the microorganisms. In purifying and restoring the environment, the growth of the microorganism can be achieved, for example, by providing the substrate and / or growth factors required for the growth of the microorganism multiple times, or by changing the already provided growth conditions. And / or regrowth, thereby optimizing and continuing the decomposition activity of the target microorganism, and effectively and continuously repairing the environment contaminated by aromatic compounds and / or organochlorine compounds. It becomes possible.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In general, the growth of microorganisms requires a carbon source, a nitrogen source, a phosphate source, and many other substances essential for growth. Deficiency in any one of these nutrient substrates, even if all others are present in sufficient amounts, will reduce the maximum number of bacteria reached. Such nutrient substrates that are rate-limiting for growth are called restriction substrates. This is why the maximum number of bacteria that can be reached differs depending on the type of carbon source in the actual culture of microorganisms. For example, when Escherichia coli is cultured using ammonium salt as a nitrogen source, if the carbon source is citrate, pyruvate, and glucose, the maximum number of bacteria that can be reached increases in this order, so that the carbon source is the limiting substrate. I understand. Also, when nitrate is used as the nitrogen source, it can be seen that the maximum number of bacteria reached does not depend on the carbon source, that is, the nitrogen source is the limiting substrate.

When the microorganism is cultured at a temperature lower than the optimum culture temperature, the maximum number of bacteria that can be reached is increased by shifting the culture temperature to the optimum culture temperature. Others
Similar effects may be observed with the addition of mineral components and the like, and it is clear that these substrates or factors are limiting.

Here, the enzyme for decomposing volatile organic chlorine compounds such as TCE is strongly expressed in the middle to late stages of logarithmic growth.
At steady state, the production of new enzymes is insignificant. In addition, these enzymes are irreversibly damaged by decomposing volatile organic chlorine compounds such as TCE, so that the amount of generated enzyme determines the decomposable amount. That is, degradation in the steady state only consumes the enzyme stock that has already been expressed and generated, and if the stock is exhausted, the degradation stops.

In order to continue the decomposition in such a situation, it is necessary to additionally express and generate a decomposing enzyme in the region where the decomposition treatment is being performed. Under normal circumstances, it is necessary to restart the growth of the microorganism to continue the degradation. In addition, when considering efficient decomposition, it is necessary to control the growth suitable for the concentration of volatile organic chlorine compounds such as TCE. For example, when the concentration of the decomposition target is low, it is more advantageous for the continuation of decomposition to continuously grow the number of bacteria corresponding to the amount of the decomposition target than to start a large number of bacteria from the beginning. It is. In addition, when the target has a higher concentration, it may be more efficient to start a larger number of bacteria.

Further, it is extremely important from the viewpoint of cost to provide a technique capable of controlling the growth of microorganisms and continuing / optimizing the decomposition in accordance with the situation at the site. More specifically, when a culture solution is provided together with the injection of microorganisms into the site, first, a certain carbon source A and a nitrogen source A are used to maximize the growth of microorganisms in the nutrient environment, and the maximum To perform the minimum disassembly. Here, in order to continue the decomposition further, regrowth is essential as described above, and it is most effective to administer a factor which is a limiting substrate in the nutritional environment. Here, regrowth is possible by applying not only a substrate such as a carbon source, a nitrogen source, and a mineral component, but also a physical or chemical factor that releases restrictions.

Variations in the selection and administration of these limiting factors are as follows: 1) Resupply of carbon source A Effective up to the concentration of carbon source A where other factors are limiting factors 2) Supply of carbon source B New maximum growth Setting of limit 3) Resupply of nitrogen source A Effective up to the concentration of nitrogen source A where other factors are limiting factors 4) Supply of nitrogen source B Setting of new maximum growth limit 5) Supply of mineral component A Other factors Is effective up to the concentration of the mineral component A, which is a limiting factor. 6) Heating of the treatment position 7) Supply of oxygen 8) Adjustment of pH 9) Physical disturbance and the like, or a combination thereof.

As described above, the growth of microorganisms is generally defined by some kind of restriction substrate and the like, and there are many variations as described above as means for gradually releasing the definition by these restriction substrates and the like.
Of these, a method of contacting a microorganism with a microorganism in a plurality of times is disclosed in Japanese Patent Application Laid-Open No. 9-10794, but no other variations are mentioned.

The microorganisms that can be used in the present invention include:
A microorganism capable of decomposing an aromatic compound and / or an organic chlorine compound, preferably a microorganism expressing oxygenase can be used. Such microorganisms include, for example, genus Pseudomonas, genus Acinetocbacter, and Alcaligene
s) Genus, Vibrio, Nocardi
a) Genus, Bacillus genus, Lactobacillus (Lac
tobacillus), Achromobacter
Genus, Arthrobacter, Micrococcus, Mycobac
terium), Methylosinus (Methylosinus), Methylomonas (Methylomonas), Berchia (Welchia),
Methylocystis genus, Nitrosomonas genus, Saccharomyce
s), Candida, Torulopsis
psis), microorganisms belonging to the genus Moraxella, the genus Burkholderia, the genus Ralstonia and the genus Comamonas.

Among these, more preferred are:
For example, J1 strain, JM1 strain, Bulkholderia cepacia KK01 strain, Burkholderia cepacia KK01 strain, Pseudomonas sp. TL1 strain (Pseudomonas sp.
TL1), Pseudomonas alkaligenes KB2 strain (P
seudomonas alcaligenes KB2), Alcaligenes sp. TL2 strain (Alcaligenes sp. TL2), Vibrio sp. KB1 strain (Vibrio sp. KB1) and the like.

