JPH0965875A - New bacterium decomposing pcbs - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new bacterium having resistance to organic solvents, excellent in ability to decompose poly-chlorinated biphenyls(PCBs), capable of decomposing PCBs in a short time and having also excellent ability to decompose a slightly decomposable coplanar PCBs having strong toxicity. SOLUTION: This new bacterium is Pseudomonas putida SN-4992 (FERM- P-15072) having resistance to organic solvents, excellent in decomposing property of poly-chlorinated biphenyls (PCBs), capable of decomposing in a short time and having high decomposing ability to PCBs having high concentration (about 1,000ppm) and having also excellent ability to decompose slightly decomposable coplanar PCBs having strong toxicity. The new bacterium is obtained by culturing bacteria separated from soil collected in Kanagawa prefecture in a culture medium containing biphenyl, screening a bacterium decomposing biphenyl, culturing the bacterium in a culture medium, selecting a strain having decomposing ability of PCBs and further selecting bacterial strain growing in toluene.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention has organic solvent resistance,
The present invention relates to a novel microorganism that assimilates and decomposes polychlorinated biphenyls (hereinafter referred to as PCBs), in particular, it decomposes highly toxic coplanar PCBs and provides high concentration of P.
The present invention relates to a novel microorganism that decomposes CBs in a short time. This novel microorganism can be used for decomposing and removing PCBs contaminating soil or water in the environment or detoxifying stored PCBs.

[0002]

2. Description of the Related Art PCBs are often used in capacitors, transformers, etc. because they are chemically stable and have good thermal characteristics. However, due to their toxicity to the human body, their production was prohibited as a harmful environmental pollutant in the 1970s. It was And
The PCBs manufactured or used so far will be collected and stored. However, some have already been released into the environment and some have been dissipated over the past 20 years. Such PCBs are stable and difficult to decompose even in the environment, and the concentration of PCBs detected in the environment has not decreased. Therefore, a method for efficiently decomposing and treating PCBs in the environment is required. Incineration is also performed as a disposal method for PCBs stored. However, there is a risk that harmful by-products such as dioxins may be generated during incineration, and the treatment is limited.

Under such circumstances, it has been considered to decompose PCBs by using microorganisms, and some bacteria having the ability to decompose PCBs have been reported. According to these, Pseudomonas spp, Flavobacterium spp, and Alcalzenes spp are known as microorganisms that decompose PCBs.

[0004]

However, the conventionally known PCBs-degrading microorganisms have the following problems. Since PCBs are fat-soluble, it takes a long time to decompose in ordinary liquid culture, but if the microorganisms have resistance to an organic solvent, PCBs can be dissolved in this organic solvent and continuously decomposed. It will be possible. However, aromatic hydrocarbons such as toluene and xylene are generally harmful to microorganisms, and PCBs-degrading microorganisms having strong resistance to these organic solvents have not been reported so far.

In addition, in the conventionally known microorganisms, P
The decomposition ability of CBs is weak, and high concentration of PCBs (for example, 1
There is a problem that it takes a long time to decompose (about 00 ppm). Furthermore, among PCBs, no microorganism has been reported that decomposes highly toxic coplanar PCBs in a short time.

The present invention has been made in view of the above problems, and an object thereof is to grow in the presence of a large amount of organic solvent and to decompose high-concentration PCBs. Coplanar PC with strong toxicity
It is to provide a novel microorganism excellent in the ability to decompose Bs.

[0007]

In order to solve the above problems, the present invention provides a novel microorganism, Pseudomonas putida FERMP-15072, which has organic solvent resistance and decomposes polychlorinated biphenyls. .

