JP2010130950A - Organic solvent-degrading microorganism and method for degrading organic solvent therewith - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an organic solvent-degrading microorganism which can efficiently degrade many kinds of solvents such as aromatic hydrocarbon-based solvents, petroleum-based hydrocarbon solvents, ester-based solvents, and alcoholic solvents with a kind of microorganism. <P>SOLUTION: There are provided Pseudomonas sp.14-N-1(NITE P-458), and a method for degrading the organic solvent comprising contacting the Pseudomonas sp.14-N-1(NITE P-458) with at least one organic solvent selected from the aromatic hydrocarbon-based solvents, the petroleum-based hydrocarbon solvents, the ester-based solvents, and the alcoholic solvents or a liquid or gas containing the same. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an organic solvent degrading bacterium and an organic solvent decomposing method using the same.

  Conventionally, many studies have been made on the action of microorganisms to decompose compounds such as hydrocarbons from the viewpoint of preventing environmental pollution. For example, when aromatic compounds are decomposed by microorganisms, the aromatic compounds are directly contained in the medium. Suspended in water to cause microbial degradation, or a method in which a small amount of an aromatic compound is dissolved in a solvent for microbial degradation. However, in the former method, since the aromatic compound does not dissolve, the contact with the microorganism is insufficient and the decomposition efficiency is poor. In the latter case, the contact with the aromatic compound is improved, but the resistance to the organic solvent is improved. Therefore, there is a problem that the reactivity of microorganisms is suppressed and the decomposition efficiency is extremely low.

  Therefore, in Patent Document 1, an organic solvent such as benzene, toluene and xylene is used to efficiently decompose hardly-decomposable polycyclic aromatic hydrocarbons contained in a large amount of petroleum using microorganisms having resistance to these. Is disclosed.

  On the other hand, in an environment where various organic solvents such as a coating plant are mixed, alcohols, ketones, ethers, esters, organic acids, and the like are generated. As a method for treating an aqueous waste liquid containing these, microorganisms that efficiently decompose them are disclosed. Patent Document 3 discloses that malodorous gas containing a mixed organic solvent such as toluene, xylene, methyl ethyl ketone, etc. generated from chemical factories, paint factories, etc., microorganisms having toluene resolution, microorganisms having xylene resolution, and methyl ethyl ketone resolution. A method for efficiently decomposing by using a combination of a microorganism and / or a microorganism having methyl ethyl ketone resolution has been disclosed.

  However, the microorganism described in Patent Document 1 cannot decompose solvents such as alcohols and esters, and the microorganism described in Patent Document 2 cannot decompose aromatic hydrocarbons. Furthermore, when various organic solvents are mixed, as in Patent Document 3, it is necessary to use a plurality of types of microorganisms at a time, and the growth state changes or the facilities become large. There are problems such as.

JP-A-7-155175 JP 10-33163 A

  The object of the present invention is an organic solvent-degrading bacterium capable of efficiently degrading various solvents such as aromatic hydrocarbon solvents, petroleum hydrocarbon solvents, ester solvents and alcohol solvents with one kind of microorganism. Is to provide.

  Another object of the present invention is to provide a method for decomposing an organic solvent using the organic solvent degrading bacterium.

As a result of various searches for microorganisms that decompose organic solvents, the present inventors have found that aromatic hydrocarbon solvents and petroleum hydrocarbons have been extracted from the soil in Hiratsuka City, Kanagawa Prefecture (Kansai Paint Co., Ltd. Hiratsuka Office). The present invention has been completed by separating and identifying a specific Pseudomonas microorganism having the ability to decompose and assimilate at least one organic solvent selected from a solvent based system, an ester based solvent and an alcohol based solvent as a carbon source. And using this Pseudomonas genus microorganism, its culture or processed product, at least one organic solvent selected from aromatic hydrocarbon solvents, petroleum hydrocarbon solvents, ester solvents and alcohol solvents or A method for decomposing organic solvents by bringing them into contact with the contained liquid or gas was established.

  Thus, the present invention relates to Pseudomonas sp. 14-N-1 (NITE P-458) is provided.

  The present invention also provides Pseudomonas sp. 14-N-1 (NITE P-458), its culture or treated product, at least one organic solvent selected from aromatic hydrocarbon solvents, petroleum hydrocarbon solvents, ester solvents and alcohol solvents Alternatively, the present invention provides a method for decomposing an organic solvent, wherein the organic solvent is brought into contact with a liquid or gas containing the organic solvent.

  According to the present invention, one kind of microorganism can efficiently decompose various solvents such as an aromatic hydrocarbon solvent, a petroleum hydrocarbon solvent, an ester solvent, and an alcohol solvent. Very useful.

BEST MODE FOR CARRYING OUT THE INVENTION

  According to the present invention, Pseudomonas sp. 14-N-1 (NITE P-458) is provided.

  These microorganisms are collected and isolated from soil in Hiratsuka City, Kanagawa Prefecture (Kansai Paint Co., Ltd. Hiratsuka Office), and the culture can be performed using a general medium used for aerobic bacteria. it can.