All of these strains have been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3-3 Higashi, Tsukuba, Ibaraki (postal code 305), and their deposit numbers (BP: under the Budapest Treaty) International deposit) and the date of deposit are as follows. J1 strain: FERM BP-5102: May 25, 1994
Japan JM1 strain: FERM BP-5352: January 1, 1995
0 KK01 strain: FERM BP-4235: March 11, 1992 TL1 strain: FERM P-14726: January 1, 1995
0 TL2 strain: FERM P-14642: November 15, 1994 KB1 strain: FERM P-14643: November 15, 1994 KB2 strain: FERM BP-5354: November 15, 1994

The strains J1 and JM1 were initially deposited.
Corynebacterium belonging to the genus Corynebacterium
msp. J1 "and" Corynebacteriu "
m sp. JM1 ", but it was confirmed by the subsequent examination that the strain did not belong to the genus Corynebacterium. Therefore, the indication of identification was changed to" J1 strain "and" JM1 strain ".
It has been changed. The JM1 strain is obtained by mutating a J1 strain capable of decomposing an aromatic compound or a chlorinated ethylene compound by using an inducer by a mutagenesis using a mutagen, and using an organic compound without using an inducer. Is a mutant strain capable of decomposing.

In the case of using an oxygenase-producing microorganism, one or more factors selected from, for example, temperature shift, supply of nutrients, supply of oxygen, stirring, and adjustment of pH are preferable as factors affecting the decomposition action. Can be cited. Examples of the nitrogen source include a nitrate and an ammonium salt as shown in Examples, but any substance may be used as long as it is suitable for the purpose. Similarly, the mineral component includes a magnesium salt, but any substance may be used as long as it is suitable for the purpose. Further, it is more effective to perform at least one of the above-described temperature shift, supply of a nutrient source, supply of oxygen, stirring, and adjustment of pH when the stationary phase is reached.

As a medium used for obtaining a culture of a microorganism used in the present invention, a medium capable of growing the microorganism used can be used as appropriate. For example, it is also possible to culture on an inorganic salt medium such as M9 medium with an appropriate carbon source added. The composition of the M9 medium (in 1 liter of the 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 (pH 7.0)

The setting of conditions such as aeration and agitation in the cultivation of the microorganism can be selected according to the microorganism to be cultured, and may be liquid culture or solid culture. Culture temperature is 30 ° C
Before and after is desirable.

The decomposition method according to the present invention comprises a method for decomposing at least one selected from aromatic compounds and volatile organic chlorine compounds contained in various media and environments such as aqueous media, aqueous environments, soil, air and gas. It can be applied to decomposition processing of target compounds (contaminants). In the method of the present invention, the contact between the compound to be degraded and a microorganism that degrades the compound is performed by introducing a microorganism into a region where the compound to be degraded is present, or by introducing a compound to be degraded into a region holding the microorganism. Can be done by The region in which the microorganism is brought into contact with the decomposition target compound is adjusted so as to satisfy the conditions under which the microorganism can exhibit the decomposition activity, and these contacts can be performed 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 by immobilization on a suitable carrier. The object to be treated such as waste liquid, soil, gas phase, etc. may be subjected to various treatments as necessary.

The decomposition treatment of the compound to be decomposed in the aqueous medium in the present invention can be carried out by bringing the compound to be decomposed into contact with a microorganism in the aqueous medium. The main use forms are described below, but the present invention is not limited to these forms, and can be used for purification treatment (environmental restoration) by decomposing a decomposition target compound (contaminant) in any aqueous medium.

For example, as the simplest method, there is a method in which microorganisms are directly introduced into an aqueous medium contaminated with a compound to be decomposed. In this case, the pH, salt concentration, temperature, concentration of contaminants, etc. of the aqueous medium may be optimized depending on the type of microorganism.

As another application form, there is a form in which a culture tank is provided, microorganisms are cultured, and an aqueous medium contaminated with the compound to be decomposed is introduced into the culture tank at a predetermined flow rate to be decomposed. The introduction and drainage of the aqueous medium may be carried out continuously, but it is also possible to carry out the treatment intermittently or batchwise according to the treatment capacity. Such control may be optimized by performing system control in accordance with the concentration of the decomposition target compound.

Further, there is a form in which microorganisms are adhered to a carrier, for example, soil particles and the like, filled in a reaction layer, and a contaminated aqueous medium containing a compound to be decomposed is introduced into the reaction tank to perform a decomposition treatment. In this case, the carrier used is not limited to soil particles, and any carrier can be used. However, a carrier that has excellent ability to retain microorganisms and does not impair air permeability is more desirable. For example, as a material that provides a space for living microorganisms, various carriers for immobilizing microorganisms, which are widely used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment systems, and the like, can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, inorganic particulate carriers such as anthracite,
Gel carrier such as starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid, polyacrylamide, carrageenan, agarose, gelatin, ion-exchangeable cellulose, ion-exchange resin, cellulose derivative, glutaraldehyde, polyacrylic acid, polyurethane, polyester, etc. Is mentioned. In addition, cotton,
Cellulose-based materials such as hemp and paper, and lignin-based materials such as wood flour and bark can also be used.

The decomposition treatment of the compound to be decomposed in the soil according to the present invention can be carried out by contacting the microorganism to be decomposed with the compound present in the soil with a microorganism. The main use forms are described below, but the present method is not limited to these forms, and can be used for purification treatment (environmental restoration) by decomposition of any decomposition target compound in soil.

For example, the simplest method is to introduce microorganisms directly into soil contaminated by the compound to be decomposed. As a method of introduction, not only a method of spraying on the soil surface but also a method of introducing from a well inserted into the ground in the case of treatment in a relatively deep stratum. Furthermore, when pressure is applied by air, water, or the like, the microorganisms spread over a wide area, which is more effective. In this case, various conditions in the soil may be adjusted so as to be suitable for the microorganism.

