JPH0910794A - Biodegradation and purification method of pollutants released into the environment - Google Patents

Abstract

(57) [Abstract] [Purpose] Environmental purification by efficient biodegradation of aromatic compounds or organochlorine compounds released into the environment. [Structure] A microorganism to be utilized is brought into contact with a microorganism that is competitive with a carbon source capable of selectively improving the growth and decomposition activity to cause decomposition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for biodegrading aromatic compounds and / or organochlorine compounds, and in particular purification of aqueous media such as wastewater and waste liquid containing aromatic compounds and / or organochlorine compounds. The present invention relates to a biodegradation and purification method useful for restoration of contaminated soil and purification of air (gas phase) contaminated by organic chlorine compounds.

[0002]

2. Description of the Related Art In recent years, environmental pollution by organic chlorine compounds which are harmful to living organisms and are hardly decomposable has become a serious problem. In particular, in the soil of high-tech industrial areas such as domestic and overseas IC factories, tetrachlorethylene (PCE), trichlorethylene (TCE), dichloroethylene (DCE)
It is considered that the contamination by organochlorine compounds such as is spreading to a considerable extent, and many cases actually detected in environmental surveys have been reported. It is said that those 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 surrounding gas phase by removing and decomposing organic chlorine compounds is an important issue from the viewpoint of environmental conservation. Necessary technology 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, microbial decomposition of organic chlorine compounds such as TCE, which is stable in the environment, has been reported in recent years.
Research towards the practical application of this is beginning. In other words, in biodegradation treatment using microorganisms, it is possible to decompose organochlorine compounds to harmless substances by selecting microorganisms to be used, basically no special chemicals are required, and labor and cost for maintenance can be reduced. There are advantages such as. For example, TCE-degrading bacteria include Welchia alkeno as a report isolated from microorganisms capable of degrading organochlorine compounds.
phila sero 5 (USP 4877736, ATCC 53570), Welchia alke
nophila sero 33 (USP 4877736, ATCC 53571), Methyloc
ystis sp. strain M (Agric.Biol.Chem., 53,2903 (1989)
, Biosci. Biotech. Biochem., 56,486 (1992), 56,736.
(1992)), Methylosinus trichosporium OB3b (Am.Che
m.Soc.Natl.Meet.Dev.Environ.Microbiol., 29,365 (198
9), Appl.Environ.Microbiol., 55,3155 (1989), Appl.B
iochem.Biotechnol., 28,877 (1991), JP 02-92274 A, JP 03-292970 A), Methylomonas sp.MM
2 (Appl.Environ.Microbiol., 57,236 (1991)), Alcali
genes denitrificans ssp.xylosoxidans JE75 (Arch.m
icrobiol., 154,410 (1990)), Alcaligenes eutrophus
JMP134 (Appl.Environ.Microbiol., 56,1179 (1190)) Al
caligenes eutrophus KS01 (JP-A-7-123976, Mycobact
erium vaccae JOB5 (J.Gen. Microbiol., 82,163 (1974)
, Appl.Environ.Microbiol., 54,2960 (1989), ATCC 29
678), Pseudomonas putida BH (Sewer Association Journal, 24, 27
(1987)), Acinetobactor sp. Strain G4 (Appl.Enviro
n.Microbiol., 52,383 (1986), 53,949 (1987), 54,95
1 (1989), 56,279 (1990), 57,193 (1991), USP 49258
02, ATCC53617, which was originally classified as Pseudomonas cepacia, but was changed to Acinetobactor sp.),
Pseudomonas mendocina KR-1 (Bio / technol., 7,282 (198
9)), Pseudomonas putida F1 (Appl.Environ.Microbio
l., 54,1703 (1988), ibid.54,2578 (1988)), Pseudomonas f
luorescens PFL12 (Appl.Environ.Microbiol., 54,2578
(1988)), Pseudomonas fluorescens NRRL-B-18296 (US
P 4853334), Pseudomonas putida KWI-9 (JP 06-707
53), Pseudomonas cepacia KK01 (Japanese Patent Laid-Open No. 06-227)
769), Nitrosomonas europaea (Appl.Enviro
n.Microbiol., 56,1169 (1990)), Lactobacillus fruct
ivorans RE (lnt.J.Syst.Bacteriol., 30,313 (1980),
J.Appl.Bacteriol., 34,541 (1971)), Lactobacillus va
ginalissp.nov (lnt.J.Syst.Bacteriol., 39,368 (198
9), ATCC49540), etc. are known.

[0006]

However, when these degrading bacteria are used for actual environmental purification treatment, the very problem is the presence of microorganisms that originally live in the environment. In particular, it is said that there are 10 ⁷ to 10 ⁸ microorganisms, mainly bacteria, in 1 g of soil, and when the decomposing bacteria are scattered and an attempt is made to grow by adding a carbon source, the indigenous microorganisms also grow at the same time. As a result, a competitive relationship appears in the substrate (carbon source) and space, and the problem arises that the desired growth and decomposition activity of the degrading bacterium cannot be achieved sufficiently.

An object of the present invention is to efficiently improve the growth and activity of such a target degrading bacterium, thereby efficiently decomposing aromatic compounds and / or organic chlorine compounds, especially drainage / waste liquid. -To provide a method for efficiently decomposing and purifying an aromatic compound and / or an organic chlorine compound contained in an environment such as an aqueous medium such as groundwater, a soil, and a gas phase.

[0008]

The above object is achieved by the present invention described below.

That is, the present inventors have made a microorganism capable of decomposing pollutants such as aromatic compounds and / or organic chlorine compounds selectively grow the microorganism with respect to a competitive microorganism originally present in the environment. , And selectively providing a carbon source capable of selectively improving decomposition activity twice or more times, and contacting it with an aqueous medium containing an aromatic compound and / or an organic chlorine compound, soil, and a gas phase. Have found a method for efficiently decomposing and purifying aromatic compounds and / or organic chlorine compounds in an aqueous medium, soil, and gas phase.

Generally, various media have been devised for the culture of microorganisms depending on the type of microorganism and the purpose of the experiment, but basically, the carbon source, nitrogen source, inorganic salts, necessary for growth, In addition, it must contain all special required substances (vitamins, amino acids, nucleic acid bases, etc.).

Generally, in order to grow microorganisms such as bacteria in a short period of time, yeast extract, meat extract, peptone, casamino acid, CSL (Corn steep liquor), SVP
Natural media such as (Soluble vegetable protein), malt extract, waste molasses, etc., and complete nutrient media such as 2xYT medium and LB medium in which they are combined are effective and are often used as growth media in pure culture. . By its very nature, such a complete nutrient medium contains a wide variety of amino acids and sugars,
It contains a carbon source such as an organic acid and can be used as a substrate by numerous microorganisms.

[0012] Therefore, when added to the environment together with the target degrading bacteria, various microorganisms originally present in the environment uniformly proliferate. This means that the environmental microorganisms that grow uniformly with the target degrading bacteria compete with each other for various factors such as substrate, space, and oxygen, so that the target degrading bacteria can sufficiently grow and decompose. It may result in not being able to do it.

On the other hand, when the growth substrate is single or limited, the microorganisms that can efficiently utilize the growth substrate are limited as compared with the case of the complete nutrient natural medium. Such a medium is called a selective medium, and is used, for example, when performing accumulation culture or screening of microorganisms according to a certain purpose from the environment in which various microorganisms exist.

Therefore, a carbon source is selected so that the target degrading bacterium can be efficiently assimilated and is less likely to be assimilated by microorganisms in the target environment, and the carbon source is selected twice or more times. By adding to the environment separately, the growth of microorganisms existing in the environment is suppressed as a whole, and the target degrading bacteria are selectively grown, resulting in aromatic compounds and / or organic chlorine in the environment. It becomes possible to efficiently decompose and purify the compound.

