JPS61282066A - Production of novel bacterium - Google Patents

Abstract

PURPOSE:To product 1-acenaphthenol or to remove acenaphthenone as an impurity, by using a novel bacterium growing in a medium containing only acenaphthenone as a carbon source, capable of producing 1-acenaphthenol. CONSTITUTION:A strain such as Pseudomonas sp.A-5 (FERM-P 8300), belonging to the genus Pseudomonas, growing in a medium containing only acenaphthenone as a carbon source, capable of producing 1-acenaphthenol, is cultivated in the medium containing only acenaphthenone as a carbon source under an aerobic condition such as shaking culture, etc., at 6.0-9.0pH of the medium a t 25-35 deg.C for 20-70hr and 1-acenaphthenol is collected from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for obtaining a microorganism belonging to the genus Pseudomonas that grows in a medium containing only acenaphthene as a carbon source and produces 1-acenaphthenol.

[Conventional technology]

App, a shows that 1-acenaphthenol can be obtained by oxidizing acenaphthene in the presence of succinic acid.
nd Env, Hicrobiology, Jul
y, 1984. It is described in plo. However, this report is an example of oxidation by Co-oxidation, and there is no report that bacteria grow using acenaphthene as the sole carbon source.

[Problem that the invention seeks to solve]

The present invention grows in a medium containing only acenaphthene, and
The aim is to efficiently obtain microorganisms that produce acenaphthenol.

[Means for solving problems]

The present invention relates to a microorganism belonging to the genus Pseudomonas,
Grown in a medium containing only acenaphthene as a carbon source, 1
- A method for obtaining new microorganisms by isolating strains that produce acenaphthenol.

The microorganisms obtained in the present invention belong to the genus Pseudomonas and selectively produce 1-acenaphthenol by assimilating acenaphthene, and one example is Pseudomonas SP.

A-5 (Feikoken Bibori No. 8.300) is mentioned.

This strain has the following mycological properties.

(a) Morphology 1) Cell shape and size: Bacillus, 0.375 μm x 1.25 μm 2) Pleomorphism: None 3) Motile bi-motile, with polar flagella 4) Spores: Not formed 5) Durum Stainability: Negative 6) Acid-fastness: Negative (b) Growth status in each medium 1) Juicy agar plate culture: Growth is abundant, and colonies are brownish-yellow and shiny 2) Juicy agar slant culture: Growth is abundant and glossy 3) Meat juice liquid culture: A thick film is formed on the surface, resulting in turbidity 4) Meat juice gelatin puncture culture: No growth 5) Lidomus milk: Alkaline, no peptonization observed (C) Physiological properties 1) Nitrate reduction: positive 2) Denitrification reaction: negative 3) MR test: negative 4) VP test: negative 5> Indole formation: negative 6) Hydrogen sulfide formation (TSI agar): Positive 7) Hydrolysis of starch: Negative 8) Utilization of citric acid: Simmons' medium: utilized Christensen's medium: utilized “9) Utilization of inorganic nitrogen sources: Nitrate: utilized Ammonium salt: utilized 10) Pigment Production: None 11) Urease: Negative 12) Oxidase: Negative 13) Catalase: l!Ji 14) Growth range: Temperature: 26.5-37°C pH: 6.o-9.0 15) Attitude towards oxygen :Aerobic 16) OF test type 20 (oxidized type) (Note) 10% peptone water, 30"C, static culture for 114 days, indicator: For bacteria with mycological properties of BCP (bromcresol purple) or higher Bergey's Manual of Deter
As a result of a search based on the 8th edition (1975) of Pseudomonas min-tive Bacter+o+ogy, this strain was recognized as a new strain belonging to Pseudomonas 1, and was named Pseudomose SP, A-5.

In addition, this acenaphthene assimilating bacteria, strain A-5, can assimilate acenaphthylene, acenaphthenequinone, 1,2-acenaphthene diol, 1,8-monophthalic acid, benzoic acid, etc. in addition to 7-cenaphthene. I can do it.

