JPH0367581A - Phenol assimilating bacterium - Google Patents

Abstract

NEW MATERIAL:Pseudomonas putida WAS-2 having resistance to highly concentrated phenol and ability of assimilating or decomposing phenol contained in a solution. USE:For cleaning phenol-containing waste water. PREPARATION:For example, active sludge is accumulated and cultured in a medium of inorganic salt containing highly concentrated phenol and at an advanced stage of purification of bacteria, a single colony is formed on a phenol agar flat plate medium, a phenol assimilating strain is isolated and one strain having the highest phenol decomposition ability is selected to give phenol assimilating Pseudomonas putida WAS-2 (FERM P-10924) having resistance to highly concentrated phenol and capable of growing in phenol as the only carbon source.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel bacterial strain belonging to Pseudomonas putida and isolated from activated sludge of an oil refinery, and its artificial mutant strain. . More specifically, it is resistant to high concentrations of phenol and grows in wastewater containing phenol,
The present invention relates to bacteria Pseudomonas putida WAS-2 and Pseudomonas putida WAS-20, which are characterized by assimilating or degrading Pseudomonas putida WAS-20.

[Prior Art] Phenol is contained in wastewater from refineries, coal gas and coke manufacturing plants, phenol resin manufacturing plants, organic drug factories, etc., and has a growth inhibiting or bactericidal effect on many microorganisms. Therefore, it is counted as one of the difficult-to-biodegradable pollutants.

Treatment of wastewater containing phenol traditionally involves chemical oxidation,
Methods such as solvent extraction and activated carbon adsorption have been adopted, but
Today, biological methods using activated sludge and phenol-degrading bacteria are considered to be advantageous in terms of operability and cost as a method for treating wastewater that is discharged in large quantities.

Examples of microorganisms having the ability to decompose phenol include Pseudomonas genus, Nocardia genus,
Bacillus genus, Acinetobacter (
Acinetobacter) bacteria such as horses, Aureobasidium (＼ureobasidius) J
ll, fungi such as Fusarium trap, and Trichos oron.
Yeasts such as Candida l and Candida II are known. In particular, the known Pseudomonas bacterium according to the present invention includes Pseudomonas putida 8 mouths (m
Book and Fujita, Journal of the Sewerage Association.

Vol. 24, No. 273@, pp. 27-33, 1987), Pseudomonas QT31 (C, Maskew et al., Biotechnology Letters, Vol. 9, No. 9, No. 655)
-660 pages, 1987).

[Problems to be Solved by the Invention] The above-mentioned Pseudomonas bacteria, which are known to have the ability to decompose phenol, have a lag phase (lap phase) in which the maximum concentration of phenol that can be decomposed in an inorganic salt medium is 1000 to 1300 q/II. ), it takes a long time to complete the decomposition, and a long period of acclimatization to phenol is required. This means that the Pseudomonas bacteria have a low resistance to phenol. Therefore, a large amount of time must be spent on culturing and decomposing phenol.
For industrial implementation, microorganisms with high phenol decomposition ability and high resistance are desired.

Therefore, it is significant for industrial and environmental pollution prevention to efficiently and stably assimilate or decompose a large amount of industrial wastewater containing phenol using phenol-resistant and highly active 71-nol-accumulating bacteria. It is.

The present invention involves isolating phenol-assimilating bacteria that are resistant to high 11f [phenol and have the ability to assimilate or decompose phenol from activated sludge of an oil refinery, and further uses a mutation method using ultraviolet irradiation. By creating a mutant strain with high phenol tolerance, we created ■1i! containing 1 degree phenol. Y! ! The purpose of the present invention is to provide a phenol-assimilating bacterium belonging to Pseudomonas putida that enables industrial implementation of oxidative decomposition treatment of water.

[Means for solving the problem] In order to achieve the above objectives, we have developed a plant that is resistant to high concentrations of phenol and grown using phenol as the sole carbon source from the activated sludge of Tonen Corporation's Wakayama Plant refinery. Possible phenol-assimilating bacteria were screened.

Activated sludge was enriched (4 times) in an inorganic salt medium containing phenol at a concentration of 500 μ/I, and when the bacteria had been purified, it was cultured on a phenol agar plate medium (phenol Kinkon 5).
00■/j) I: was allowed to form a single colony, and 8 strains of phenol-assimilating bacteria were isolated. Furthermore, one strain with the highest phenol degrading ability was selected from among them.

This phenol-assimilating bacterium has the mycological properties shown in Table 1, and based on these properties, BerQey'SManu
al of 5ystea+atic Bacteri
ology Volume 1 (Williams & Wilki
ns, 1984), Shootmonas
Pseudomonas putida was identified as Pseudomonas putida WAS.
-2 (Deposited on August 3, 1989, Microtechnical Research Institute No. 1092)
No. 4, FERN P-10924).