The microorganisms used in the present invention may be any microorganisms capable of decomposing aromatic compounds and / or organic chlorine compounds, and specifically, the genus Corynebacterium , Pseudomonas ( Pseudo )
monas) genus, Acinetobacter (Acinetobacter) genus,
Alcaligenes (Alcaligenes) genus Vibrio (Vibri
o) the genus Nocardia (Nocardia) the genus Bacillus (Bacillu
s ) genus, Lactobacillus genus, Achromobacter genus, Arthrobacter genus, Micrococcus
Genus Mycobacterium (Mycobacterium) genus, Mechiroshinasu (Methylosinus) genus, Methylomonas (Methylomon
as ), Belchia ( Welchia ), Methylosis ( Me )
thylocystis ), Nitrosomonas
Genus Saccharomyces (Saccharomyces) genus Candida (Candida) genus, include a microorganism belonging to the genus Torulopsis (Torulopsis), for example, Corynebacterium sp. J1 strain (Corynebacterium sp.J1, the Ministry of International Trade and Industry Life Institute of Advanced Industrial Science and Technology Accession No. : FERM BP-5102), Corynebacterium spice JM1 strain ( Corynebacterium sp.J)
M1, Ministry of International Trade and Industry, Institute of Biotechnology, Contract number: FERM P-1
4727), Pseudomonas cepacia KK01 strain ( Pseudomonas
cepacia KK01, Ministry of International Trade and Industry, Institute of Bioengineering and Technology, Contract number: FERM BP-4235), Pseudomonas species TL1
Strains ( Pseudomonas sp. TL1, Ministry of International Trade and Industry, Biotechnology Industrial Technology Research Center deposit number: FERM P-14726), Pseudomonas alcaligenes KB2 strain ( Pseudomonas alcaligenes KB2 , Ministry of International Trade and Industry Biotechnology Engineering Research Center trust number: FERM P-14644), Alcaligenes Species TL2 strain ( Alcaligenes sp.TL
2, Ministry of International Trade and Industry, Biotechnology Institute of Technology Contract No .: FERM P-14
642), Vibrio species KB1 strain ( Vibrio sp.KB1
, Ministry of International Trade and Industry, National Institute of Biotechnology, Engineering number: FERM P-14
643) and the like.

Specific selective carbon sources include, for example, pyruvic acid, citric acid, gluconic acid,
Examples thereof include organic acids such as α-ketoglutaric acid, itaconic acid, sodium malate, sodium glutamate and salts thereof, but any substance may be used as long as it is a compound capable of selectively increasing the growth and activity of the target degrading bacterium. .

As a method of adding the carbon source, in order to logarithmically grow the pollutant-degrading microorganism introduced into the medium,
At the time of introducing the microorganism, it is preferable to first contact the carbon source.

The timing of the second and subsequent contact is preferably at least one of the logarithmic growth phase and the stable growth phase of the growth of microorganisms, and the additional contact during the logarithmic growth phase stabilizes the pollutants in particular. It is particularly preferable for decomposition.

Further, the concentration of the carbon source is preferably slightly lower than the optimum concentration for the microorganism.

The inorganic salt medium used for culturing the microorganism used in the present invention may be any medium as long as it is necessary for the growth of ordinary microorganisms and the microorganisms can grow, for example, selective to M9 medium. It is also possible to culture with the addition of a carbon source.

The composition of M9 medium is shown below.

Na ₂ HPO ₄ : 6.2 g KH ₂ PO ₄ : 3.0 g NaCl: 0.5 g NH ₄ Cl: 1.0 g (in 1 l of medium; pH 7.0) Cultivation can be carried out under aerobic conditions Either culture or solid culture may be used. The culture temperature is preferably about 15 to 30 ° C.

The aromatic compound and / or organochlorine compound in the present invention is decomposed by contacting the aromatic compound and / or organochlorine compound in an aqueous medium, soil and gas phase with the degrading microorganism. Can be done. The microorganisms can be contacted with the aromatic compound and / or the organochlorine compound by any method as long as the microorganisms can exhibit decomposition activity, using various methods such as a batch method, a semi-continuous method, and a continuous method. Can be implemented. The microorganism can be used in a semi-fixed state or by immobilization on a suitable carrier. The waste liquid, the soil, the gas phase, or the like to be treated may be subjected to various treatments as necessary.

The decomposition treatment of the aromatic compound and / or the organic chlorine compound in the aqueous medium in the present invention is carried out by contacting the aromatic compound and / or the organic chlorine compound present in the aqueous medium with the decomposing microorganism. You can The main usage forms are described below, but the present invention is not limited to these usage forms, and can be used for purification treatment of aromatic compound and / or organic chlorine compound contamination in any aqueous medium.

For example, the simplest method is to introduce the decomposing microorganism directly into an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound. In this case, it is necessary to adjust the pH, salt concentration, temperature of the aqueous medium, the concentration of pollutants, etc., but the degrading microorganisms maintain their degrading activity unless they are extremely acidic or alkaline and have a high salt concentration.

As another utilization form, a culture tank is provided to cultivate the decomposing microorganism, and an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound is introduced into the culture tank at a predetermined flow rate to decompose the microorganism. There is a form. The introduction of the aqueous medium and the drainage may be carried out continuously, but it is also possible to carry out the treatment intermittently or in a batch manner depending on the treatment capacity. Such control may be optimized by system control according to the concentration of the aromatic compound and / or the organic chlorine compound.

Further, the degrading microorganisms are adhered to a carrier such as soil particles, the reaction layer is filled with the degrading microorganisms, and an aromatic compound and / or organic chlorine compound-contaminated aqueous medium is introduced into the reaction vessel for decomposing treatment. There is a form to do. 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, various materials commonly used in bioreactors conventionally used in the pharmaceutical industry, the food industry, the wastewater treatment system, and the like can be used as a material that provides a living space for microorganisms. More specifically, porous glass, ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, inorganic particulate carriers such as anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid. Gel carriers such as polyacrylamide, carrageenan, agarose and gelatin, ion exchangeable cellulose, ion exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester. As natural products, cellulose-based products such as cotton, hemp, and paper, and ligulin-based products such as wood flour and bark can also be used.

The decomposition treatment of the aromatic compound and / or the organic chlorine compound in the soil in the present invention can be carried out by contacting the aromatic compound and / or the organic chlorine compound present in the soil with the decomposing microorganism. . The main uses are described below, but the present strain is not limited to these forms, and can be used for purification treatment of aromatic compounds and / or organochlorine compounds in any soil.

For example, the simplest method is to introduce the decomposing microorganism directly into the soil contaminated with an aromatic compound and / or an organic chlorine compound. The method of introduction includes not only the method of spraying on the soil surface, but also the method of introducing from a well inserted into the ground in the case of treatment in a relatively deep formation. Furthermore, when pressure is applied by air, water, etc., the decomposing microorganisms spread over a wide area, which is more effective. in this case,
Although it is necessary to adjust various conditions in the soil to be suitable for the degrading microorganism, the degrading microorganism is allowed to grow faster in the presence of a carrier such as soil particles, and in that sense, the condition in the soil is convenient. .

Further, the degrading microorganisms are attached to a carrier, which is filled in a reaction tank, and the reaction tank is filled with an aromatic compound and / or
Alternatively, there is a form in which soil contaminated with an organic chlorine compound is introduced mainly into the aquifer and decomposed. The form of the reaction tank is preferably fence-shaped or film-shaped so that it can cover a wide range in soil. In this case, any carrier can be used, but it is more preferable that the carrier has excellent ability to retain microorganisms and does not impair air permeability.
For example, as a material that provides a living space for microorganisms,
Conventionally, various microbial carriers widely used in bioreactors used in the pharmaceutical industry, food industry, wastewater treatment system, etc. can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, inorganic particulate carriers such as anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid. Gel carriers such as polyacrylamide, carrageenan, agarose and gelatin, ion exchangeable cellulose, ion exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester. Cellulose-based materials such as cotton, hemp and paper, and lignin-based materials such as wood flour and bark can also be used as natural products.

