JP2655564B2 - New microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel microorganism, and more particularly to a novel microorganism of the genus Pseudomonas which can decompose a high concentration (75% v / v) of toluene in a solution containing salt. Toluene is a highly toxic organic solvent, but the microorganism of the present invention is resistant to aromatic hydrocarbons such as benzene and xylene in addition to 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 genera Pseudomonas, Flavobacterium, Arginogenes and the like are known as microorganisms capable of decomposing organic solvents, but these microorganisms have sufficient decomposition ability. In particular, when a hardly decomposable substance such as an aromatic hydrocarbon is contained, a long time is required for the decomposition. In addition, there is also a problem that when a relatively large amount of organic solvent flows out, it cannot be effectively handled.

[0003]

The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and as a result, the microorganisms isolated from the ocean grew in various organic solvents, and aromatic hydrocarbons such as toluene. They have the ability to decompose hydrogen and find that they meet the above objectives, and have completed the present invention.

[0004] That is, the present invention belongs to the genus Pseudomonas, has a resistance to organic solvents, and is used in a solution containing salt.
% (V / v) of a novel microorganism Pseudomonas putida strain SH-2992 (FERM P-
13564).

[0005] The microorganism of the present invention is a novel bacterium isolated from the ocean in Kanagawa Prefecture and having the following mycological properties. In the following tests, sodium chloride was used
A 2.5-supplemented medium was used. A. B. Morphological properties (1) Bacillus (2) Spores:-(3) Size: width 0.8-1.2 μm, length 1.2-4.0 μm Growth state 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) Growth in aerobic state: + (4) Catalase reaction: + (5) Oxidation of ethanol to acetic acid:-(6) Glucose Oxidative degradation of +: (7) Requirement of salt: + (8) Number of flagella:> 1 (9) Production of fluorescent dye: + (10) Production of pyocyanin:-(11) Production of carotenoid:-(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) Gelatin hydrolysis:-(21) Starch Hydrolysis of-: (22) Lecithinase (yolk):-(23) Lipase (Tween 80 hydrolysis):-(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) Utilization 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:-Hippurate: + L-sorbose:-Dextrin:-Raffinose:-Dulcit Rule:-

(34) Formation of acid from sugars Glucose: + mannitol:-adonitol:-arabinose:-inositol:-rhamnose:-sorbitol:-maltose:-sucrose:-

The above properties are described in the manual of Barjay.
Of Deterministic Bacteriology (Berge
A search was performed based on the eighth edition of the y's Manual of Determinative Bacteriology, which revealed that the bacterium belongs to the genus Pseudomonas and corresponds to Pseudomonas putida. However, the present bacterium is different from known strains belonging to Pseudomonas putida in properties such as D-arabinose availability and GC content of DNA. ) SH
The strain was named -2992 strain. This bacterium has been deposited with the National Institute of Advanced Industrial Science and Technology, and the deposit number is FERM
It is P-13564. In the present invention, in addition to the present bacterium, even mutants obtained by mutating them by natural or artificial means are included in the present invention.

Next, a method for testing the resistance and decomposition of an organic solvent in the present invention will be described. The organic solvent tolerance test is performed by inoculating the isolated strain on an LB agar plate medium, overlaying various organic solvents on the plate medium, culturing at 30 ° C. for 24 hours, observing the growth state of the bacteria, and examining the resistance ability. The presence or absence was determined. The decomposition test of the organic solvent was performed by filling a culture bottle (5 liters) with seawater to which peptone was added as a nitrogen source, sterilizing, adding a strain isolated from the organic solvent, and adding p.
H was adjusted to 7.0, the mixture was shaken (120 rpm / min), and the presence or absence of decomposition ability was determined by measuring the amount of carbon dioxide generated after completion of the culture.

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. Since this temperature varies depending on the type of the organic solvent, the composition of the medium, the pH and other conditions, it cannot be specified uniformly, but it can be set to, for example, 10 to 30 ° C, preferably 20 to 30 ° C. The pH of the reaction system may usually be set in the range of 6.0 to 7.5.

The cultivation is usually carried out under aerobic conditions, and for example, a shaking culture method, aeration stirring culture method, or the like can be used. The culture time depends on the amount and type of organic solvent,
When containing 1% of toluene, it is usually 10 days or more, preferably 15 to 30 days. Substances added as inorganic salts include phosphates, magnesium salts, calcium salts,
Iron salts and, if necessary, trace metal salts are used. The nitrogen source may be any one that can be assimilated by the test bacteria, such as peptone, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, and various amino acids. One of these nitrogen sources may be used alone, or two or more thereof may be used in appropriate combination. Further, an appropriate amount of a vitamin, yeast extract, malt extract or the like 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 test bacterium in advance in a culture solution containing a carbon source which can be assimilated by the bacterium, for example, glucose, lactose, saccharose, maltose, molasses, starch, etc. The case where the body is added to a liquid containing a substance having a composition similar to that of the above-described medium to carry out the reaction is also included.

[0015]

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 added on the plate medium, and the layers were cultured at 30 ° C. for 24 hours. After 24 hours of culture,
The growth state of the bacteria was visually observed, and the presence or absence of the resistance ability was determined. The results are shown in Table 1.

[0016]

[Table 1]

Example 2 A 200 ml shake flask was charged with 100 ml of LB medium supplemented with 2% NaCl, and various organic solvents were added to a predetermined concentration to obtain a viable cell count medium. LB in this medium
Organic solvent resistant bacterium Pseudomonas putida (Bioval A) SH-2992 strain grown and cultured for 24 hours in a medium
100 μl of a culture of (FERM P-13564) was added, a silicon stopper was attached, and the cells were cultured with shaking at 25 ° C. and 120 rpm / min. The cultivation was performed until the number of bacteria reached the stationary phase (10 ⁹ ), and the time was measured at a predetermined time from 24 hours to 240 hours. For comparison, the same test was performed when the cells were cultured 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 a cylindrical glass bottle. In other words, this bottle contains an inorganic medium (composition?) 1500 m
1 ml of Pseudomonas putida (Bioval A) SH-2992 strain (FERM P-13564) pre-cultured for 24 hours.
Was added thereto, toluene was added at a predetermined concentration, a Teflon-made rotor (5 × 2 cm) was put in, and all three sides of the culture bottle were sealed, followed by shaking culture at 25 ° C. and 180 rpm / min for 10 days. Confirmation of the decomposition of the organic solvent was performed by measuring the concentration of carbon dioxide generated by decomposition of the organic solvent. The measurement was performed by analyzing a sample in which a certain amount of gas was collected 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.
Has the ability to decompose toluene. in this way,
Since this bacterium can decompose organic solvents such as toluene in sewers and seawater containing salt, it is expected to be used for treatment of sewers and seawater contaminated with organic solvents and oils.

Claims (2)

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