Further, there is a form in which microorganisms are adhered to a carrier, filled in a reaction tank, and the reaction tank is introduced into the aquifer of soil contaminated with the compound to be decomposed, mainly into an aquifer to perform a decomposition treatment. The form of the reaction tank is fence-like or film-like,
Those that can cover a wide range in soil are desirable. In this case, any carrier can be used,
It is more desirable to use one that has an excellent ability to retain microorganisms and does not impair air permeability. For example, as a material that provides a space for living microorganisms, various carriers for immobilizing microorganisms, which are widely used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment systems, and the like, can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, inorganic particulate carriers such as kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid , Polyacrylamide,
Gel-like carriers such as carrageenan, agarose and gelatin,
Examples include ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane, polyester, and the like. As natural products, cellulosic materials such as cotton, hemp, paper, wood flour,
Lignin-based materials such as bark are also available.

The decomposition treatment of the compound to be decomposed in the gas phase in the present invention can be carried out by bringing the compound to be decomposed present in the gas phase into contact with a microorganism. The main use modes are described below, but the present method is not limited to these modes, and can be used for purification treatment (environmental restoration) by decomposition of any decomposition target compound in the gas phase.

For example, there is a form in which a microorganism is cultured by providing a culture tank, and a gas contaminated with the compound to be decomposed is introduced into the culture tank at a predetermined flow rate to decompose the microorganism. There is no particular limitation on the gas introduction method, but it is more preferable that the introduction of the gas agitates the culture solution and promotes aeration. The introduction and exhaust of the gas may be performed continuously, but it is also possible to perform the processing intermittently or batchwise according to the processing capacity. Such control may be optimized by controlling the system according to the concentration of the volatile organic chlorine compound.

As another mode of use, a microorganism is used as a carrier,
For example, it is attached to soil particles, etc.
There is a form in which a contaminated gas containing a compound to be decomposed is introduced into the reaction tank to perform a decomposition treatment. In this case, the carrier used is
Anything is available, not limited to soil particles,
It is more desirable to use one that has an excellent ability to retain microorganisms and does not impair air permeability. For example, as a material that provides a space for living microorganisms, various carriers for immobilizing microorganisms that have been widely used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment systems, and the like can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, inorganic particulate carriers such as kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid , Polyacrylamide,
Gel-like carriers such as carrageenan, agarose and gelatin,
Examples include ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane, polyester, and the like. As natural products, cellulosic materials such as cotton, hemp, paper, wood flour,
Lignin-based materials such as bark are also available.

Further, as a material which can serve both as a microorganism holding and as a nutrient supply, there can be mentioned many examples of composts used in agriculture, forestry and fisheries. That is, straws and sawdust of cereals such as wheat straw, dried products derived from plants such as rice bran, snow cabbage, and sugarcane marc, and marine wastes such as crab and shrimp shells can be used.

To purify the pollutant gas, microorganisms may be introduced after being filled in advance with a substance to be a carrier. In order to perform the decomposition reaction more efficiently, the above-described nutrients, water content, oxygen concentration, and the like may be maintained at desired conditions. In addition, the ratio of the amount of water to the carrier in the reaction tank may be appropriately selected depending on the amount and concentration of the gas to be treated, based on the growth and air permeability of the microorganism. Care should be taken to facilitate contact with, for example,
Columns, tubes, tanks, and boxes can be used. Furthermore, a unit having such a shape may be unitized with an exhaust duct, a filter, or the like, or some units may be connected in accordance with the capacity.

In some cases, the contaminant gas is initially adsorbed on the carrier material, and there are rare cases where the effect of using microorganisms is not well observed. However, after a certain period of time, the contaminants adhering to the carrier material are decomposed, It is said that the resorption of the contaminant on the surface of the decomposed material regenerates the adsorptivity to the carrier material. In this way, a constant decomposition can always be expected without saturating the decontamination ability.

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

[0046]

EXAMPLES Examples will be shown below, but these examples do not limit the scope of the present invention in any way.

Example 1 [Effect of Temperature Increase on Decomposition of Aromatic Compounds by JM1 Strain (Liquid Culture System)] A 500 ml Sakaguchi flask was prepared by removing a colony of the J1 strain on an M9 agar medium containing 0.2% of yeast extract. 200 ml of M9 medium containing 0.2% yeast extract
And cultured with shaking at 25 ° C. for 24 hours.

Here, the medium prepared in the same manner as above
Phenol, o-cresol, m
-200 ppm each of cresol, p-cresol and toluene (100 ppm of toluene only),
0.1 ml of the culture solution of the JM1 strain was inoculated, and cultured with shaking at 15 ° C. for 72 hours. Here, phenol and cresol are analyzed by liquid chromatography (spectrophotometer).
Toluene was measured by gas chromatography (FID detector) to determine the residual ratio with respect to the initial concentration.

After the measurement, each of the culture solutions was divided into two portions.
The temperature was further increased to 48 ° C. for another 48 hours with shaking culture, and the respective residual rates were determined as described above. Table 1 shows the results.