The decomposition treatment of the aromatic compound and / or the organic chlorine compound in the gas phase in the present invention is carried out by bringing the aromatic compound and / or the organic chlorine compound existing in the gas phase into contact with the above-mentioned decomposing microorganism. be able to. The main use forms are described below, but the present strain is not limited to these forms, and can be used for purification treatment of any gas phase contamination of aromatic compounds and / or organochlorine compounds in the gas phase.

For example, there is a mode in which a culture tank is provided to cultivate the degrading microorganism, and a gas contaminated with an aromatic compound and / or an organic chlorine compound is introduced into the culture tank at a predetermined flow rate to decompose the gas. The method of introducing the gas is not particularly limited, but a form in which the culture solution is agitated by the method of introducing the gas to promote aeration is more preferable. The introduction and exhaust of the gas may be performed continuously, but it is also possible to perform the treatment intermittently or in a batch manner depending on the treatment capacity.
It is advisable to optimize such control by controlling the system according to the concentration of the organic chlorine compound.

As another utilization form, the degrading microorganisms are attached to a carrier such as soil particles and the like is filled in a reaction layer, and an aromatic compound and / or organic chlorine compound-contaminated gas is introduced into the reaction tank. There is a form in which the decomposition process is performed.
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 habitat for microorganisms, various microbial carriers that have been widely used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment system, etc. can be used. More specifically, porous glass,
Inorganic particulate carriers such as ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid, polyacrylamide, carrageenan, agarose, gelatin, etc. Examples of the gel carrier, ion-exchangeable cellulose, ion-exchange resin, cellulose derivative, glutaraldehyde, polyacrylic acid, polyurethane, polyester and the like. Cellulose-based materials such as cotton, hemp and paper, and lignin-based materials such as wood flour and bark can also be used as natural products.

For purification of the polluted gas, the substance to be a carrier may be pre-filled and then the bacterium may be introduced, or pre-culture may be performed. In order to carry out the decomposition reaction more efficiently, it is advisable to maintain the above-mentioned carbon source, water content ratio, oxygen concentration and the like under 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. Consideration should be given to promote contact with, for example, a column, a tube, a tank, and a box type 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.

The polluted gas may initially be adsorbed on the carrier material, and in some cases the effect of microbial utilization is not well observed, but after a certain period of time, the pollutant adhered to the carrier material is decomposed, and By re-adsorbing the pollutant on the surface of the material in which the pollutant is decomposed, the adsorptivity to the carrier material is regenerated. In this way, decontamination capacity is not saturated and constant decomposition can always be expected.

The method of the present invention can be applied to waste liquid treatment of both closed and open systems, soil treatment, and air treatment. It should be noted that the microorganism may be immobilized on a carrier or the like, or various methods for promoting growth may be used in combination.

[0037]

[Embodiment 1 ] Effect of pyruvate on growth of J1 strain and growth control of environmental microorganisms (liquid culture system) M9 containing 0.2% of yeast extract M9 containing 0.2% of yeast extract in a 500 ml Sakaguchi flask was used for colony of J1 strain on agar medium. 200 ml of the medium was inoculated and cultured at 25 ° C for 24 hours with shaking.

Next, 200 ml of water sampled from a fairly polluted reservoir in Atsugi City, Kanagawa Prefecture was put into four 500 ml Sakaguchi flasks.
Yeast extract was added to two of them as carbon sources, and pyruvic acid was added to the other two at 0.1%, and then 1 J1 strain was cultured in the above manner to obtain 10 ⁴ cells. / Ml, and the cells were cultivated with shaking at 25 ° C and 120 rpm, and the number of bacteria was measured over time. Pyruvic acid was added at an interval of 12 hours at a rate of 0.1% three times. The number of bacteria was measured by colony counting on M9 agar medium containing 0.2% of yeast extract. The results are shown in FIG.

In the case of yeast extract, strain J1 and environmental microorganisms grew equally. In contrast, in the case of pyruvic acid, J1
The strain showed good results in the final bacterial count and its persistence compared to the case of yeast extract, but the growth of environmental microorganisms was clearly inferior, and it was possible to selectively grow the J1 strain. Met.

Embodiment 2 FIG . Effect of Pyruvate on Degradation of Aromatic Compounds by J1 Strain (Liquid Culture System) In a medium prepared in the same manner as in Example 1, phenol, o-cresol, m-cresol, p-cresol and toluene were used as decomposition target substances, respectively. 300ppm (toluene only 100p
pm) was added and 0.1 ml of the bacterial solution of the J1 strain cultured as described above was inoculated, followed by shaking culture at 25 ° C. and 120 rpm, and the decrease of each compound was measured with time. Pyruvate was prepared in Example 1
Was added 3 times as above. For the measurement, phenol and cresol were measured by liquid chromatography (spectrophotometric detector) and toluene was measured by gas chromatography (FID detector), and the residual ratio with respect to the initial concentration was shown. The results are shown in Figure 2 (phenol), Figure 3 (o-cresol), and Figure 4.
(M-cresol), FIG. 5 (p-cresol), and FIG. 6 (toluene), respectively.

In the case of yeast extract, the initial decomposition of each aromatic compound was early, but the decomposition stopped in the middle, whereas almost complete decomposition was observed in the system containing pyruvic acid. .

Embodiment 3 FIG . Effect of Pyruvic Acid on TCE Degradation by J1 Strain (Liquid Culture System) To the medium used in Example 1, phenol as a TCE degradation inducing substance was added to 200 ppm and TCE was added to 10 ppm, and then 5 ml was added to 25 ml. After inoculating 0.1 ml of the J1 strain cultivated as described above into a vial vial and sealing it completely with a butyl rubber stopper and aluminum cap, shake culture at 25 ° C and 120 rpm to reduce TCE over time. It was measured. Note that pyruvic acid was added three times as in Example 1. The amount of TCE was determined by gas chromatography (FI
The amount of TCE was also quantified in the same experimental system without the addition of the JM1 strain as a control, and the residual rate relative to the TCE amount of the control was determined. FIG. 7 shows the results.

The initial decomposition was better in the yeast extract-containing system, but the decomposition stopped halfway, and finally, the decomposition proceeded significantly in the pyruvic acid-containing system with few competing microorganisms.

Example 4. Effect of pyruvic acid on DCE degradation by strain J1 (liquid culture system) cis-1,2-dichloroethylene (ci
s-1,2-DCE), trans-1,2-dichloroethylene (trans-1,
2-DCE) and 1,1-dichloroethylene (1,1-DCE) were adjusted to 10 ppm each by the same method as in Example 3 except that the respective amounts were 10 ppm.
The decrease in E was measured. The results are shown in FIG. 8 (cis-1,2-DCE), FIG. 9 (trans-1,2-DCE) and FIG. 10 (1,1-DCE), respectively.

In all cases, the same results as in the case of TCE decomposition in Example 3 were obtained, showing the effect of pyruvic acid.

Example 5. Effect of sodium malate on growth of JM1 strain and growth control of environmental microorganisms (liquid culture system) Colonies of JM1 strain on M9 agar medium containing 0.5% of sodium malate were added to 1.0% sodium malate in a 500 ml Sakaguchi flask. 200 ml of M9 medium containing the above was inoculated, and shake culture was carried out at 25 ° C for 24 hours.

Water similar to that used in Example 1 was added to four 50
200 ml of each was added to a 0 ml Sakaguchi flask, and yeast extract was added to two of the carbon sources at 0.1% and sodium malate to the other two at 0.1%. 1 more each
10 ⁴ cells / m of JM1 strain cultured as above
l, and culture by shaking at 25 ° C and 120 rpm.
The number of bacteria was measured over time. The number of bacteria was measured by colony counting on M9 agar medium containing 0.2% of yeast extract. In addition, 0.1% of sodium malate was added every 8 hours four times. Figure 1 shows the results
It is shown in FIG.