This microorganism is isolated from nature and is extracted by the following steps. Approximately 200 samples of soil, landfill leachate, sludge, seawater, etc. were collected from 125 locations nationwide and the bacteria contained in these samples were searched. First, 10 ml of the modified PAS medium and 0.1 g of biphenyl are put in a test tube, and 1 g or 1 ml of collected data after sterilization is added and stirred. This is 30
After static culture for a day, observe the changes in the color and turbidity of the culture solution in a test tube to search for bacteria that decompose biphenyl. The modified PAS medium will be described later. The bacteria extracted in the above culture unify the colonies on LB agar medium. This single bacterium was precultured in LB liquid medium for 24 hours, and the above culture solution 10 was added to a culture flask (volume 200 ml) containing 100 ml of modified PAS medium and 10 ppm of PCBs.
Add 0 μl and incubate with shaking. PCB from these strains
56 strains were obtained as those having s-degradability. From these, 11 strains with high ability to decompose PCBs were selected, and inoculated into a petri dish overlaid with an organic solvent and examined for growth. When the organic solvent was hexane, most of the bacteria were found to grow. However, when toluene was used as the organic solvent, growth was observed only in 4 out of 11 strains. These four
Among the strains, the one having the highest degradability of PCBs is the novel microorganism according to the present invention, and Pseudomonas putida SN-4
Name it 992. This strain was isolated from a sample (soil) collected in Kanagawa prefecture, and has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, and its deposit number is FERM P-15072.

The bacteriological properties of this bacterium are shown below. A. Morphology (1) Bacillus (2) Spore: (-) (3) Size (μm): width 0.8 to 1.2, length 1.2
~ 4.0 B. Growth state in culture medium (1) Growth temperature range: The optimum growth temperature range is 20 to 30 ° C. Physiological and biochemical properties (1) Gram reaction: Negative (2) Motility: + (3) Aerobic growth: + (4) Anaerobic growth:-(5) Catalase reaction: + (6) Oxidase reaction: + (7) Fluorescent dye production: + (8) OF medium: Oxidation (9) Growth at 41 ° C .: − (10) Growth at 4 ° C .: + (11) GC content (mol%): 63 0.0 (12) Production of acid from sugar Glucose: + Mannitol: − Adonitol: − Arabinose: − Inositol: − Rhamnose: − Sorbitol: − Maltose: − Sucrose: −

The above properties are described in the manual of Barjay.
Of Determinating Bacteriology Eighth Edition
(Bergey's Manual of Determinative Bacteriology, 8t
Based on the above properties, this bacterium belongs to the genus Pseudomonas and is considered to correspond to Pseudomonas putida. However, this strain is different from known strains belonging to Pseudomonas putida in sugar degradability, GC
It is considered to be a new strain due to differences in the content. In addition,
The bacterium can be stored, for example, under the following conditions. L
After growing in B medium, 100 μl of the growing bacteria was added to 200 μl of glycerin, and the mixture was well stirred-
Store at 80 ° C. Alternatively, add skim milk, freeze-dry and store.

[0011]

EXAMPLES The properties of the novel microorganism according to the present invention will be described below based on the test results. (1) Organic solvent resistance The organic solvent resistance was judged by the following test. The strain was inoculated on an LB agar plate medium, and each solvent was added to the plate medium with 20
After overlaying with ml, the mixture is incubated at 30 ° C. for 96 hours. After culturing for 96 hours, the quality of the growth of the bacteria is observed, and the presence or absence of resistance ability is judged.

The test results are shown in Table 1.
The strain Pseudomonas according to the invention
Although putida) SN-4992 is not observed to grow in benzene, it is observed to grow in n-hexane, cyclohexane, p-xylene, o-xylene, toluene or n-heptanol. Therefore, it is understood that it has resistance to most organic solvents and has superior organic solvent resistance to other already known bacteria. In Table 1, Log Pow is a logarithmic representation of the partition coefficient of each organic solvent.

[0013]

[Table 1]

(2) Degradability of PCBs Conditions for performing a PCBs degradation test are set as follows. The temperature is set within the range of the growth temperature of the microorganism used, preferably within the range of the optimum growth temperature. Although it cannot be specified uniformly because it depends on the type of PCBs, the composition of the medium, pH and other conditions, it can be set to, for example, 10 to 30 ° C, preferably 20 to 30 ° C. The pH of the reaction system may be usually set in the range of 6.0 to 7.5. The culture is usually carried out under aerobic conditions, and a shaking culture method, aeration stirring method, or the like can be used. The culturing time varies depending on the amount and type of PCBs, but when PCBs are contained in an amount of 10 ppm or more, it is usually 10 days or more, preferably 15 to 30 days. As substances added to the medium as inorganic salts, phosphates, magnesium salts, calcium salts, iron salts and, if necessary, trace metal salts are used. The substance added as a nitrogen source may be any substance that can be assimilated by the bacteria used, and examples thereof include urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, and various amino acids.
These nitrogen sources may be used alone or in combination of two or more. Further, vitamins, yeast extract, malt extract, and the like may be added in appropriate amounts as a nutrient source for promoting the growth of the bacteria used.