[0050]

[Table 1]

Example 2 [Effect of Temperature Increase on Decomposition of Volatile Organic Chlorine Compounds by J1 Strain (Liquid Culture System)] Phenol was added to the medium used in Example 1 as a decomposition inducer to a final concentration of 200 ppm. 10 ml of this culture was inoculated with 0.1 ml of the bacterial solution of the pre-cultured J1 strain and added to a 27.5 ml vial. This was sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TCE), cis-1,
2-dichloroethylene (cis-1,2-DCE), t
trans-1,2-dichloroethylene (trans-
After 1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) were added at 10 ppm, shaking culture was performed at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

The amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector). As a control, the amount of volatile organic chlorine compounds was measured in the same experimental system but without the J1 strain. The residual rate was determined with respect to the amount of the volatile organic chlorine compound as a control. 2 after measurement
A series of the samples was shake-cultured for an additional 48 hours, with the culture temperature maintained at 15 ° C. and the culture temperature maintained at 5 ° C. for the other, and the residual ratio of each was determined as described above. Table 2 shows the results.

[0053]

[Table 2]

Example 3 [Effect of Temperature Increase on Decomposition of Aromatic Compound by KK01 Strain (Liquid Culture System)] Sodium Glutamate 0.2%
Colonies of the KK01 strain on M9 agar medium containing
200 ml of M9 medium containing 0.2% of sodium glutamate in a 0 ml Sakaguchi flask was inoculated and cultured with shaking at 30 ° C. for 24 hours.

Here, the medium prepared in the same manner as above
Phenol, o-cresol, m
-200 ppm each of cresol, p-cresol and toluene (100 ppm of toluene only),
0.1 ml of a culture solution of the KK01 strain was inoculated and incubated at 15 ° C for 72 hours.
Shaking culture was performed for hours. Here, phenol and cresol were measured by liquid chromatography (spectrophotometric detector), and toluene was measured by gas chromatography (FID detector), and the residual ratio to the initial concentration was determined. After the measurement, each culture solution was bisected. One was kept at a culture temperature of 15 ° C., the other was kept at a culture temperature of 30 ° C., and further shake-cultured for an additional 48 hours, and the remaining ratio of each was determined as described above. Was. Table 3 shows the results.

[0056]

[Table 3]

Example 4 [Effect of Temperature Rise on Decomposition of Volatile Organochlorine Compounds by Strain KK01 (Liquid Culture System)] Phenol was added to the medium used in Example 3 as a decomposition inducer to a final concentration of 200 ppm. K obtained by pre-culturing 10 ml of this culture solution
0.1 ml of the bacterial solution of the K01 strain was inoculated and added to a 27.5 ml vial. This is sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TCE), ci
s-1,2-dichloroethylene (cis-1,2-DC
E), trans-1,2-dichloroethylene (tra
After adding ns-1,1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) at 10 ppm, shaking culture was performed at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the KK01 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.
After the measurement, duplicate samples were shake-cultured for an additional 48 hours, with the culture temperature kept at 15 ° C. and the culture temperature kept at 30 ° C. for the other, and the residual ratio of each was determined as described above. Table 4 shows the results.

[0059]

[Table 4]

Example 5 [Effect of Temperature Increase on Decomposition of Aromatic Compound by JM1 Strain (Liquid Culture System)] A 500 ml Sakaguchi colony of the JM1 strain colony on M9 agar medium containing 2.0% sodium malate was used. The flask was inoculated into 200 ml of M9 medium containing 2.0% sodium malate in a flask, and cultured at 15 ° C. for 72 hours with shaking.

Here, the medium prepared in the same manner as above
Phenol, o-cresol, m
-200 ppm each of cresol, p-cresol and toluene (100 ppm of toluene only),
0.1 ml of the culture solution of the JM1 strain was inoculated, and cultured with shaking at 15 ° C. for 72 hours. Here, phenol and cresol are analyzed by liquid chromatography (spectrophotometer).
Toluene was measured by gas chromatography (FID detector) to determine the residual ratio with respect to the initial concentration.

After the measurement, each of the culture solutions was divided into two portions.
The temperature was further increased to 48 ° C. for another 48 hours with shaking culture, and the respective residual rates were determined as described above. Table 5 shows the results.

[0063]

[Table 5]

Example 6 [Effect of Temperature Rise on Decomposition of Volatile Organic Chlorine Compounds by JM1 Strain (Liquid Culture System)] Culture solution 1 used in Example 5
0 ml was inoculated with 0.1 ml of the bacterial solution of the precultured JM1 strain, and added to a 27.5 ml vial. This was sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-1,2-DCE), trans-1,2-dichloroethylene (trans-1,2-DCE), And 1,1-dichloroethylene (1,1-DCE) were added at 10 ppm each, followed by shaking culture at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the JM1 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined. After the measurement, the stoppers of all the samples were opened, and the remaining components of the decomposition target compound were removed. This was sealed again with a butyl rubber stopper and an aluminum cap, and re-added so that each of the above-mentioned compounds to be decomposed was 10 ppm each. Shaking culture was performed for 48 hours, and the respective residual rates were determined as described above. Table 6 shows the results.

[0066]

[Table 6]

Example 7 [Effect of Temperature Rise on Decomposition of Aromatic Compounds by J1 Strain (Sand System)] The resolution was compared in a system in which 50 g of Sawara sand was added to a 67.5 ml vial bottle. First, 0.2
5 ml of M9 medium containing 5% yeast extract was inoculated with 0.1 ml of the precultured J1 strain. Here, preculture was performed using M9 medium containing 0.2% yeast extract,
Shaking culture was performed at 24 ° C. for 24 hours.

Further, phenol, o-cresol, m-cresol, p-cresol, and toluene were added as the compounds to be decomposed so as to have a final concentration of 200 ppm (toluene alone: 100 ppm). Static culture was performed at 72 ° C for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed. Phenol and cresol were measured by liquid chromatography (spectrophotometric detector), and toluene was measured by gas chromatography (FID detector), and the residual ratio to the initial concentration was determined.