In the case of yeast extract, the JM1 strain and the environmental microorganisms grew equally. On the other hand, in the case of sodium malate, the JM1 strain showed good results in the final number of cells and its persistence as compared with the case of yeast extract, whereas
The growth of environmental microorganisms was clearly inferior to this, and it was possible to selectively grow the JM1 strain.

Embodiment 6 FIG . Effect of Sodium Malate on Degradation of Phenol by JM1 Strain (Liquid Culture System) To a medium prepared in the same manner as in Example 5, 300 ppm of phenol as a substance to be decomposed was added and cultured as above.
After inoculating 0.1 ml of the bacterial solution of 1 strain, the culture was carried out at 25 ° C. with shaking at 120 rpm, and the decrease of each compound was measured with time. Note that sodium malate was added four times as in Example 5. The measurement was performed by liquid chromatography (spectrophotometric detector), and the residual ratio with respect to the initial concentration was shown. The result is shown in FIG.

The initial decomposition was better in the yeast extract-added system, but the decomposition stopped in the middle, whereas almost complete decomposition was observed in the system containing sodium malate.

Example 7. Effect of Sodium Malate on Degradation of TCE by JM1 Strain (Liquid Culture System) TCE was added to the medium used in Example 5 to a concentration of 10 ppm, and then 5 ml was injected into a 25 ml vial bottle and cultured as described above. After inoculating 0.1 ml of the bacterial solution of the JM1 strain described above, it was completely sealed with a butyl rubber stopper and an aluminum cap, and cultured by shaking at 25 ° C. and 120 rpm, and the decrease in TCE was measured with time. Note that sodium malate was added four times as in Example 5. The amount of TCE was quantified by gas chromatography (FID detector) by the headspace method, and as a control, the amount of TCE was also quantified in the system in which the JM1 strain was not added in the same experimental system. I asked for the rate. FIG. 13 shows the results.

As in the case of the phenol decomposition of Example 6,
Initial decomposition was better with the yeast extract addition system,
Finally, the decomposition proceeded in the sodium malate addition system with few competing microorganisms.

Example 8. Effect of Sodium Malate on Degradation of DCE by JM1 Strain (Liquid Culture System) cis-1,2-dichloroethylene (ci
s-1,2-DCE), trans-1,2-dichloroethylene (trans-1,
2-DCE) and 1,1-dichloroethylene (1,1-DCE) were each adjusted to 10 ppm by the same method as in Example 7, except that
The decrease in E was measured. The results are shown in Fig. 14 (cis-1,2-DCE),
FIG. 15 (trans-1,2-DCE) and FIG. 16 (1,1-DCE) are shown respectively.

In the case of cis-1,2-DCE and trans-1,2-DCE, as in the case of TCE decomposition in Example 7, the initial decomposition was better with the yeast extract-containing system, but the final decomposition was Degradation progresses in a system in which sodium malate is added, which has few competitive microorganisms.
In 1-DCE, the sodium malate addition system was superior to the initial stage of decomposition.

Example 9. Effect of sodium glutamate on growth of KK01 strain and growth control of environmental microorganisms (liquid culture system) KK on M9 agar medium containing 0.2% sodium glutamate
The 01 strain colony was inoculated into 200 ml of M9 medium containing 0.2% sodium glutamate in a 500 ml Sakaguchi flask at 30 ° C.
The culture was carried out with shaking for 18 hours.

Water similar to that used in Example 1 was added to four 50
200 ml each was added to a 0 ml Sakaguchi flask, and yeast extract was added to two of the carbon sources at 0.1%, and sodium glutamate was added to the other two at 0.05%. Furthermore, one strain each of the KK01 strains cultivated as described above was subjected to 10 ⁴ cel
The cells were added at about ls / ml, shake-cultured at 30 ° C. and 120 rpm, and the number of bacteria was measured over time. In addition, 0.05% of sodium glutamate was added 4 times every 6 hours. The number of bacteria is measured by M9 containing 0.2% yeast extract.
This was done by colony counting on agar.
The results are shown in FIG.

In the case of yeast extract, strain KK01 and environmental microorganisms grew equally. On the other hand, in the case of sodium glutamate, the KK01 strain showed good results in the final bacterial count and its persistence as compared with the case of the yeast extract, whereas the growth of environmental microorganisms was clearly inferior to this. It was possible to grow the KK01 strain selectively.

Example 10. Effect of Sodium Glutamate on Degradation of Phenol by KK01 Strain (Liquid Culture System) To a medium prepared in the same manner as in Example 9, 600 ppm of phenol as a substance to be decomposed was added, and KK was cultured as described above.
After inoculating 0.1 ml of the bacterial solution of the 01 strain, it was cultured with shaking at 30 ° C. and 120 rpm, and the decrease of each compound was measured with time. Note that sodium glutamate was added four times as in Example 9. The measurement was performed by liquid chromatography (spectrophotometric detector), and the residual ratio with respect to the initial concentration was shown.
The results are shown in Fig. 18.

Degradation was stopped in the yeast extract-added system, whereas it was almost completely degraded in the sodium glutamate-added system, showing that the sodium glutamate-added system was superior to the yeast extract-added system in degrading phenol.

Example 11. Effect of Sodium Glutamate on TCE Degradation by KK01 Strain (Liquid Culture System) TCE was added to the medium used in Example 9 at 10 ppm,
After adding phenol as a TCE decomposition inducing substance to 200 ppm, inject 5 ml into a 25 ml vial bottle and inoculate 0.1 ml of the KK01 strain culture cultivated as described above, then complete with a butyl rubber stopper and aluminum cap. Sealed, 30 ℃, 120rpm
The cells were cultivated with shaking at 25 ° C, and the decrease in TCE was measured over time. Note that sodium glutamate was added four times as in Example 9. The amount of TCE was quantified by gas chromatography (FID detector) by the headspace method. As a control, TC in the system in which the KK01 strain was not added in the same experimental system was used.
The amount of E was also quantified and the residual rate relative to the amount of TCE in the control was calculated. The results are shown in Fig. 19.

Similar to the phenol decomposition of Example 10, the superiority of TCE decomposition in the sodium glutamate-added system over the yeast extract-added system was shown.

Example 12 Effect of Sodium Glutamate on DCE Degradation by KK01 Strain (Liquid Culture System) cis-1,2-dichloroethylene (ci
s-1,2-DCE), trans-1,2-dichloroethylene (trans-1,
2-DCE) and 1,1-dichloroethylene (1,1-DCE) were each changed to 10 ppm by the same method as in Example 11 except that
The decrease in DCE was measured. The results are shown in Fig. 20 (cis-1,2-DCE
), FIG. 21 (trans-1,2-DCE), FIG. 22 (1,1-DCE).
Are shown below.

Similar to the TCE degradation of Example 11, the superiority of each DCE degradation in the sodium glutamate-added system over the yeast extract-added system was shown.

Example 13. Effect of pyruvic acid on the growth of J1 strain and growth control of indigenous microorganisms (15 ℃, brown forest soil) 1 ml of M9 medium containing 0.1% pyruvic acid or 0.1% yeast extract was injected into a 15 ml sterile tube, Atsugi City, Kanagawa Prefecture. 4 g of brown forest soil collected from the above was added, and half of the soil was inoculated with 10 μl of JM1 strain cultivated as in Example 1, and then cotton plugged at 15 ° C, which is close to the actual temperature in soil. After static culture, the numbers of JM1 strain and indigenous microorganisms were measured over time. In addition, pyruvic acid was added in an amount corresponding to 0.1% after 24 hours and 36 hours. The number of bacteria was measured by adding 5 ml of sterilized water to the tube, stirring and extracting for 1 minute, and diluting the supernatant one by one, but performing colony counting on M9 agar medium containing 0.2% of yeast extract. The results are shown in FIG.