The present bacterial strain can be isolated from soil or the like as an isolation source using a medium containing only acenaphthene as a carbon source. For example, first, the soil in which this strain exists is added to a suitable medium containing only acenaphthene as a carbon source and cultured, and then 1 platinum of the above culture solution is added to a similar plate medium with agar added. The ears are streaked, and each colony that has emerged is further suspended in the optimal liquid A medium, and then streaked again on a plate medium. Repeat this operation until uniform colonies are obtained on the plate.
Once uniform colonies are obtained, this can be carried out by confirming that the resulting colonies are not different from each other.

The culture medium used for the isolation of the above strains includes acenaphthene as a carbon source, ammonia as a nitrogen source, inorganic and organic ammonium salts such as ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, ammonium succinate, etc., such as urea, peptone, etc. Nitrogen-containing organic matter such as yeast extract, etc., as an inorganic salt, for example, KH2
Those containing various phosphates such as PO4, Na2HPO4.12H20, magnesium sulfate, ferrous sulfate, calcium chloride, etc. can be used.

Cultivation is usually preferably carried out under aerobic conditions such as shaking culture. The pH of the medium is 6.0-9.0, and the culture temperature is 25-3.
A culture period of 20 to 70 hours is usually appropriate at 5°C.

The novel microorganism obtained by the method of the present invention can be used for producing useful organic compounds such as 1-acenaphthenol. That is, the strain of the present invention is cultured in a medium containing acenaphthene as a carbon source, and 1-acenaphthenol, which is the target substance, is collected from the resulting culture solution and purified. A method for collecting and purifying 1-acenaphthenol from this culture solution can be performed according to a method for collecting and purifying general organic compounds. For example, a filtrate obtained by removing Kokutai and other substances from a culture solution is made acidic, and then extracted with an organic solvent such as ether, and this extract is analyzed using TLC, HPLC, etc.
- Isolate acenaphthenol.

Furthermore, the above-mentioned bacterial strain can also be used to remove acenaphthene and the like which this bacterial strain can be responsible for. For example, acenaphthene and the like contained as impurities and acenaphthene and the like contained in waste can be removed.

〔Example〕

Hereinafter, the method of the present invention will be specifically explained based on Examples.

Example 1 An acenaphthene-containing liquid medium (I>10-) having the following composition and pH was placed in a test tube, 0.1 g of the collected soil was added thereto, and the culture was incubated at 30° C. for 7 days.

Composition and pH of liquid separation medium (I) Acenaphthene 2gNH4NO34
g KH2PO41,5g Na HPO-12HO1,5g Mg50 ・lH2O10jI! F Fe50 ・7H205JIy Ca C1210'9 Yeast extract 10jIy Deionized water 1.0OOdpH7,2 (5NNaO
H) Approximately 3 loopfuls of the above culture solution were added to 101d of the same fresh separation liquid medium (I) as above, and cultured again at 30°C for 3 to 7 days. When visible growth was observed, one platinum loop of the culture solution was streaked onto a plate medium prepared by adding 1.7% agar to a liquid separation medium having the above composition.

One platinum loop of each colony generated was further suspended in 10 m of liquid separation medium (I) having the above composition, and the suspension was repeated on a plate until uniform colonies were obtained. It was confirmed visually and microscopically that the multiple colonies that were generated were not different from each other. The properties and physiological properties of the above-mentioned strain on each medium were as described above, and it was named Pseudomonas SP, A-5.

Next, an example in which 1-acenaphthenol was produced and an example in which acenaphthene was removed using this strain are listed below as reference examples.

Reference Example 1: Production of 1-acenaphthenol Strain A-5 was cultured in a slant medium prepared by adding 1.7% agar to a liquid medium (III) having the following composition and pH, and then cultured in a test tube. 10 d' of an acenaphthene-containing liquid medium (■) having the following composition and pH was added, two loopfuls of the above strain A-5 were inoculated into this liquid medium (Ir), and cultured under shaking at 30°C for 4 days.