After the measurement, duplicate samples were subjected to static cultivation for an additional 48 hours while maintaining the culture temperature at 15 ° C. and the other at the culture temperature of 25 ° C. I asked. Table 7 shows the results.

[0070]

[Table 7]

Example 8 [Effect of Temperature Rise on Decomposition of Volatile Organic Chlorine Compounds by J1 Strain (Sand System)] In the same system as in Example 7, phenol was used as a decomposition-inducing substance at a final concentration of 200 ppm, and bacteria were inoculated. The obtained culture solution was added to the sand in the vial bottle, and then sealed with a butyl rubber stopper and an aluminum cap. In addition to this, trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-1,2-DCE), transns-1,
2-dichloroethylene (trans-1,2-DC
E), and 1,1-dichloroethylene (1,1-DC
E) to give 10 ppm each,
Static culture was performed for 2 hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the J1 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.

After the measurement, duplicate samples were subjected to static cultivation for an additional 48 hours while maintaining the culture temperature at 15 ° C. and the other at the culture temperature of 25 ° C. I asked. Table 8 shows the results.

[0074]

[Table 8]

Example 9 [Effect of Temperature Rise on Decomposition of Aromatic Compounds by KK01 Strain (Sand System)]
The resolution was compared in a system to which 0 g was added. First, 0.2%
5 ml of M9 medium containing sodium glutamate was prepared, and 0.1 ml of the pre-cultured KK01 strain was inoculated thereto. Here, the preculture was performed by shaking culture at 30 ° C. for 24 hours using an M9 medium containing 0.2% sodium glutamate.

Further, phenol, o-cresol, m-cresol, p-cresol and toluene were added as compounds to be decomposed to a final concentration of 200 ppm (toluene alone: 100 ppm). Static culture was performed at 72 ° C for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed. Phenol and cresol were measured by liquid chromatography (spectrophotometric detector), and toluene was measured by gas chromatography (FID detector), and the residual ratio to the initial concentration was determined.

After the measurement, duplicate samples were subjected to static cultivation for an additional 48 hours while maintaining the culture temperature at 15 ° C. and the other at the culture temperature of 30 ° C. I asked. Table 9 shows the results.

[0078]

[Table 9]

Example 10 [Effect of Temperature Increase on Decomposition of Volatile Organic Chlorine Compounds by Strain KK01 (Sand System)] In the same system as in Example 9, phenol was used as a decomposition-inducing substance at a final concentration of 200 ppm, and bacteria were inoculated. The obtained culture solution was added to the sand in the vial bottle, and then sealed with a butyl rubber stopper and an aluminum cap. Trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-1,2-DCE), trans
-1,2-dichloroethylene (trans-1,2-D
CE), and 1,1-dichloroethylene (1,1-D
CE) was added at 10 ppm each, and static culture was performed at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector). As a control, the amount of volatile organic chlorine compounds in the same experimental system but without the KK01 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.

After the measurement, duplicate samples were subjected to static cultivation for an additional 48 hours while maintaining the culture temperature at 15 ° C. and the other at the culture temperature of 30 ° C. I asked. Table 10 shows the results.

[0082]

[Table 10]

Example 11 [Effect of Temperature Rise on Decomposition of Aromatic Compound by JM1 Strain (Sand System)] The resolution was compared in a system in which 50 g of Sahara sand was added to a 67.5 ml vial. First, 2%
5 ml of M9 medium containing sodium malate
This was inoculated with 0.1 ml of the precultured JM1 strain. Here, the pre-culture was performed by shaking culture at 15 ° C. for 72 hours using an M9 medium containing 2% sodium malate.

Further, phenol, o-cresol, m-cresol, p-cresol and toluene were added as compounds to be decomposed to a final concentration of 200 ppm (toluene alone: 100 ppm). Static culture was performed at 72 ° C for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed. Phenol and cresol were measured by liquid chromatography (spectrophotometric detector), and toluene was measured by gas chromatography (FID detector), and the residual ratio to the initial concentration was determined.

After the measurement, duplicate samples were subjected to static cultivation for an additional 48 hours while maintaining the culturing temperature at 15 ° C. and the other at the culturing temperature of 25 ° C. I asked. Table 11 shows the results.

[0086]

[Table 11]

Example 12 [Effect of Temperature Rise on Decomposition of Volatile Organic Chlorine Compounds by JM1 Strain (Sand System)] In the same system as in Example 11, a culture solution inoculated with bacteria was added to sand in a vial bottle. After that, it was sealed with a butyl rubber stopper and an aluminum cap. Trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-1,2-DCE), trans1,2-
Dichloroethylene (trans-1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) were added at 10 ppm each, and the mixture was allowed to stand at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the JM1 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.

After the measurement, the stoppers of all the samples were opened to remove the remaining compounds to be decomposed. This was sealed again with a butyl rubber stopper and an aluminum cap, and re-added so that each of the above-mentioned compounds to be decomposed was 10 ppm each. Then, the culture temperature was kept at 15 ° C for a series, and the culture temperature was set at 25 ° C for another series. Static culture was performed for 48 hours, and the residual ratio of each was determined as described above. Table 12 shows the results.

[0090]

[Table 12]

Example 13 [Effect of Mineral Component on Decomposition of Volatile Organic Chlorine Compounds by J1 Strain (Liquid Culture System)] Phenol was added to the medium used in Example 1 as a decomposition inducer to a final concentration of 200 ppm. The preculture was performed on 10 ml of this culture.
0.1 ml of the bacterial solution of one strain was inoculated and added to a 27.5 ml vial. This was sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TCE), cis-
1,2-dichloroethylene (cis-1,2-DC
E), trans-1,2-dichloroethylene (tra
ns-1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) were added at 10 ppm each, followed by shaking culture at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

The amount of these volatile organochlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organochlorine compounds was measured in the same experimental system but without the J1 strain. The residual rate was determined with respect to the amount of the volatile organic chlorine compound as a control.