In the present specification, medium A (Yeast extract 0.5 g / l, MgSO4 · 7H ₂ O 0.1 g / l, CaCl ₂ · 2H ₂ O 0.1 g / l, ferric
Citrate 0.002 g / l, Vitamin solution 1.0 ml / l, Metal solution 10.0 ml / l) and medium B (K ₂ HPO ₄ 1.0 g / l, KH ₂ PO ₄ 0.5 g / l) 4 : 1 and mixed and sterilized is called "BM liquid medium". Medium A and medium B are mixed at a ratio of 4: 1 and further sterilized by adding 1.5% Ager. This is referred to as “BM agar medium”.

The above metal solutions are CaCl ₂ · 2H ₂ O 0.4 g / l, H ₃ PO ₄ 0.5 g / l, CuSO ₄ · 5H ₂ O 0.04 g / l, KI 0.1 g / l, FeSO ₄ · _{7H 2 O 0.2g / l, MnSO} 4 · 4~7H 2 O 0.4g / l, ZnSO 4 · 7H 2 O 0.1g / l, NaMoO 4 · 2H 2 O 0.1g / l, 12N HCl 2 Vitamin solution consists of Ca-Panthotheneate 0.4 g / l, Inositol 0.2 g / l, P-Aminobenzoate 0.2 g / l, P-Aminobenzonate 0.2 g / l, Pyridoxin 0.4 g / l. l, Thiamin 0.4 g / l, Biotin 0.002 g / l, Vitamin B ₁₂ 0. 0005 g / l, Naiacin 0.4 g / l.

  In addition, the microorganism of the present invention was isolated and identified as follows.

Culture method:
In the case of a liquid medium, a screw-cap test tube is used. After adding the substrate, the culture is performed with shaking at 30 ° C. and 150 rpm. In the case of an agar medium, the culture is performed at 30 ° C. using a sterile petri dish.

Isolation method (serial dilution method / 100-fold dilution method):
100 μL of a culture solution containing microorganisms collected from soil is added to 0.85% physiological saline (9.9 mL) prepared in advance, and a culture solution diluted to 10 ⁻² times the concentration from the stock solution is prepared. About 100 μL of this diluted culture solution is added to fresh physiological saline (9.9 mL) to prepare a diluted culture solution having a concentration of 10 ⁻⁴ times that of the stock solution. This is repeated to prepare diluted culture solutions of 10 ⁻⁶ times and 10 ⁻⁸ times concentration, and after vigorous stirring by vortex, 100 μL of each diluted culture solution is smeared on the agar medium.

Analysis method:
Measurement of fungal growth:
Using a digital colorimeter (trade name: mini photo 518R) manufactured by Taitec Co., Ltd., the sterility control is adjusted to 0 point, and then the cell turbidity OD _{660 nm} of the diluted culture solution is measured.

Gas chromatography (GC) analysis:
Samples are extracted with 2 ml of diethyl ether, taken with a 10 μl syringe and GC analysis is performed. Perform three measurements with GC, determine the concentration from the calibration curve, and examine the solvent resolution.

GC analysis conditions:
In the case of organic solvents other than cyclohexanone and ethyl acetate, Column Packing PEG 20M-PT Uniport B
Column size 2.6mm x 2m
Flow rate 40ml / min, N ₂ gas
Injection temp. 180 ° C
Column temp. 100 ° C
Detection FID

In case of cyclohexanone Column Packing PEG 20M-PT Uniport B
Column size 1.6mm × 2m
Flow rate 40ml / min, N ₂ gas
Injection temp. 180 ° C
Column temp. 100 ° C
Detection FID

In the case of ethyl acetate, Column Packing PEG 20M-PT Uniport B
Column size 2.6mm x 2m
Flow rate 40ml / min, N ₂ gas
Injection temp. 180 ° C
Column temp. 80 ° C
Detection FID

As the substrate, mineral spirits (mixture of C _9-12 aliphatic hydrocarbon and C _9-10 aromatic hydrocarbon) and Swazol 1000 (mixture of C ₉ aromatic hydrocarbon) were used.

  The primary screening was performed as follows.

  Soil was added to each 5 mL of BM liquid medium containing 200 ppm mineral spirits or Swazol 1000. The culture was performed with shaking at 30 ° C. and 300 rpm for 7 days, and planting was performed 4 times. The culture solution was streaked on a BM agar medium and left to stand for 5 days. All the bacteria grown from this agar medium were isolated and cultured in slant for 5 days. The isolated bacterium was added to 5 mL of BM liquid medium containing 200 ppm mineral spirits or Swazol 1000, and cultured with shaking at 30 ° C. and 150 rpm for 10 days. From the results of GC analysis, several types of mineral spirits and swazol 1000-degrading bacteria candidates were obtained by selecting a culture solution showing high resolution.

  Secondary screening was performed as follows.

