JPH06277045A - New microorganism - Google Patents

Abstract

PURPOSE:To provide a new microorganism belonging to the genus Pseudomonas, having organic solvent resistance and an ability to decompose toluene of a prescribed concentration in a salt-containing solution, and suitable for treating sewerages contaminated with salts, organic solvents, etc. CONSTITUTION:This new microorganism is a strain, Pseudomonas putida SH-2992 (FERM-P-13564), belonging to the genus Pseudomonas, having organic solvent resistance against organic solvents containing at least one kind of benzene, toluene, and xylene, and having an ability to decompose 75% (v/v) toluene in a solution containing salts. The microorganism has following mycological properties. Morphological properties: bacillus; spore size: 0.8-1.2mum wide and 1.2-4.0mum long; growth state in a medium: optimal growth temperature range of 20-30 deg.C; physiological properties: gram reaction is negative, mobility is +, growth in an aerobic state is +, catalase reaction is +, the oxidation of ethanol into acetic acid is -.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism of the genus Pseudomonas which is capable of decomposing high concentration (75% v / v) of toluene in a solution containing sodium chloride. Toluene is highly toxic among organic solvents, but the microorganism of the present invention is resistant to aromatic hydrocarbons such as benzene and xylene as well as toluene. Therefore, the microorganism of the present invention can be used for removing organic solvents in the ocean, decomposing organic solvents such as sewers, and the like.

[0002]

2. Description of the Related Art Generally, microorganisms belonging to the genus Pseudomonas, Flavobacterium, Arginogenes, etc. are known as microorganisms capable of decomposing organic solvents. In particular, when a hardly decomposable substance such as aromatic hydrocarbon is contained, the decomposition takes a long time. In addition, there is also a problem that when a relatively large amount of organic solvent flows out, it is not possible to effectively cope with it.

[0003]

The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, microorganisms isolated from the ocean grow in various organic solvents, resulting in aromatic carbonization such as toluene. The present invention has been completed by finding that it has the ability to decompose hydrogen and meets the above purpose.

That is, the present invention belongs to the genus Pseudomonas, has resistance to organic solvents, and is 75 in a solution containing salt.
% (V / v) of a novel microorganism Pseudomonas putida SH-2992 (FERM P-
13564).

The microorganism of the present invention is a novel bacterium which is isolated from the ocean in Kanagawa prefecture and has the following mycological properties. In the following tests, sodium chloride was used.
2.5 supplemented medium was used. A. Morphological Properties (1) Bacillus (2) Spores :-( 3) Size: Width 0.8-1.2 μm, Length 1.2-4.0 μm B. Growth condition in culture medium (1) Growth temperature range: Optimal growth temperature range is 20-30 ° C

C. Physiological properties (1) Gram reaction: Negative (2) Motility: + (3) Aerobic growth: + (4) Catalase reaction: + (5) Oxidation of ethanol to acetic acid: − (6) Glucose Degradation: + (7) Salt requirement: + (8) Number of flagella:> 1 (9) Fluorescent dye production: + (10) Pyocyanin production:-(11) Carotenoid production:-(12 ) Production of other pigments :-( 13) OF medium: + (14) Growth at 41 ° C :-( 15) Growth at 4 ° C: +

(16) Production of levan from sucrose :-( 17) Arginine dihydrolase: + (18) Oxidase reaction: + (19) Denitrification reaction:-(20) Hydrolysis of gelatin:-(21) Starch Hydrolysis of :-( 22) Lecithinase (yolk) :-( 23) Lipase (hydrolysis of Tween 80) :-( 24) GC content of DNA (mol%): 66.8% (25) Lysine decarboxylase:- (26) Ornithine decarboxylase:-(27) Urease:-(28) Indole production:-(29) VP test: + (30) Hydrogen sulfide production:-

(31) ONPG hydrolysis :-( 32) Phenylalanine decarboxylase:-(33) Availability of carbon source: Glucose: -Trehalose: -DL-Arginine: + Acetate: + Succinate: + Fumaric acid Salt: + L-malate: + Lactate: + Citrate: + Pyruvate: + Glycerol:-D-arabinose:-Maltose:-Cellobiose:-Lactose:-Starch:-Inulin:-Phthalate : -Mannitol: -D-xylose: -L-arabinose: -rhamnose: -D-galactose: -sucrose: -trehalose: -gluconate: + malonate: -D-malate: -L-tartaric acid Salt:-Sorbitol: -Adonitol:-Ethanol: -Naphthalene: -Hiprate: + L-sorbose: -Dextrin: -Raffinose: -Dulcit :-

(34) Production of Acid from Sugar Glucose: + Mannitol: -Adonitol: -Arabinose: -Inositol: -Rhamnose: -Sorbitol: -Maltose: -Sucrose:-

The above various properties are described in Berjay's manual
Of deterministic bacteriology (Berge
A search based on the y's Manual of Determinative Bacteriology) 8th edition revealed that this bacterium belongs to the genus Pseudomonas and corresponds to Pseudomonas putida. However, since this bacterium exhibits properties different from known strains belonging to Pseudomonas putida in terms of utilization of D-arabinose and GC content of DNA, it is recognized as a new strain, and this bacterium is identified as Pseudomonas putida (Biovar A ) SH
The strain was named −2992 strain. This bacterium has been deposited at the Institute of Biotechnology, Institute of Biotechnology, and the deposit number is FERM.
It is P-13564. In the present invention, in addition to the bacterium, mutant strains obtained by mutating the bacterium by natural or artificial means are also included in the present invention, as long as they have the above-mentioned ability.