After the measurement, magnesium sulfate was added to one of the duplicate samples so that the final concentration was 10 mM. Additional 4 for these
Shaking culture was performed for 8 hours, and the residual ratio of each was determined as described above. Table 13 shows the results.

[0094]

[Table 13]

Example 14 [Effect of Mineral Component on Decomposition of Volatile Organic Chlorine Compounds by Strain KK01 (Liquid Culture System)] The medium used in Example 3 was supplemented with phenol as a decomposition inducer at a final concentration of 200 pp.
m, and 10 ml of this culture was inoculated with 0.1 ml of the bacterial culture of the pre-cultured KK01 strain, and 27.5 ml was added.
Added to vials. This is sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TCE),
cis-1,2-dichloroethylene (cis-1,2-
DCE), trans-1,2-dichloroethylene (t
trans-1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) were added at 10 ppm each, followed by shaking culture at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the KK01 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.

After the measurement, magnesium sulfate was added to the duplicate samples so that one of the samples was left as it was and the other of the samples had a final concentration of 10 mM. Additional 4 for these
Shaking culture was performed for 8 hours, and the residual ratio of each was determined as described above. Table 14 shows the results.

[0098]

[Table 14]

Example 15 [Effect of Mineral Component on Decomposition of Volatile Organic Chlorine Compounds by JM1 Strain (Liquid Culture System)] The bacterial solution of the JM1 strain obtained by pre-culturing 10 ml of the culture solution used in Example 5 was added. 1 ml
And added to a 27.5 ml vial. This was sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-1,2-DCE), trans-1,2
-Dichloroethylene (trans-1,2-DCE),
And 1,1-dichloroethylene (1,1-DCE) were added at 10 ppm each.
Shaking culture was performed for hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the JM1 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.

After the measurement, the stoppers of all the samples were opened to remove the remaining compounds to be decomposed. This was sealed again with a butyl rubber stopper and an aluminum cap, and re-added to the above-mentioned compound to be decomposed to 10 ppm each, and then magnesium sulfate was added to the series as it was and another series to a final concentration of 10 mM. These were further shake-cultured for 48 hours, and the residual ratio of each was determined as described above. Table 15 shows the results.

[0102]

[Table 15]

Example 16 [Effect of Nitrogen Source on Decomposition of Volatile Organic Chlorine Compounds by Strain J1 (Liquid Culture System)] In the medium used in Example 1, ammonium chloride in M9 medium was replaced with 0.5 g / l potassium nitrate. The test was performed in a system with exactly the same conditions except for the above. Phenol is used as a decomposition inducer at a final concentration of 200 pp.
Then, 10 ml of the culture was inoculated with 10 ml of the bacterial solution of the strain Л, which had been pre-cultured, and added to a 27.5 ml vial. This was sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TCE), cis-
1,2-dichloroethylene (cis-1,2-DC
E), trans-1,2-dichloroethylene (tra
ns-1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) were added to each at 10 ppm, followed by shaking culture at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

The amount of these volatile organochlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organochlorine compounds was measured in a similar experimental system without adding strain J1. The residual rate was determined with respect to the amount of the volatile organic chlorine compound as a control.

After the measurement, ammonium chloride was added to duplicate samples so that one of the samples was left as it was and the other had a final concentration of 0.5 g / l. These were further shake-cultured for 48 hours, and the residual ratio of each was determined as described above. Table 16 shows the results.

[0106]

[Table 16]

Example 17 [Effect of Nitrogen Source on Decomposition of Volatile Organic Chlorine Compounds by Strain KK011 (Liquid Culture System)] In the medium used in Example 3, ammonium chloride in M9 medium was replaced with 0.5 g / l potassium nitrate. The test was performed in a system with exactly the same conditions except for the above. Phenol is used as a decomposition inducer at a final concentration of 20.
0 ppm, and 10 ml of this culture solution was inoculated with 0.1 ml of the bacterial solution of the pre-cultured KK01 strain.
Added to 5 ml vial. This is sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TC
E), cis-1,2-dichloroethylene (cis-
1,2-DCE), trans-1,2-dichloroethylene (trans-1,2-DCE), and 1,1-
10p each of dichloroethylene (1,1-DCE)
pm, followed by shaking culture at 15 ° C for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the KK01 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.

After the measurement, ammonium chloride was added to duplicate samples so that one of the samples was left as it was and the other had a final concentration of 0.5 g / l. These were further shake-cultured for 48 hours, and the residual ratio of each was determined as described above. Table 17 shows the results.

[0110]

[Table 17]

Example 18 [Effect of Nitrogen Source on Decomposition of Volatile Organic Chlorine Compounds by JM1 Strain (Liquid Culture System)] In the medium used in Example 5, ammonium chloride in M9 medium was replaced with 0.5 g / l potassium nitrate. The test was performed in a system with exactly the same conditions except for the above. Preliminary culture of 10 ml of the culture solution used in Example 5
1 was inoculated and added to a 27.5 ml vial. This was sealed with a butyl rubber stopper and an aluminum cap, and trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-1,2-DCE), trans-1,
2-dichloroethylene (trans-1,2-DC
E), and 1,1-dichloroethylene (1,1-DC
E) was added at 10 ppm each, followed by 15 ° C.
For 72 hours with shaking.

Here, each compound to be decomposed was 2
The test was performed in series.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the JM1 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.