In the case of yeast extract, the indigenous microorganisms grew better than in the J1 strain, whereas in the case of pyruvic acid,
The growth of indigenous microorganisms was clearly inferior to that of the J1 strain.
It was possible to grow the strain selectively.

Example 14 Effect of pyruvic acid on decomposition of phenol in soil by J1 strain (15 ℃, brown forest soil) Phenol 600ppm and 0.1% Pyruvic acid or 0.1%
1 ml of M9 medium containing yeast extract was infused in a 15 ml sterile tube, 4 g of brown forest soil similar to that used in Example 13 was added, and half of the mixture was cultivated as in Example 1 to obtain 10 μl of bacterial solution of strain J1.
After inoculating l with cotton plug, close to the temperature in the actual soil 15
The culture was allowed to stand at ℃, and the phenol concentration was measured over time. Note that pyruvic acid was added twice more as in Example 13. Phenol amount is 5 ml in a tube
The sterilized water was added and the phenol in the supernatant extracted by stirring for 1 minute was detected by the JIS detection method using aminoantipyrine (JIS K0102-1993, 28.1). The result is shown in FIG.
Shown in

Although the yeast extract-added system initially decomposed, the decomposition stopped halfway, and finally the pyruvate-added system obviously decomposed.

Example 15 Effect of pyruvic acid on decomposition of TCE in soil by J1 strain (15 ℃, brown forest soil) TCE50ppm, phenol 300ppm as TCE decomposition inducer,
And 0.1% pyruvic acid or 0.1% yeast extract
1 ml of M9 medium was poured into a screw vial with a 15-ml Teflon inner lid, 4 g of brown forest soil similar to that used in Example 13 was added, and 0.1 ml of a bacterial solution of the J1 strain cultured as in Example 1 was added.
After inoculation, it is completely sealed and close to the temperature in the actual soil
Static culture was performed at 15 ° C. Pyruvate was prepared in Example 13
Was added twice more as above.

The amount of TCE was extracted by stirring with 5 ml of n-hexane for 3 minutes, diluted with 10 times in some cases, and quantified by gas chromatography (ECD detector).
The decrease in TCE was measured. As a control, we also quantified the amount of TCE in the same experimental system without the addition of strain J1.
The residual ratio to the control TCE amount was calculated. The results are shown in FIG.

Similar to the phenol decomposition of Example 14, the yeast extract-added system was initially decomposed, but the decomposition was stopped halfway, and finally the pyruvate-added system was clearly decomposed. Advanced.

Example 16. Effect of sodium malate on growth of JM1 strain and growth control of indigenous microorganisms (15
℃, fine sand soil) Inject 1 ml of M9 medium containing 0.1% sodium malate or 0.1% yeast extract into a 15 ml sterilized tube, add 4 g of Sawara Tsusa, and cultivate half of them as in Example 5. After inoculating 10 μl of the cultivated JM1 strain, a cotton stopper was applied, and static culture was performed at 15 ° C, which is close to the actual temperature in the soil.
The numbers of strains and indigenous microorganisms were measured. In addition, sodium malate was 0.1% every 12 hours for 4 times.
Added considerable. The number of bacteria was measured by adding 5 ml of sterilized water to the tube, stirring and extracting for 1 minute, and diluting the supernatant one by one, but performing colony counting on M9 agar medium containing 0.2% of yeast extract. The results are shown in FIG.

In the case of yeast extract, the indigenous microorganisms grow better than the JM1 strain, and the number of bacteria in the JM1 strain is not maintained. On the other hand, in the case of sodium malate, the indigenous microorganisms grow more than the JM1 strain. Obviously, it was inferior to this, and it was possible to grow JM1 strain selectively.

Example 17 Effect of Sodium Malate on Degradation of Soil Phenol by Strain JM1 (15
℃, fine sand soil) Inject 1 ml of M9 medium containing 600 ppm of phenol and 0.5% sodium malate or 0.1% yeast extract in a sterile volume of 15 ml, add 4 g of Sahara Tsuchisa, and add half of them as in Example 5. After inoculating 10 μl of the bacterial solution of the JM1 strain that had been cultivated in the above, a cotton plug was applied, and static culturing was performed at 15 ° C, which is close to the actual temperature in soil, and the phenol concentration was measured over time. In addition,
Sodium malate was added an additional 4 times as in Example 16. Phenol was quantified by adding 5 ml of sterilized water to the tube and detecting the phenol in the supernatant obtained by stirring and extracting for 1 minute by the detection method by JIS method using aminoantipyrine (JIS K0102-1993, 28.1). The results are shown in FIG.

In the yeast extract-added system, the decomposition stopped halfway, and finally the sodium-malate-added system clearly showed the decomposition.

Example 18. TEC in soil by JM1 strain
Effect of Sodium Malate on Decomposition Treatment of Soybean (15 ℃,
Fine sand) TCE 50ppm and 0.5% sodium malate or 0.1%
1 ml of M9 medium containing yeast extract was injected into a screw vial with a 15 ml Teflon inner lid, 4 g of Sawara Tosago was added, and 0.1 ml of a JM1 strain bacterial solution cultivated as in Example 5 was inoculated. Sealed and close to the actual temperature in the soil 15 ℃
The culture was allowed to stand still. In addition, sodium malate is used in Example 1.
Similar to 6, added 4 more times. TCE amount is 5ml
Gas chromatography (ECD detector) after diluting and extracting with n-hexane for 3 minutes, depending on the case, 10-fold dilution
The decrease in TCE was measured over time. As a control, in the same experimental system, the system without JM1 strain was added.
The TCE amount was also quantified, and the residual rate relative to the control TCE amount was calculated. The results are shown in FIG.

Initially, the yeast extract-added system was more degraded, but the degradation almost stopped from about the second day, and finally the sodium malate-added system was clearly degraded.

Example 19 Effect of Sodium Glutamate on Degradation of Phenol in Soil by Strain KK01 (15 ℃, Fine Sand Soil) 1 ml of M9 medium containing 600 ppm of phenol and 0.05% sodium glutamate or 0.1% peptone was injected into a 15 ml sterile tube, and Sahara Tsuchisa After inoculating 10 μl of the bacterial solution of the KK01 strain, which had been cultured as in Example 9, to the half of the mixture, a cotton plug was placed and static culture was performed at 15 ° C., which is close to the temperature in the actual soil, and The phenol concentration was measured.
In addition, sodium glutamate was used after 24 hours of culture and 3
After 6 hours, 0.05% equivalent was added. Phenol was quantified by adding 5 ml of sterilized water to the tube and detecting the phenol in the supernatant extracted by stirring for 1 minute by the detection method by JIS method using aminoantipyrine (JIS K0102-1993, 28.1). The results are shown in FIG.

In the yeast extract-added system, the decomposition stopped halfway, and finally, the sodium-glutamate-added system clearly showed the decomposition.

Example 20. TCE in soil from KK01 strain
Effect of Sodium Glutamate on the Degradation of Soil (15
℃, fine sand) TCE50ppm, phenol 400ppm as TCE decomposition inducer,
And 1 ml of M9 medium containing 0.05% sodium glutamate or 0.1% peptone was injected into a screw vial with a 15 ml Teflon inner lid, 4 g of Sawara Tsusa was added, and a bacterial solution of KK01 strain cultivated as in Example 9 was added. After inoculating ml, it was completely sealed and statically cultivated at 15 ° C, which is close to the temperature in the actual soil. Note that sodium glutamate was added twice more as in Example 19. TCE amount is 5 ml
What was stirred and extracted with n-hexane for 3 minutes was diluted with 10 cultures in some cases and quantified by gas chromatography (ECD detector), and the decrease in TCE was measured over time.
As a control, the amount of TCE was also quantified in a system in which the KK01 strain was not added in the same experimental system, and the residual rate relative to the amount of TCE in the control was determined. The results are shown in FIG.