  The isolated candidate bacteria were inoculated into 5 mL of BM liquid medium containing 500 ppm of mineral spirits or swazol 1000, and cultured with shaking at 30 ° C. and 150 rpm for 10 days. As a result, strain 14-N-1 was obtained as a swazole 1000-degrading bacterium.

  A part of the base sequence of the DNA encoding 16SrDNA of this strain was determined and identified by partial base sequence analysis (international base sequence database BLAST search). As a result, it was found that this strain belongs to the genus Pseudomonas, and shows different properties in organic solvent resolution from the known strains belonging to Pseudomonas. Therefore, this strain was identified as a new bacterial species, and Pseudomonas sp. . It was named 14-N-1. This strain has been deposited with the National Institute of Technology and Evaluation Patent Microorganism Deposit Center, and has been assigned a deposit number of NITE P-458.

  In order to investigate the ability to decompose and assimilate the organic solvent by the isolated strain, various organic solvents were selected as substrates, and the isolated strain was inoculated in 5 ml of each substrate-containing BM liquid medium for 72 hours at 30 ° C. And shaking culture at 150 rpm.

  As a result, not only for petroleum hydrocarbon solvents such as mineral spirits and swazol 1000, but also for aromatic hydrocarbon solvents such as toluene and xylene, ester solvents such as butyl acetate, and alcohol solvents such as n-butanol. It was found to have a high resolution.

  The decomposition of the organic solvent by the isolated strain can be carried out by a method known per se, for example, the cells are contacted with the organic solvent or a liquid or gas containing the organic solvent in the presence of minerals, moisture and air. Can be performed. As the microbial cells, any of isolated living cells, cultures or processed products thereof (for example, frozen products, lyophilized products, etc.) can be used. It is also possible to use an immobilization carrier on which these cells are immobilized.

  Immobilization of these cells on a carrier can be performed by a method known per se, and examples thereof include an entrapment method, a physical adsorption method, and a covalent bond method.

The carrier is in the form of a hollow shape, irregular shape, porous shape, etc., which has a large surface area per unit volume, or swells by absorbing water, has fluidity, and does not easily flow out of the reaction system. Those having a particle size and specific gravity are suitable, and the carrier shape may be, for example, a plate shape, a fiber shape, a special shape such as a cylindrical shape, a sponge shape, a grain / lump shape, a cubic shape, etc. And a fine granular material that is easy to ensure sufficient surface area. As the carrier material, various organic / inorganic materials conventionally used as carrier materials such as microorganisms and enzymes can be used. For example, inorganic materials such as granular activated carbon, crushed activated carbon, charcoal, zeolite, mica, sand particles, etc. Polymer materials such as photocurable resins, polyurethane, polyvinyl alcohol, polyethylene, polyacrylamide, polyester, polypropylene, agar, alginic acid, carrageenan, cellulose, dextran, agarose, ion exchange resins; porous ceramics such as silica gel; anthracite A resin material mixed with activated carbon or the like may be used, and these may be used alone or in combination of two or more.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited only to these examples.

Example 1: Decomposition of various organic solvents by 14-N-1 strain <Decomposition test>
500 ppm of various organic solvents are added to the BM liquid medium, and 50 μl of the preculture solution of strain 14-N-1 is added thereto, followed by culturing for 3 days. During this period, OD _{660 nm} and GC are measured daily to grow the bacteria. The degree and solvent resolution were evaluated. The same procedure was performed at 1000 ppm for the high degradation substrate and 200 ppm for the low substrate. The results are shown below.

Decomposition characteristics with respect to mineral spirits At 500 ppm, OD _{660 nm} exceeded 0.7 in one day, and about 80% of decomposition occurred in one day. Thereafter, the degree of growth and the degradation concentration did not change even if the culture was continued.

Degradation characteristics with respect to Swazol 1000 , at OD _{660 nm} reached a maximum of 0.6 in one day, decomposed by about 80% on the first day, and then decomposed by about 90% on the fifth day.

Decomposition characteristic with respect to toluene 200 ppm completely decomposed in 3 days.

Decomposition characteristics for industrial xylene Decomposition 40% in 3 days at 200 ppm.

Decomposition characteristic with respect to toluene 200 ppm completely decomposed in 3 days.

Decomposition characteristics with respect to n-butanol 200 ppm completely decomposed in 3 days.

Decomposition characteristics with respect to butyl acetate At 200 ppm, complete decomposition occurred in 3 days.

Claims (3)

  Pseudomonas sp. 14-N-1 (NITE P-458).   Pseudomonas sp. A carrier on which 14-N-1 (NITE P-458) is supported or immobilized.   Pseudomonas sp. 14-N-1 (NITE P-458), its culture or treated product, at least one organic solvent selected from aromatic hydrocarbon solvents, petroleum hydrocarbon solvents, ester solvents and alcohol solvents Or a method for decomposing an organic solvent, wherein the organic solvent is brought into contact with a liquid or gas containing the organic solvent.