After the measurement, the stoppers of all the samples were opened to remove the remaining compounds to be decomposed. This was sealed again with a butyl rubber stopper and an aluminum cap, and re-added to 10 ppm each of the above-mentioned decomposition target compound.
Ammonium chloride was added such that These were further shake-cultured for 48 hours, and the residual ratio of each was determined as described above. The results are shown in Table 18.

[0115]

[Table 18]

Example 19 [Effect of Stirring on Decomposition of Volatile Organochlorine Compounds by JM1 Strain (Sand System)] In the same system as in Example 11, a culture solution inoculated with bacteria was added to sand in a vial bottle. Then, it was sealed with a butyl rubber stopper and an aluminum cap. Trichlorethylene (TCE), c
is-1,2-dichloroethylene (cis-1,2-D
CE), trans-1,2-dichloroethylene (tr
ans-1,2-DCE) and 1,1-dichloroethylene (1,1-DCE) were added at 10 ppm each, and the mixture was incubated at 15 ° C. for 72 hours. Here, the test was performed in duplicate for each compound to be decomposed.

Here, the amount of these volatile organic chlorine compounds was measured by gas chromatography (FID detector), and as a control, the amount of volatile organic chlorine compounds in the same experimental system but without the JM1 strain was added. Was also measured, and the residual ratio with respect to the amount of the volatile organic chlorine compound as a control was determined.

After the measurement, the stoppers of all the samples were opened to remove the remaining components to be decomposed. A series was stirred as it was, and another series was stirred with a spatula, sealed again with a butyl rubber stopper and an aluminum cap, and re-added to 10 ppm of each of the compounds to be decomposed, followed by further static culture for an additional 48 hours. The residual ratio of each was determined as described above. The results are shown in Table 19.

[0119]

[Table 19]

[0120]

According to the decomposition method of the present invention, at least one of volatile organic chlorine compounds and aromatic compounds is selected.
More effective microbial degradation of the species is possible, and the degradation method of the present invention is applied to soil contaminated with such compounds,
By applying the present invention to the purification treatment of the environment such as water and gas, more effective environmental restoration treatment becomes possible.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shinya Furusaki 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Takeshi Nomoto 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon F term (reference) 4B065 AA12X AA41X AA55X AC20 BB02 BB03 BB12 BC02 BC03 BC06 BC08 CA54 4D040 DD01 DD11 DD31

Claims (56)