Similar to the phenol decomposition in Example 19, in the yeast extract-added system, the decomposition stopped halfway, and finally, the sodium glutamate-added system obviously proceeded to decompose.

Example 21. Effect of sodium malate on decomposition treatment of TCE in the gas phase by soil aeration using JM1 strain 0.1 ml of JM1 strain cultured in the same manner as in Example 5 was used.
It was added to 30 ml of M9 medium in a vial containing 2% yeast extract or 0.2% sodium malate, and brown forest soil was added to the surface of the water. After sealing with a butyl rubber stopper and leaving it at 20 ° C. overnight, the excess culture solution was decanted and removed. Air aerated in a saturated solution of TCE was passed through the soil at a flow rate of 60 ml / min for 30 minutes, then completely sealed with a butyl rubber stopper and an aluminum seal, and statically cultured at 20 ° C. Incidentally, sodium malate was added in an amount equivalent to 0.2% after 24 hours and 36 hours of culture. The amount of TCE was quantified by gas chromatography by the headspace method, and the amount of TCE was measured over time. The results are shown in Fig. 31.

From these results, the superiority of sodium malate in the decomposition treatment of TCE in the gas phase by soil aeration of JM1 strain was shown.

Embodiment 22 FIG . Effect of Sodium Malate on Degradation of TCE in Gas Phase by Continuous Aeration of Soil Using JM1 Strain 0.1 ml of JM1 strain cultivated in the same manner as in Example 5 was
It was added to 30 ml of M9 medium in a vial containing 0.2% yeast extract or 0.2% sodium malate, and brown forest soil was added to the surface of the water. After sealing with a butyl rubber stopper and leaving it at 20 ° C. overnight, the excess culture solution was decanted and removed.
This was completely sealed with a butyl rubber stopper and an aluminum seal, and then static culture was carried out at 20 ° C. while continuously aerated air aerated in a TCE saturated solution at a flow rate of 0.5 ml / min. It should be noted that sodium malate was used after 24 hours of culture and 4 times.
After 8 hours, an amount equivalent to 0.2% was added. The amount of TCE was determined by quantifying the amount of TCE in the air flowing out by gas chromatography, and the amount of TCE was measured over time. The results are shown in Fig. 32.

These results showed the superiority of sodium malate in the decomposition treatment of TCE in the gas phase by continuous aeration of JM1 strain.

Example 23. Effect of Citric Acid on Degradation of Phenol in Soil by TL1 Strain (15 ℃, Loamy Soil) Colonies of TL1 strain on M9 agar medium containing 0.1% citric acid and 0.02% yeast extract were cultured in a 500 ml Sakaguchi flask. 200 ml of M9 medium containing 0.1% of acid was inoculated and shake culture was carried out at 25 ° C. for 30 hours.

Next, 1 ml of M9 medium containing 300 ppm of phenol and 0.05% citric acid or 0.1% yeast extract was added to 15 ml.
The loam soil collected from Ibaraki prefecture was infused with a sterile tube.
4 g was added, and half of them were cultured as described above.
After inoculating 10 μl of the bacterial solution of one strain, a cotton plug was applied, static culture was performed at 15 ° C, which is close to the actual temperature in soil, and the phenol concentration was measured over time. In addition, citric acid was added in an amount equivalent to 0.05% three times, 24 hours after culture, 36 hours after culture, and 48 hours after culture. Phenol was quantified by adding 5 ml of sterilized water to the tube and detecting the phenol in the supernatant obtained by stirring and extracting for 1 minute by the detection method by JIS method using aminoantipyrine (JIS K0102-1993, 28.1). Results are shown in FIG.

Although the yeast extract-added system initially decomposed, the decomposition stopped halfway, and finally the citric acid-added system obviously decomposed.

Example 24. Effect of citric acid on decomposition of TCE in soil by TL1 strain (15 ℃, loamy soil) TCE20ppm, phenol 300ppm as TCE decomposition inducer,
And M containing 0.05% citric acid or 0.1% yeast extract
1 ml of 9 medium was poured into a screw vial with a 15 ml Teflon inner lid, and 4 g of loam soil similar to that used in Example 23 was added.
Furthermore, 0.1 ml of the bacterial solution of the TL1 strain cultured as in Example 23.
After inoculation, it is completely sealed and close to the temperature in the actual soil
Static culture was performed at 15 ° C. Note that citric acid was added three times as in Example 23. The amount of TCE was extracted by stirring with 5 ml of n-hexane for 3 minutes, diluted 10 times in some cases and quantified by gas chromatography (ECD detector), and the decrease in TCE was measured over time. As a control, TCE in the same experimental system without TL1 strain
The amount was also quantified, and the residual rate relative to the control TCE amount was calculated. The results are shown in Fig. 34.

As in the case of the phenol decomposition of Example 23, the yeast extract-added system was initially decomposed, but the decomposition was stopped in the middle, and the citric acid-added system was finally revealed. It has been disassembled.

Example 25. Effect of Gluconic Acid on Degradation of Phenol in Soil by Strain TL2 (15 ℃, loamy soil) Colonies of strain TL2 on M9 agar medium containing 0.2% gluconic acid contained 0.1% gluconic acid in a 500 ml Sakaguchi flask. 200 ml of M9 medium was inoculated and shake culture was carried out at 25 ° C for 24 hours.

Next, 1 ml of M9 medium containing 500 ppm of phenol and 0.05% gluconic acid or 0.1% yeast extract was added to 15 ml.
Add 4 g of loamy soil collected from Ibaraki prefecture to a sterile tube with a volume of 10 ml, and inoculate 10 μl of the bacterial solution of the TL2 strain cultured as above into half of the soil, then plug it with cotton and put it in the actual soil. C., and the phenol concentration was measured over time. Gluconic acid was added to the culture 24
After three hours, 36 hours and 48 hours, 0.
Added an amount equivalent to 05%. Phenol determination in tubes
5 ml of sterilized water was added, and the phenol in the supernatant extracted by stirring for 1 minute was detected by the JIS detection method using aminoantipyrine (JIS K0102-1993, 28.1). Results are shown in FIG.

Degradation stopped halfway in the yeast extract-added system, and apparently progressed in the gluconic acid-added system.

Example 26. Effect of gluconic acid on decomposition of TCE in soil by TL2 strain (15 ℃, loamy soil) TCE 40ppm, phenol 500ppm as TCE decomposition inducer,
And 1 ml of M9 medium containing 0.05% gluconic acid or 0.1% yeast extract was poured into a screw vial bottle with a 15 ml Teflon inner lid, 4 g of loam soil similar to that in Example 23 was added, and further cultured as in Example 25. Bacterial fluid of TL2 strain 0.
After inoculating 1 ml, it was completely sealed, and statically cultured at 15 ° C, which is close to the temperature in the actual soil. Gluconic acid was added three times as in Example 25.

The amount of TCE was extracted by stirring with 5 ml of n-hexane for 3 minutes, diluted 10 times in some cases, and quantified by gas chromatography (ECD detector).
The decrease in TCE was measured. As a control, the TCE amount was also quantified in a system in which the TL2 strain was not added in the same experimental system, and the residual ratio to the TCE amount of the control was calculated. Fig. 3 shows the results.
6 is shown.

Similar to the case of the phenol decomposition of Example 25, the decomposition was stopped halfway in the yeast extract-added system, and the decomposition was obviously advanced in the gluconic acid-added system.

Example 27. Effect of α-ketoglutarate on degradation of phenol in soil by KB1 strain (15
(° C, loamy soil) KB1 strain colonies on M9 agar medium containing 0.2% α-ketoglutarate were inoculated into 200 ml M9 medium containing 0.1% α-ketoglutarate in a 500 ml Sakaguchi flask and incubated at 25 ° C for 24 hours. Shaking culture was performed.