Table 1 (Continued) Morphology Rod Bacteria (0.8-1.2X 2.0-4.0%) Motile Flagella Durum staining Spore formation Aerobic/anaerobic growth Nitrate reduction and denitrification reaction Indole production Catalase Oxidase Urease Gelatin Liquefaction Use of citric acid A few polar flagella Negative aerobic + + + + Pigment production (Pseudomonas agar F) + (Water-soluble, fluorescent yellow pigment) Pigment production (Pseudomonas agar P 〉 0-F test (Huah-Leifson method) Temperature range for growth at 41°C. , 0 to 9.0) is good + Table 1 (continued) Ornithine ◆Decarboxylase + Accumulation of poly-β-hydroxyacetic acid Carbon source utilization Glucose Fructose Maltose Galactose Xylose Mannitol Sucrose Lactose Aesculin Citrate p-Hydroxy Benzoic acid phenol Pseudomonas putida WAS-2 is 1000■/
It is possible to grow in an inorganic salt medium containing a high concentration of phenol of 3 liters, and in an inorganic salt medium containing phenol as the sole carbon source.
Phenol at a concentration of 1000 .mu./I can be almost completely degraded in 20 to 30 hours (including a lag period of 12 hours) by batch shaking aerated cultivation at 0.degree. In addition, the WAS-2 strain has the ability to assimilate phenol at a concentration of 1000 q/1 or more in a nutrient medium (Nutriendo Broth, formerly manufactured by FCO).

Pseudomonas putida WAS-2 of the present invention is positive for nitrate reduction and resourceful for xylose, galactose and maltose as carbon sources compared to Pseudomonas putida BH described in the above-mentioned literature by Hashimoto and Fujita. The mycological properties are different in that they have chemical properties;
Furthermore, since the lag period and phenol decomposition wFIIIi are shorter, the two are judged to be very different strains.

Furthermore, the present inventor has developed a method for improving the resistance ability of Pseudomonas putida WAS-2 to phenol.
By irradiating the S-2 strain with ultraviolet light and inducing mutagenesis, 20
A mutant strain capable of growing in an inorganic salt medium containing phenol at a concentration of 00j19/R was isolated as a single colony, and this mutant strain was transformed into Pseudomonas putida W.
It was named 5-2D (deposited on August 3, 1989, FERNP-10925, FERNP-10925). The WAS-2D strain has the same mycological properties as the parent strain WAS-2, but its ability to assimilate or decompose phenol is higher than that of the WAS-2 strain, and it uses phenol as its only carbon source. Phenol at a concentration of 200011 I/l is almost completely degraded in about 48 hours by batch shaking aeration culture in mineral salts medium.

Pseudomonas ◆ putida W isolated as above
AS-2 or WAS-2D is Newt'JXnt.7
After subculturing (twice) for 24 hours at an optimal temperature of 28°C in Dirco Jl, #0003-Of-6, 50ON/I! It is possible to grow the cells by inoculating them into an inorganic salt medium containing phenol at a concentration of , and performing aeration culture at the same temperature for an additional 24 hours.

The mineral salt medium was sodium hydrogen phosphate (341814
1゜dipotassium hydrogen phosphate (641) 1), 1iQ
Il ammonium (201M), magnesium sulfate (
0.3+eH), ferrous sulfate (1 μM>, zinc chloride (1
µM) and calcium chloride (10 µM), pH
It is preferable to adjust the final concentration to 7.0, but alternatively, the raw water flowing into the aeration tank of the activated sludge equipment may be sterilized by autoclaving, and phenol may be added to the water to a final concentration of 1 g/I. can also be used as a culture medium. In addition, when cultivating a large amount of the phenol-assimilating 100% glutinous rice bran of the present invention, rice bran at a concentration of 1% (Wt/v01) with no carbon source added may be used instead of the above-mentioned inorganic salt medium containing phenol. Good too.

Next, the bacteria of the present invention obtained by culturing in large quantities according to the procedure shown above are added to the wastewater containing phenol and aerated, or the wastewater containing phenol is acclimatized to activate the bacteria of the present invention. By adding sludge to similar wastewater and aerating it, it is possible to oxidatively decompose phenol-containing wastewater.

In particular, the WAS-20 strain has a high ability to tolerate phenol, so it may be used directly for wastewater treatment without being acclimatized to phenol in advance.

Pseudomonas putida WAS-2 or WA of the present invention
S-2D can most preferably assimilate or decompose phenol, but also retains the ability to assimilate water-soluble substituted phenols such as -cresol and p-hydroxybenzoic acid, and the bacterium of the present invention is used as the parent strain. It is also possible to create mutant strains with higher tolerance and degrading ability for these phenols by mutagenesis.

[Effects of the Invention] The bacteria of the present invention are resistant to high concentrations of phenol and have a high ability to decompose phenol.