[Claims] 1. A decomposition method comprising a decomposition step of decomposing at least one compound selected from a volatile organic chlorine compound and an aromatic compound with a microorganism having a decomposing action of the compound to be decomposed. A decomposition method, wherein a factor affecting the decomposition action of the microorganism is applied to the microorganism at least once during the decomposition step. 2. The method according to claim 1, wherein the factor is a factor that affects the growth of the microorganism. 3. The method according to claim 1, wherein the microorganism is a microorganism that expresses oxygenase. 4. The method according to claim 1, wherein the factor is selected from the group consisting of temperature shift, nutrient supply, oxygen supply, stirring, and pH adjustment.
The decomposition method according to any one of claims 1 to 3, which is as described above. 5. The method according to claim 3, wherein the nutrient source is at least one selected from a substrate, a carbon source, a nitrogen source, and a mineral component that affect the growth of the microorganism. 6. The decomposition method according to claim 5, wherein the nitrogen source is at least one selected from ammonium salts and nitrates. 7. The method according to claim 1, wherein the mineral component is a magnesium salt,
The decomposition method according to claim 5, wherein the decomposition method is a ferrous salt or a molybdate. 8. The microorganism expressing the okigesinase may be a genus Pseudomonas, a genus Acinetobacter, or Alcaligen.
es), Vibrio, Nocardi
a) Genus, Bacillus genus, Lactobacillus (Lac
tobacillus), Achromobacter
Genus, Arthrobacter, Micrococcus, Mycobac
terium), Methylosinus (Methylosinus), Methylomonas (Methylomonas), Berchia (Welchia),
Methylocystis genus, Nitrosomonas genus, Saccharomyce
s), Candida, Torulopsis
The method according to any one of claims 3 to 7, which is at least one of microorganisms belonging to the genera Psis, Moraxella, Burkholderia, Ralstonia, and Comamonas. 9. The microorganism according to claim 1, wherein the microorganism is strain J1 (FERM BP-5).
The decomposition method according to any one of claims 3 to 7, which is (102). 10. The microorganism may be a JM1 strain (FREM BP).
-5352). 11. The method according to claim 11, wherein the microorganism is a Pseudomonas sp. TL1 strain (Pseudomonas sp.
14726). 12. The method according to claim 8, wherein the microorganism is Pseudomonas alcaligenes. 13. The method according to claim 11, wherein the microorganism is Pseudomonas alcaligenes KB2,
FERM P-14644). 14. The method according to claim 8, wherein the microorganism is Burkholderia cepacia. 15. The microorganism is Burkholderia cepacia KK01, F.
The decomposition method according to claim 14, which is ERM BP-4235). 16. The microorganism may be an Alcaligenes sp. TL2 strain (Alcaligenes sp. TL2, FERMP).
The decomposition method according to claim 8, wherein the decomposition method is -14642). 17. The microorganism, wherein the microorganism is Vibrio species K
B1 strain (Vibrio sp. KB1, FERM P-146)
43. The decomposition method according to claim 8, which is 43). 18. The compound to be decomposed is phenol,
18. One or more selected from o-cresol, m-cresol, p-cresol, toluene, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene and 1,1-dichloroethylene. The decomposition method according to any one of the above. 19. The decomposition method according to claim 1, wherein the decomposition of the decomposition target compound is performed in a medium. 20. The decomposition method according to claim 19, wherein the medium is a medium containing water. 21. The medium according to claim 19, wherein said medium is an aqueous medium.
The decomposition method described in 1. 22. The decomposition method according to claim 19, wherein the decomposition target compound is a volatile compound, and the medium is a gas. 23. The method according to claim 1, wherein the microorganism is carried on a carrier. 24. A decomposing step of decomposing a pollutant consisting of at least one selected from a volatile organic chlorine compound and an aromatic compound contained in a polluted environment by contacting the pollutant with a microorganism capable of decomposing the pollutant. An environmental remediation method comprising: applying a factor that affects the decomposing action of the microorganism to the microorganism at least once during the decomposition step. 25. The method of claim 24, wherein the contaminated environment is a contaminated aqueous environment. 26. The environmental restoration method according to claim 25, wherein the contaminated water collected from the contaminated aqueous environment is brought into contact with the microorganism. 27. The environmental restoration method according to claim 26, wherein the microorganism is carried on a carrier. 28. The method according to claim 28, wherein the carrier carrying the microorganism is accommodated in a container, and the contaminated water is introduced into the container to decompose contaminants contained in the contaminated water. . 29. introducing said contaminated water into said container;
3. The discharge of purified water from the container is performed continuously.
8. The environmental restoration method according to 8. 30. The environmental restoration method according to claim 24, wherein the contaminated environment is contaminated soil. 31. An aqueous medium containing said microorganisms is introduced into said contaminated soil and brought into contact therewith.
Environmental restoration method described in 1. 32. The environmental remediation method according to claim 31, wherein the factor that affects the decomposing action of the microorganism is introduced into the soil by applying pressure through an injection well provided in the soil. 33. The contaminated soil is introduced into an aqueous medium containing the microorganisms and brought into contact with the soil.
0. The environmental restoration method according to item 0. 34. The environmental restoration method according to claim 30, wherein soil is brought into contact with the microorganism while the microorganism is supported on a carrier. 35. The method according to claim 24, wherein the polluted environment is a polluted gas. 36. The contaminated gas is introduced into a liquid medium containing the microorganisms and brought into contact therewith.
Environmental restoration method described in 1. 37. The environmental restoration method according to claim 36, wherein the microorganism is carried on a carrier. 38. The environmental remediation according to claim 36, wherein a carrier carrying the microorganism is arranged in an aqueous medium in a container, and the contaminated gas is introduced into the aqueous medium and brought into contact with the contaminated gas. Method. 39. The environmental restoration method according to claim 36, wherein the introduction of the contaminated air into the aqueous medium is performed continuously. 40. The environmental restoration method according to claim 24, wherein the factor is a factor that affects the growth of the microorganism. 41. The method according to claim 39, wherein the microorganism is a microorganism that expresses oxygenase. 42. The factor selected from the group consisting of temperature shift, nutrient supply, oxygen supply, agitation and pH adjustment.
The environmental restoration method according to any one of claims 39 to 41. 43. The environmental restoration method according to claim 42, wherein the nutrient source is at least one selected from a substrate, a carbon source, a nitrogen source, and a mineral component that affect the growth of the microorganism. 44. The method according to claim 43, wherein the nitrogen source is at least one selected from ammonium salts and nitrates. 45. The method according to claim 43, wherein the mineral component is a magnesium salt, a ferrous salt or a molybdate. 46. The microorganism expressing the okigesinase may be a genus Pseudomonas, a genus Acinetobacter, or Alcaligen.
es), Vibrio, Nocardi
a) Genus, Bacillus genus, Lactobacillus (Lac
tobacillus), Achromobacter
Genus, Arthrobacter, Micrococcus, Mycobac
terium), Methylosinus (Methylosinus), Methylomonas (Methylomonas), Berchia (Welchia),
Methylocystis genus, Nitrosomonas genus, Saccharomyce
s), Candida, Torulopsis
46. The environmental restoration method according to claim 41, wherein the method is at least one of microorganisms belonging to the genera Psis, Moraxella, Burkholderia, Ralstonia, and Comamonas. . 47. The microorganism, wherein the microorganism is strain J1 (FERM BP-
The environmental restoration method according to any one of claims 41 to 45, wherein the method is 5102). 48. The microorganism may be a JM1 strain (FREM BP).
The environmental restoration method according to any one of Claims 41 to 45. 49. The microorganism is a Pseudomonas sp. TL1 strain (Pseudomonas sp.
The environmental restoration method according to claim 46, wherein the environmental restoration method is 14726). 50. The microorganism according to claim 46, wherein the microorganism is Pseudomonas alcaligenes.
Environmental restoration method described in 1. 51. The microorganism, wherein the microorganism is Pseudomonas alcaligenes KB2, F2
The environmental restoration method according to claim 46, which is ERM P-14644). 52. The method according to claim 46, wherein the microorganism is Burkholderia cepacia. 53. The microorganism is Burkholderia cepacia KK01, F.
The environmental restoration method according to claim 46, which is ERM BP-4235). 54. The microorganism, wherein the microorganism is Alcaligenes sp. TL2 (Alcaligenes sp.
The environmental restoration method according to claim 46, wherein the environmental restoration method is -14642). 55. The microorganism, wherein the microorganism is Vibrio sp.
B1 strain (Vibrio sp. KB1, FERM P-146)
43. The environmental restoration method according to claim 46, wherein the method is 43). 56. The decomposition target compound is phenol,
55. One or more selected from o-cresol, m-cresol, p-cresol, toluene, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene and 1,1-dichloroethylene. The environmental restoration method according to any one of the above.