The test may be carried out after culturing the bacterium in the above-mentioned medium, and the bacterium used for the test may be a carbon source capable of assimilating it, such as glucose, lactose, sucrose, maltose, molasses molasses. The test may be performed by pre-culturing in a culture solution containing starch, etc., and adding the obtained cells to a solution containing a substance having the same composition as the above-mentioned medium.

(A) PCB of the bacterium according to the present invention and a standard bacterium
Comparison of s-degradability Pseudomonas putida SN-4992 of the present invention
A test for comparing the degradability of PCBs was carried out using the following bacteria as follows. Take 3 platinum loops from the petri dish and preculture for 48 hours using LB liquid medium. On the other hand, 100 ml of modified PAS medium and 10 ppm of PCBs are put into a culture flask having a volume of 200 ml, and 100 μl of the above culture solution is added. This is 3 at 30 ℃
Shake culture (120 rpm) for 0 days.

The above-mentioned improved PAS medium contains K in 1 liter.
₂ HPO ₄ : 0.45 g, KH ₂ PO ₄ : 0.20 g,
NH ₄ Cl: 0.25 g, MgSO ₄ .7H ₂ O: 0.
_{020g, ZnSO 4 · 7H 2 O} : 0.005g, Fe
SO ・ 7H ₂ O: 0.0010g, CaCl ₂・ 2H ₂
O: 0.0005 g, L-ascorbic acid: 0.001
It contains 0 g. PCBs added to this are the only carbon sources.

After culturing for 30 days, the culture broth was 10,000.
Centrifuge for 15 minutes at rpm to separate into supernatant and precipitate. PCBs are extracted by adding 25 ml of n-hexane to the supernatant, and this is repeated 3 times. 2 ml of nitric acid and 2 ml of sulfuric acid were placed in the precipitation portion to completely decompose the cells, and 25 ml of n-hexane was added in the same manner as the supernatant to prepare PC.
Extract Bs (repeat 3 times). The respective extracts are combined and concentrated with an evaporator to make 2 ml. PCBs in this liquid were analyzed by a gas chromatograph with an electron capture detector (ECD-GC) [manufactured by Shimadzu Corporation 15
A-GC, 2 m column OV-17, column temperature 200
C., N ₂ gas 40 ml / min) to determine the PCBs concentration. The amount of decomposition is calculated by comparing this with the amount of PCBs added to the culture solution. The results of such a test are shown in Table 2.

[0019]

[Table 2] Put on this table, KC-200, KC-300, KC-
400 and KC-500 indicate the types of PCBs (Kanechlor, manufactured by Kanegafuchi Chemical Co., Ltd.), each mainly containing 2, 3, 4, 5 chlorine atoms in the molecule. I'm out.

As shown in this table, Pseudomonas putida SN-4992, which is a bacterium according to the present invention, has a remarkably high degradability of PCBs as compared with other already known bacteria. Pseudomonas Petida ST-299
2 has a considerably high degradability with respect to KC-200, but the degradability with respect to KC-500 is greatly reduced, which is significantly different from the degradability of Pseudomonas putida SN-4992.

(B) Decomposition test of high-concentration PCBs This test was conducted in order to investigate the decomposability of PCBs existing at high concentrations, and the procedure is as follows. Three platinum loops of the bacterium according to the present invention (Pseudomonas putida SN-4992) were collected from a petri dish and added to a culture flask (volume 100 ml) containing 50 ml of LB liquid medium. This is 30 ℃
Culture with shaking (120 rpm) for 72 hours. This culture solution is centrifuged at 10,000 rpm for 15 minutes to collect the bacterial cells, and the cells are washed 3 times with physiological saline. And improved P
Culture flask containing 100 ml of AS medium (volume 200
ml), 100 ppm PCBs as the sole carbon source
And add the above cells. This is shake-cultured at 30 ° C. for 10 days.

The amount of decomposed PCBs after culturing is measured in the same manner as the test for comparing the decomposability of PCBs described in (a). The results of this test are as shown in Table 3, and it is understood that the higher the number of chlorine in the molecule, the lower the decomposition rate, but the higher the decomposability.