Furthermore, compared to known phenol-assimilating bacteria, the induction period and phenol decomposition time are shorter. In particular, the mutant strain Pseudomonas putida WAS-29 can absorb phenol at a high concentration of 2000119/1 in an inorganic salt medium, and does not necessarily need to be acclimated in phenol-containing wastewater. This bacterium has superior phenol resistance compared to known microorganisms.

Therefore, when the l111M of the present invention is introduced into activated sludge, it is expected to improve the purification of phenol-containing wastewater. Furthermore, it is expected that it will be useful as a host bacterium or a gene donor when genetically engineering the l[Im of the present invention].

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Mutagenesis by ultraviolet light (creation of Pseudomonas putida WAS-2D): The parent strain Pseudomonas putida WAS-2 was
M's Nutriendo Broth (formerly FC.

Company, #0003-01-6) Medium, 28℃ Te 2x1
G8m/#1e (After shaking culture until D seedling density is reached, the bacterial cells are centrifuged, and 0.IH80304 of 2I11 is grown.
Suspend in aqueous solution.

This suspension was heated for 3 hours using a 15W germicidal lamp as an ultraviolet source.
Ultraviolet rays are irradiated for 15 seconds from five different distances. At this time,
The survival rate of bacterial cells was 1 to 5%. After irradiation with ultraviolet rays, the bacterial cells are centrifuged and cultured with shaking at 28°C for 24 hours. next,
Collect the culture solution of 0.1ad and give 500 to 150019/
The cells are inoculated onto Nutriendo Agar medium (Difco, #0001-01-8) containing phenol at a concentration of 1 and incubated at 28°C for 24 hours. The mutant strain that grew best on the above medium containing phenol at a concentration of 1000q# compared to the non-UV irradiated control was isolated as a mutant strain, and this was called Pseudomonas putida WAS.
It was named -2D.

Example 2 Growth of phenol-assimilating bacteria: Phenol-assimilating bacterium Pseudomonas putida WAS
A single colony of -2 or WAS-20 is inoculated into 5 m of Nutriendo broth and incubated with shaking at 28°C for 24 hours. Furthermore, the obtained bacterial cells were subcultured into 50-ml fresh nutrient broth and cultured for 24 hours at 28°C. Of this, 1150 volumes of culture were
Contains 500q/l of phenol (r 1000jd!
+7) Mineral salt medium [34e14 NaH2PO4.

6418K HPO4, 201N(N
Mouth,) 2 So 4.

0.318 MQSO4, 1μHFe50, 1μ
MZnCj2, 10 μHCaCj2: pH 7.0
Inoculate the cells once and culture with shaking aerobically at 28°C for 24 hours.

As a result, Pseudomonas putida WAS-2 or W
The final bacterial cell concentration of As-2D was 5.8×100 cells/Id or 5.6×100 cells/d, respectively.

Example 3 Phenol assimilation by Pseudomonas putida WAS-2: An example in which 1150 volumes of a culture of Pseudomonas putida WAS-2 grown as described in Example 2 was grown with 1000 j19/j of phenol as carbon source. 2
The same inorganic salt medium 50d as above was inoculated and cultured aerobically at 30°C with shaking (150 rpm). After a 12-hour induction period, a good assimilation growth rate was exhibited, and 25 hours after the start of culture, the residual phenol concentration in the culture supernatant was 0.5 ■/j! At the same time, the bacterial cells reached the maximum growth amount.

The residual amount of phenol was measured according to JIS K 0102-1971 (factory wastewater test method) using 4-aminoantipyrine.

Example 4 Phenol assimilation by Pseudomonas putida WAS-2D: An example in which 1150 volumes of a culture of Pseudomonas putida WAS-20 grown as described in Example 2 was grown with phenol 20004/1 as carbon source. Inoculate the same inorganic salt medium 50- as in 2 and shake aerobically at 28℃!
1 (150 rpm) culture. After a lag period of 15 hours, it showed a good assimilation growth rate, and 48 hours after the start of culture,
The residual phenol concentration in the culture supernatant is 0.5 III/II
At the same time, the bacterial cells reached the maximum growth amount.

Claims (4)

[Claims] (1) Pseudomonas putida, a bacterium that is resistant to high concentrations of phenol and has the ability to assimilate or decompose phenol contained in liquid.
utida￥) WAS-2. (2) Pseudomonas putida WAS-2 according to claim 1, which is capable of assimilating phenol at a concentration of 1000 mg/l in an inorganic salt medium. (3) Pseudomonas putida, a bacterium that is resistant to high concentrations of phenol and has the ability to assimilate or decompose phenol contained in liquid.
utida￥) WAS-2D. (4) Pseudomonas putida WAS-2D according to claim 3, which is capable of assimilating phenol at a concentration of 2000 mg/l in an inorganic salt medium.