Next, the test method for resistance and decomposition of the organic solvent in the present invention will be explained. The organic solvent resistance test was carried out by inoculating the isolated strain on an LB agar plate medium, overlaying various organic solvents on the plate medium, and culturing at 30 ° C. for 24 hours, observing the growth state of the bacteria, and measuring the resistance ability. The presence or absence was judged. In addition, the decomposition test of the organic solvent was carried out by filling a culture bottle (5 liters) with seawater added with peptone as a nitrogen source, sterilized, and then added with the strain separated from the organic solvent.
The presence or absence of decomposition ability was determined by adjusting H to 7.0, shaking (120 rpm / min), and measuring the amount of carbon dioxide generated after the culture was completed.

The oxidative decomposition conditions in this decomposition method are set within the range of the growth temperature of the microorganism used, preferably within the range of the optimum growth temperature. This temperature cannot be uniformly specified because it varies depending on the type of organic solvent, the composition of the medium, the pH, and other conditions, but 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 the shaking culture method, aeration and stirring culture method and the like can be used. The culture time varies depending on the amount and type of organic solvent,
When it contains 1% of toluene, it is usually 10 days or longer, preferably 15 to 30 days. Substances added as inorganic salts include phosphates, magnesium salts, calcium salts,
Iron salts and other trace metal salts are used if necessary. The nitrogen source may be any one that can be assimilated by the test bacterium, and examples thereof include peptone, 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 kinds. Furthermore, an appropriate amount of vitamins, yeast extract, malt extract, etc. may be added as a nutrient source for promoting the growth of the test bacteria.

In the present invention, a bacterium obtained by culturing the above-mentioned test bacterium in advance in a culture medium containing a carbon source that can be assimilated by the bacterium, for example, glucose, lactose, saccharose, maltose, molasses, starch, etc. It also includes the case where the body is added to a liquid containing a substance having the same composition as the above-mentioned medium to carry out the reaction.

[0015]

EXAMPLES Next, the present invention will be described in detail with reference to Examples. Example 1 Separated strains and preserved strains were inoculated on an LB agar plate medium, 30 ml of various organic solvents were placed on this plate medium, and after overlaying, the mixture was cultured at 30 ° C. for 24 hours. After culturing for 24 hours,
The growth state of the bacteria was visually observed, and the presence or absence of tolerance was judged. The results are shown in Table 1.

[0016]

[Table 1]

Example 2 100 ml of 2% NaCl-added LB medium was placed in a 200 ml shake flask, and various organic solvents were added so as to have a predetermined concentration, which was used as a viable cell count medium. LB in this medium
Organic solvent-resistant bacterium Pseudomonas putida (Biobar A) SH-2992 strain grown and precultured in a medium for 24 hours
100 μl of a culture of (FERM P-13564) was added, a silicon stopper was attached, and the culture was carried out with shaking at 25 ° C. and 120 rpm / min. Culturing was performed until the number of bacteria reached the stationary phase (10 ⁹ cells), and the time was determined by measuring at a predetermined time from 24 hours to 240 hours. For comparison, the same test was carried out when the culture was performed without adding an organic solvent. The results are shown in Tables 2 and 3.

[0018]

[Table 2]

[0019]

[Table 3]

Example 3 Total volume 5250 ml, diameter 200 mm, height 330 mm
The organic solvent decomposition test was carried out using the cylindrical glass bottle of. That is, 1500m of inorganic medium (composition?) In this bottle
1 ml of Pseudomonas putida (Biovar A) SH-2992 strain (FERM P-13564) pre-cultured for 24 hours
Was added, toluene of a predetermined concentration was added, a Teflon rotor (5 × 2 cm) was put therein, all three mouths of the culture bottle were closed, and shake culture was carried out at 25 ° C. and 180 rpm / min for 10 days. The decomposition of the organic solvent was confirmed by measuring the carbon dioxide concentration generated by the decomposition of the organic solvent. The measurement was performed by analyzing a sample obtained by collecting a certain amount of gas from the gas phase portion of the culture bottle with a microsyringe by gas chromatography. The results are shown in Table 4.

[0021]

[Table 4]

[0022]

INDUSTRIAL APPLICABILITY The novel microorganism of the present invention is resistant to various organic solvents and is 75% (v / v) in a solution containing salt.
It has the ability to decompose toluene. in this way,
Since this bacterium can decompose organic solvents such as toluene in sewerage containing salt and seawater, it is expected to be used for treating sewerage and seawater contaminated with organic solvents and oil.

Claims (2)

[Claims] 1. A novel microorganism Pseudomonas putida SH-2992 strain (FERM) belonging to the genus Pseudomonas, which is resistant to organic solvents and has the ability to decompose 75% (v / v) of toluene in a solution containing sodium chloride. P-13564). 2. The novel microorganism according to claim 1, wherein the organic solvent contains at least one of benzene, toluene and xylene.