[0023]

[Table 3]

(C) Decomposition test of coplanar PCBs Coplanar PCBs are isomers with a particularly strong toxicity and have the following molecular structures.

Embedded image The degradability of the coplanar PCBs was tested using the bacterium of the present invention and the bacterium already known, and the results are shown below.

3 platinum loops of bacteria were collected in LB liquid medium, and 48
Pre-culture for hours. Then, this culture solution is centrifuged to collect proliferating bacteria. This fungus was placed in a culture bottle (capacity 200 m
l) suspended in 50 ml of modified PAS medium in
PCBs of 0 ppm (Kanecrawl K manufactured by Kanegafuchi Chemical Co., Ltd.)
5 ml of toluene in which 1 kg of C-400 contains about 4 g of coplanar PCBs) is added, and shaking culture (100 rpm) is performed at 30 ° C. After culturing, centrifugation is performed to separate into a supernatant liquid portion and a precipitation portion. For the supernatant, undigested PCBs were prepared by adding an equal amount of n-hexane.
The operation of extracting is repeated three times. Regarding the precipitated portion, 10 ml of concentrated sulfuric acid and 5 ml of concentrated nitric acid are added to completely decompose the cells, and then 35 ml of distilled water is added and cooled.
Then, the operation of adding an equal amount of n-hexane and extracting undecomposed PCBs is repeated three times. The n-hexane extracted from the supernatant and the precipitated portion are combined and concentrated with an evaporator to a final volume of 2 ml.

On the other hand, 0.5 g of an activated carbon / silica gel mixture was packed in a column for chromatography by a wet method using n-hexane, and 2.0 g of anhydrous sodium sulfate was laminated. 2m of analytical sample concentrated in the above step on this column
1 is introduced, and the operation of injecting 1 ml of n-hexane while washing the inside of the test tube and the column, which is the container for the analytical sample, is carried out twice, and the entire amount of the analytical sample is introduced into the column. And 200m of 2% benzene / n-hexane mixture
1 is introduced, and low polarity interfering substances such as PCBs other than coplanar PCBs are eluted and removed. After this, 30 ml of toluene
Is added to elute the coplanar PCBs. N for this sample
-Add 0.2 ml of decane and 50 ° with an evaporator.
After maintaining at C to remove toluene, n-hexane is added as a solvent to make a 3 ml sample.

The measurement of the decomposition rate of coplanar PCBs is performed by magnetic field type gas chromatography / mass spectrometry (GC / MS).
The analysis is performed using [JMS-SX102A manufactured by JEOL Ltd.]. For this analysis, ULTRA1 (40 m × 0.2) manufactured by Hewlett-Packard Co. was used as a column.
mm × 0.11 μm) and the temperature is raised under the following conditions to use the spletless method. Coplanar PCBs
A commercially available standard product can be used for the identification and quantification of the decomposition components of. Temperature rising condition ・ Hold at 100 ° C for 1 minute. -The temperature is raised from 100 to 200 ° C at 25 ° C / min. -The temperature is raised at 2 ° C / min from 200 to 280 ° C. -Then, maintain at 280 ° C for 5 minutes.

The results of the above test are shown in Table 4, and the bacterium according to the present invention is coplanar PCBs.
It is understood that also has a high degradability.

[Table 4]

[0029]

As described above, the novel microorganism of the present invention has strong organic solvent resistance and high P
Since it has the ability to decompose CBs, PCBs dissolved in an organic solvent can be decomposed more quickly. Therefore, PC that pollutes river water, seawater, sewage, etc.
It can be used for the decomposition and treatment of Bs and the purification of soil contaminated with PCBs. In addition, this novel microorganism has a high degradability even for PCBs having a high concentration, for example, about 100 ppm, and can be used for treating highly contaminated water or contaminated soil.
It is also possible to use it for the processing of. Furthermore, it can also be used for the decomposition treatment of coplanar PCBs that have high toxicity and are difficult to decompose.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C02F 3/34 ZAB B09B 3/00 ZABE (C12N 1/20 C12R 1:40) (72) Inventor Michio Hayashi 2-16-9 Kabukicho, Shinjuku-ku, Tokyo Shinjuku TK Building Sumicon Celtech Co., Ltd.

Claims (1)

[Claims] 1. Pseudomonas putida FERM P-, which has organic solvent resistance and decomposes polychlorinated biphenyls.
15072.