Next, 800 ppm of phenol and 0.05% α-
M9 medium containing ketoglutaric acid or 0.1% yeast extract
Inject 1 ml into a 15 ml sterilized tube, add 4 g of loamy soil collected from Ibaraki prefecture, and inoculate 10 μl of the bacterial solution of KB1 strain cultivated as described above into half of that, then plug with cotton,
Static culture was performed at 15 ℃, which is close to the actual temperature in soil, and the phenol concentration was measured over time. It should be noted that α-ketoglutaric acid was added after 3 hours of culturing for 24 hours, 36 hours and 48 hours.
0.05% equivalent was added over the course of time. Phenol was quantified by adding 5 ml of sterilized water to the tube and detecting phenol in the supernatant by stirring and extracting for 1 minute by the detection method by JIS method using aminoantipyrine (JIS K0102-1933, 28.1). The results are shown in Fig. 37.

Degradation stopped halfway in the yeast extract-added system, and apparently progressed in the α-ketoglutaric acid-added system.

Example 28. Effect of α-ketoglutaric acid on decomposition of TCE in soil by KB1 strain (15 ℃, loamy soil) TCE20ppm, phenol 600ppm as TCE decomposition inducer, and M9 medium 1ml containing 0.05% α-ketoglutaric acid or 0.1% yeast extract Was poured into a screw vial with a 15 ml Teflon inner lid, and loam soil similar to that in Example 23 was added.
4 g was added, and 0.1 ml of the bacterial solution of KB1 strain cultivated as in Example 25 was further inoculated, completely sealed, and statically cultivated at 15 ° C. close to the actual temperature in soil. Note that α-ketoglutaric acid was added three more times as in Example 27. The amount of TCE was extracted by stirring with 5 ml of n-hexane for 3 minutes, diluted 10 times in some cases and quantified by gas chromatography (ECD detector), and the decrease in TCE was measured over time. As a control, KB in a similar experimental system
The amount of TCE in the system without the addition of one strain was also quantified, and the residual ratio to the amount of TCE in the control was calculated. The results are shown in FIG.

Similar to the case of the decomposition of phenol in Example 27, the decomposition of the yeast extract-added system stopped halfway and the decomposition obviously progressed in the α-ketoglutarate added system.

Example 29. Effect of itaconic acid on degradation of phenol in soil by KB2 strain (15 ℃, loamy soil) Colonies of KB2 strain on M9 agar containing 0.2% itaconic acid contained 0.1% itaconic acid in a 500 ml Sakaguchi flask. M9
200 ml of the medium was inoculated and shake culture was carried out at 25 ° C for 20 hours.

Then, 1 ml of M9 medium containing 200 ppm of phenol and 0.05% itaconic acid or 0.1% yeast extract was added to 15 ml.
After injecting it into a sterile sterile tube containing 4 ml of loam soil collected from Ibaraki prefecture, and inoculating 10 μl of the bacterial solution of KB2 strain, which had been cultivated as described above, into one half of the loam soil, cotton plug it and put it in the actual soil. C., and the phenol concentration was measured over time. In addition, itaconic acid was used in the culture 24
After hours and 36 hours, 0.05% equivalents were added. Phenol was quantified by adding 5 ml of sterilized water to the tube and stirring and extracting for 1 minute. Phenol in the supernatant was detected by the JIS method using aminoantipyrine (JISK0102-199).
3,28.1). The results are shown in Fig. 39. Degradation stopped halfway in the yeast extract-added system, and apparently progressed in the itaconic acid-added system.

Example 30. TCE in soil with KB2 strain
Effect of itaconic acid on the decomposition treatment of TCE (15 ℃, loamy soil) TCE20ppm, phenol 200ppm as a TCE decomposition inducer, and M containing 0.05% itaconic acid or 0.1% yeast extract
1 ml of 9 medium was poured into a screw vial with a 15 ml Teflon inner lid, and 4 g of loam soil similar to that used in Example 23 was added.
Further, 0.1 ml of bacterial solution of KB1 strain cultured as in Example 25.
After collecting, it is completely sealed and close to the temperature in the actual soil
Static culture was performed at 15 ° C. Note that itaconic acid was added twice more as in Example 29.

The amount of TCE was extracted by stirring with 5 ml of n-hexane for 3 minutes, diluted 10 times in some cases and quantified by gas chromatography (ECD detector), and the decrease of TCE was measured over time. As a control, in the same experimental system, the amount of TCE in the system in which KB2 strain was not added was also quantified, and the residual ratio to the TCE amount of the control was obtained. Fig. 4 shows the results.
0 is shown.

Similar to the case of the phenol decomposition of Example 29, in the yeast extract addition system, the decomposition stopped halfway, and in the itaconic acid addition system, the decomposition obviously progressed.

[0106]

The method of the present invention enables efficient biodegradation treatment in an aqueous medium containing an aromatic compound and / or an organic chlorine compound, soil, and a gas phase.

[Brief description of the drawings]

FIG. 1 is a diagram showing the growth of a bacterium (J1 strain) in Example 1.

FIG. 2 is a diagram showing the growth of a bacterium (J1 strain) in Example 2.

FIG. 3 is a diagram showing the growth of a bacterium (J1 strain) in Example 2.

FIG. 4 shows the growth of the bacterium (J1 strain) in Example 2.

FIG. 5 shows the growth of the bacterium (J1 strain) in Example 2.

FIG. 6 shows the growth of the bacterium (J1 strain) in Example 2.

FIG. 7 is a diagram showing the growth of the bacterium (J1 strain) in Example 3.

FIG. 8 shows the growth of the bacterium (J1 strain) in Example 4.

FIG. 9 shows the growth of the bacterium (J1 strain) in Example 4.

FIG. 10 shows the growth of the bacterium (J1 strain) in Example 4.

FIG. 11 shows the growth of the bacterium (JM1 strain) in Example 5.

FIG. 12 shows the growth of the bacterium (JM1 strain) in Example 6.

FIG. 13 shows the growth of the bacterium (JM1 strain) in Example 7.

FIG. 14 shows the growth of the bacterium (JM1 strain) in Example 8.

FIG. 15 shows the growth of the bacterium (JM1 strain) in Example 8.

FIG. 16 shows the growth of the bacterium (JM1 strain) in Example 8.

FIG. 17 shows the growth of the bacterium (KK01 strain) in Example 9.

FIG. 18 shows the growth of the bacterium (KK01 strain) in Example 10.

FIG. 19 shows the growth of the bacterium (KK01 strain) in Example 11.

FIG. 20 shows the growth of the bacterium (KK01 strain) in Example 12.

FIG. 21 shows the growth of the bacterium (KK01 strain) in Example 12.

FIG. 22 shows the growth of the bacterium (KK01 strain) in Example 12.

FIG. 23 shows the growth of the bacterium (J1 strain) in Example 13.

FIG. 24 shows the growth of the bacterium (J1 strain) in Example 14.

FIG. 25 shows the growth of the bacterium (J1 strain) in Example 15.

FIG. 26 shows the growth of the bacterium (JM1 strain) in Example 16.

FIG. 27 shows the growth of the bacterium (JM1 strain) in Example 17.

FIG. 28 shows the growth of the bacterium (JM1 strain) in Example 18.

FIG. 29 shows the growth of the bacterium (KK01 strain) in Example 19.

FIG. 30 shows the proliferation of the bacterium (KK01 strain) in Example 20.

FIG. 31 shows the growth of the bacterium (JM1 strain) in Example 21.

FIG. 32 shows the growth of the bacterium (JM1 strain) in Example 22.

FIG. 33 shows the growth of the bacterium (TL1 strain) in Example 23.

FIG. 34 shows the growth of the bacterium (TL1 strain) in Example 24.

FIG. 35 shows the growth of the bacterium (TL2 strain) in Example 25.

FIG. 36 shows the growth of the bacterium (TL2 strain) in Example 26.

FIG. 37 shows the growth of the bacterium (KB1 strain) in Example 27.

FIG. 38 shows the growth of the bacterium (KB1 strain) in Example 28.