Composition and pH of liquid medium (Ir) Acenaphthene 2g Urea
3gKH2PO41,5g Na HPO-12H01,5g MgSO4・7H2010ay FeS0・7H205IIg CaCl2 10jly yeast extract 10#F deionized water
1.000d! pH 7,2 (5N NaOH) Composition and pH of liquid medium (I[[) Acenaphthene 2gNH4NO34
g K) I2PO41,5g Na HPO-12H01,5g Mg504zl-12010ay FeSO4・7H205N! J CaC1210jIy yeast extract 10ay deionized water
1,0OOal! pH7.2 (5N NaO
H) Next, the entire volume of this culture solution was added to 100 m of the above liquid medium (I).
The cells were cultured together in a flask with shaking for 24 hours.

Furthermore, the entire amount of the obtained culture solution was added to the above liquid medium (II).
Shaking culture was carried out for 24 hours in a flask with 1.000 IrR.

The culture solution obtained in this way was centrifuged to remove bacterial cells, etc., and then the 5H 1-4C, I* bath solution was adjusted to pH 3.
and extracted three times with 1.0001 d of diethyl ether.

The obtained ether layer was concentrated under reduced pressure and dried to give 1-
A product containing acenaphthenol was obtained. This product is
Purification was performed by PLC to obtain 1-acenaphthenol 5q. This 1-acenaphthenol was identified by GC-MS analysis and UV absorption analysis.

Reference Example 2: Removal of acenaphthene A liquid medium (rV
)10ad! strain A cultured in Reference Example 1.
Two platinum loops of -5 were inoculated and cultured with shaking at 30°C for 24 to 48 hours.

Composition of liquid medium (IV) and l) H benzoic acid 2g urea
3gKH2PO41,59 Na HPO-12H01,5g Mg5O・7H20110ll1 FeSO4-71-1205* Ca CI 2 10 sF yeast extract 10η deionized water
1.000dpH 7.2 (5N NaOH) Next, the total amount of the above culture solution was diluted with the amount of acenaphthene added to 50%.
Liquid medium 100#d which is the same as the above liquid medium (It) except that it is 0#F! Put it in a flask with ff1 for 5 days.
Culture was carried out with shaking at 130°C. When the obtained culture solution was examined for the presence of acenaphthene by TLC and GC, it was confirmed that acenaphthene had completely disappeared.

〔Effect of the invention〕

The novel microorganism obtained by the method of the present invention grows in a medium containing only acenaphthene as a carbon source, and can be used to produce useful 1-acenaphthenol using this acenaphthene as a raw material. Alternatively, it can also be used to remove acenaphthene contained in waste.

Furthermore, by using Pseudomonas SP and A-5 as bacterial strains, it can be used to remove not only acenaphthene but also acenaphthylene, acenaphthenequinone, 1,2-acenaphthendiol, 1,8-naphthalic acid, etc. .

Patent applicant: Representative of Nippon Steel Chemical Co., Ltd.
Patent Attorney Katsuo Naruse (2 others) Procedural amendment September 24, 1985 Director General of the Patent Office Michibe Uga 1, Indication of the case 1985 Patent Application No. 122600 2, Name of the invention Novel Microorganisms Acquisition method 3, relationship with the case of the person making the amendment Patent applicant address 5-13-16 Ginza, Chuo-ku, Tokyo Name (6
64) Nippon Steel Chemical Co., Ltd. 4, Agent 105 Telephone 03 (433) 4420
Address: Shinm3-8WrB, Minato-ku, Tokyo, 5th Floor, Building 5, Date of amendment order: Voluntary amendment 6, Number of inventions increased by amendment: None 7, Subject of amendment: 111, page 2 of the statement of contents "Grows in a medium containing only acenaphthene and produces 1-acenaphthenol" written in lines 4 and 5 is corrected as follows.

“Grows in a medium containing only acenaphthene as a carbon source,
``Produce 1-acenaphthenol from acenaphthene''

Claims (1)

[Claims] A method for obtaining a novel microorganism, which comprises isolating a strain of a microorganism belonging to the genus Pseudomonas that grows in a medium containing only acenaphthene as a carbon source and produces 1-acenaphthenol.