FIG. 39 shows the growth of the bacterium (KB2 strain) in Example 29.

FIG. 40 shows the growth of the bacterium (KB2 strain) in Example 30.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area // (C12N 1/20 C12R 1:15) (C12N 1/20 C12R 1:38) (C12N 1 / 20 C12R 1:05) (C12N 1/20 C12R 1:63) (72) Inventor Yukitoshi Okubo 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc.

Claims (42)

[Claims] 1. A method of introducing a microorganism capable of decomposing the pollutant into the medium in a method for purifying a pollutant-contaminated medium; and at least one of growth of the microorganism and decomposition activity of the pollutant, Selectively select the pollutant by contacting the microorganism with a carbon source capable of selectively improving against a competitive microorganism that originally exists in the environment in which the pollutant exists, by contacting the microorganism with the microorganism twice or more times. A purification method comprising the step of decomposing into: 2. The purification method according to claim 1, wherein the first contact between the microorganism and the carbon source is performed when the microorganism is introduced into the medium. 3. The method according to claim 1, wherein the contact between the microorganism and the carbon source is carried out in a logarithmic growth phase of the microorganism.
Purification method according to any one of. 4. The purification method according to claim 1, wherein the contact between the microorganism and the carbon source is carried out when the growth of the microorganism is in a steady state. 5. The purification method according to claim 3, wherein the contact between the microorganism and the carbon source is performed when the growth of the microorganism is in a steady state. 6. The purification method according to claim 1, wherein the contaminant is at least one of an aromatic compound and an organic chlorine compound. 7. The method according to claim 1, wherein the microorganism is a microorganism expressing an oxidase, oxygenase. 8. Expressing an oxygenase
Corynebacterium (Corynebacterium )
Genus, Pseudomonas (Pseudomonas ) Genus, Acinetobacter
Tar (Acinetobacter ) Genus, Alcaligenes (Alcalige
nes ) Genus, vibrio (Vibrio) Genus, Nocardia (Nocard
ia) Genus Bacillus (Bacillus) Genus Lactobacillus (La
ctobacillus ) Genus, Achromobacter (Achromobacter
 ) Genus, Arthrobacter (Arthrobacter) Genus, micro
Coccus (Micrococcus ) Genus, mycobacterium (My
cobacterium ) Genus, Methylosynas (Methylosinus) Genus,
Methylomonas (Methylomonas) Genus, Berchia (Welchia
 ) Genus, methylocystis (Methylocystis ) Genus, nitro
Zomonas (Nitrosomonas) Genus, Saccharomyces (Saccha
romyces ) Genus, Candida (Candida ) Genus, Torlopsis
(Torulopsis) At least one of the microorganisms belonging to the genus
The method according to claim 7, wherein the method is a class. 9. The method according to claim 8, wherein the microorganism is Corynebacterium sp. 10. The method according to claim 9, wherein the microorganism is Corynebacterium sp. Strain J1 ( Corynebacterium sp. J1, Ministry of International Trade and Industry, Institute of Biotechnology, Industrial Technology, Accession No .: FERM BP-5102). . 11. The method according to claim 9, wherein the microorganism is Corynebacterium sp. Strain JM1 ( Corynebacterium sp. JM1, Ministry of International Trade and Industry, Institute of Life Science and Technology, deposit number: FERM P-14727). . 12. The method according to claim 8, wherein the microorganism is Pseudomonas sp. 13. The method according to claim 12, wherein the microorganism is Pseudomonas sp. TL1 strain ( Pseudomonas sp. TL1, Ministry of International Trade and Industry, Institute of Life Science and Technology, deposit number: FERM-P14726). The method according to claim 8, characterized in that 14. A microorganism is Pseudomonas alkali monocytogenes (Pseudomonasalcaligenes). 15. The method according to claim 14, wherein said microorganism is Pseudomonas alcaligenes KB2 ( Pseudomonas alcaligenes KB2 , Ministry of International Trade and Industry, Institute of Life Science and Technology, deposit number: FERM P-14644). 16. The method according to claim 8, wherein the microorganism is Pseudomonas cepacia . 17. The microorganism is Pseudomonas cepacia KK.
The method according to claim 16, which is strain 01 ( Pseudomonascepacia KK01, Ministry of International Trade and Industry, Institute of Biotechnology, Industrial Technology, Accession No .: FERM BP-4235). 18. The method according to claim 8, wherein the microorganism is Alcaligenes sp. 19. The method according to claim 18, wherein the microorganism is Alcaligenes sp. TL2 strain ( Alcaligenes sp. TL2, Ministry of International Trade and Industry, Institute of Life Science and Technology, deposit number: FERM-14642). 20. The microorganism is Vibrio species ( Vi)
brio sp.). 21. The microorganism is Vibrio species KB1.
21. The method according to claim 20, wherein the strain is a strain ( Vibrio sp. KB1, Ministry of International Trade and Industry, Institute of Biotechnology, Engineering number: FERM P-14643). 22. The method according to claim 1, wherein the carbon source is an organic acid or a salt thereof. 23. The method according to claim 22, wherein the organic acid or a salt thereof is a carboxylic acid or a salt thereof. 24. The carboxylic acid or a salt thereof is pyruvic acid, citric acid, gluconic acid, α-ketoglutaric acid,
24. The method according to claim 23, which is one or more of itaconic acid, malic acid, glutamic acid, or a sodium salt thereof. 25. The method according to claim 1, wherein the contaminated medium is an aqueous medium. 26. The method according to claim 25, wherein the contacting comprises contacting the carrier supporting the microorganism with an aqueous medium containing an aromatic compound and / or an organic chlorine compound. 27. A method of contacting, in which a carrier supporting the microorganism is contained in a container, and an aqueous medium containing an aromatic compound and / or an organic chlorine compound is introduced from one of the containers and discharged from the other. 27. The method of claim 26, wherein 28. The biodegradation and purification method according to claim 26, wherein the aromatic compound is at least one of phenol, toluene and cresol. 29. The biodegradation and purification method according to claim 26, wherein the organic chlorine compound is at least one of trichlorethylene (TCE) and dichloroethylene (DCE). 30. The method according to claim 1, wherein the polluting medium is soil. 31. Decomposition / purification is carried out by introducing an aqueous medium containing the microorganism into a contaminated soil and growing the microorganism in the soil by applying the selective carbon source and / or oxygen. 31. The method of claim 30 characterized. 32. The method according to claim 31, wherein the introduction of the microorganism into the soil is carried out by pressure from an injection well provided in the soil. 33. The method according to claim 30, wherein the contacting introduces a soil containing an aromatic compound and / or an organic chlorine compound into a liquid phase containing the microorganism. 34. The method according to claim 30, wherein the contacting is carried out by contacting a soil containing an aromatic compound and / or an organic chlorine compound with a carrier supporting the microorganism. 35. The biodegradation and purification method according to claim 33, wherein the aromatic compound is at least one of phenol, toluene and cresol. 36. The biodegradation and purification method according to claim 33, wherein the organic chlorine compound is at least one of trichlorotyrene (TCE) and dichloroethylene (DCE). 37. The method according to claim 1, wherein the polluting medium is air. 38. The method according to claim 37, wherein the contacting introduces air containing an aromatic compound and / or an organic chlorine compound into a liquid phase containing the microorganism. 39. The method according to claim 37, wherein the contacting is carried out by contacting the carrier supporting the microorganism with air containing an aromatic compound and / or an organic chlorine compound. 40. The contact is characterized in that a carrier supporting the microorganism is contained in a container, and air containing an aromatic compound and / or an organic chlorine compound is introduced from one of the containers and discharged from the other. 39. The method of claim 38. 41. The method according to claim 38, wherein the aromatic compound is at least one of phenol, toluene and cresol. 42. The method according to claim 38, wherein the organic chlorine compound is at least one of trichlorethylene (TCE) and dichloroethylene (DCE).