JP2922842B2 - Method for imparting phenol resistance to microorganisms that degrade polycyclic aromatic compounds and method for treating polycyclic aromatic compound-containing wastewater using the phenol resistant microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a polycyclic aromatic compound
Decomposing Pseudomonas spp. And Rhodo
a method for imparting phenol resistance to a genus coccus; and
Polycyclic aromatic compound-containing wastewater using the phenol-resistant bacterium
, And Pseu for decomposing polycyclic aromatic compounds
domonas and Rhodococcus
Add to activated sludge and acclimatize in SRT more than 10 days
A method of acclimating bacteria which is characterized and a condition acclimated by the method.
The present invention relates to a method for treating polycyclic aromatic compound-containing wastewater using a culture .

[0002]

2. Description of the Related Art Polycyclic aromatic compounds are hardly biodegradable compounds, and are difficult to regulate by wastewater regulations such as BOD and COD. It is considered that they are released into the environment without being decomposed in the treatment facility. When these compounds are released, they are accumulated as biodegradable environmental pollutants in river basins and in bays into which rivers flow, so it is necessary to take measures to prevent them. In recent years, marine pollution by these compounds contained in petroleum fractions due to petroleum distilling accidents and the like has become a problem, and attention has been paid to environmental restoration by microorganisms.

[0003]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for decomposing a biodegradable polycyclic aromatic compound.
Udomonas spp. or Rhodococcus spp.
For imparting phenolic resistance to wood, biodegradable polycyclic
Pseudomonas spp. That degrades aromatic compounds
And Rhodococcus spp.
It is to provide a method of processing waste water containing RT10_nichiijode馴養Suruhoho and polycyclic aromatic compounds.

[0004]

The above object is achieved by the following invention. That is, the present invention is resistant to Pseudomonas and Rhodococcus spp. That degrade polycyclic aromatic compounds.
A method of imparting phenolic properties, and separation of polycyclic aromatic compounds
Pseudomonas spp. And Rhodoco
ccus spp. added to activated sludge, SRT more than 10 days
The method of acclimatization and the phenol resistance obtained by the above method
Bacteria imparted and / or acclimated bacteria acclimated by the above method
This is a method for treating polycyclic aromatic compound-containing wastewater using

[0005]

According to the present invention, a polycyclic aromatic compound is decomposed.
Pseudomonas spp. And Rhodococcu
Mutation caused by ultraviolet irradiation etc. to s sp.
By acclimating the mutant in a phenol-supplemented medium,
Pseudomonas spp. That degrades aromatic aromatic compounds
And Rhodococcus spp.
Can be given. P which decomposes polycyclic aromatic compounds
pseudomonas spp. and Rhodococcus
Add genus bacteria to activated sludge and acclimatize in SRT 10 days or more
Thus, an acclimated bacterium can be obtained. Biodegradable polycyclic aromatic
In the treatment of compound-containing wastewater,
Phenol-resistant bacteria and / or
The acclimatized bacteria obtained after nourishment settle in the activated sludge and become active
As it is a priority species of sludge, the phenol-resistant bacteria, and / or
Or該馴Yokin depending on the use of the polycyclic aromatic in waste water
Group compounds can be decomposed and removed effectively.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described in more detail with reference to embodiments of the present invention. Polycyclic aromatic compound of the present invention
Decomposing Pseudomonas spp. And Rhodo
Examples of coccus spp, Pseudomonas pauc
imobilis (Pseudomonas poshimovirus) bacteria and Rh
odococcus ruber bacteria
Can be More specifically, Pseudomonas paucimob
ilis 421Y strain (FERM BP-5122) (hereinafter referred to as KF-1 strain) and Rhodococcus ruber
TA 0902 strain (FERM BP-5121) (hereinafter referred to as KF-2 strain) .

[0007] The above microorganisms are obtained by culturing a large number of microorganisms detected from 120 kinds of soil up to the third order in one cycle of 7 days, and accumulating up to the third order. Was spread on an agar medium and separated from the formed colonies. The microbiological properties of the obtained microorganism were examined, and it was confirmed that the microorganism was a novel strain . The obtained mycological properties are shown below.

[0008]

[0009]

[0010]

[0011]

[0012] (+): Weak positive

From the above results, the KF-1 strain was found to be BERGEY'S MA
NUAL OF Systematic Bacteriology, Vol.1, p214 (1984)
Same and strains belonging to Pseudomonas paucimobilis on the basis of
Was decided. For the KF-2 strain, cell wall analysis was performed. As a result, it was found that the diamino acid was meso-diaminopimelic acid and mycolic acid was present. Structure of the fatty acid is a straight chain saturated fatty acids and branched unsaturated fatty acids of C _18, was Tuberculostearic acid marked with a methyl group at position to C ₁₀ of the branched unsaturated fatty acids. From this and the above results, the KF-2 strain was BERGEY'S MANUAL OF
Based on Systematic Bacteriology, Vol. 2, pp. 1479-1480 (1989), it was identified as a strain belonging to Rhodococcus ruber.
Was. As a result of measuring the quinone composition and the G + C content of the KF-1 strain and the KF-2 strain, the quinone composition of the KF-1 strain was ubiquinone-8 (composition ratio: 99%), and G +
The C content was 62.0 mol% G + C, and the quinone composition of the KF-2 strain was menaquinone-8 (H2) (composition ratio: 98%).
And the G + C content was 67.0 mol% G + C. The KF-1 strain has been deposited as FERM BP-5122, and the KF-2 strain has been deposited as FERMBP-5121.

The above microorganism is a novel microorganism that degrades polycyclic aromatic compounds. The polycyclic aromatic compound referred to in the present invention is an aromatic having two aromatic rings, and includes a compound in which an aromatic ring forms a heterocyclic ring with an oxygen atom, a sulfur atom, or a nitrogen atom. Polycyclic aromatic compound referred to in the present invention
The substance is biphenyl, dibenzofuran, dibenzothiophene, fluorene, carbazole, phenanthrene, anthracene, pyrene and the like, and their substituted products .

By the way, the effluent of a petroleum refinery or coke plant contains a high concentration of phenol together with a polycyclic aromatic compound. Therefore, the polycyclic aromatic compound-degrading bacteria KF-1 and KF-2 of the present invention also need to have the ability to withstand highly biotoxic phenol. There
Thus, the present inventors have repeatedly studied a method for imparting phenol resistance to the KF-1 strain and the KF-2 strain, and
Invented. That is, the mutant strain obtained by irradiating ultraviolet rays to the cultured cells of the logarithmic growth phase of these bacteria or by treating the cultured cells with a substance such as ethyl methanesulfonate, etc. the by acclimatization with phenol added medium was found to be capable to impart resistance to phenolic in KF-1 strain and KF-2 strain. The improved KF- having such phenolic resistance.
By using one strain and the KF-2 strain, polycyclic aromatic compounds can be decomposed and removed in high-concentration phenol.
The mycological properties of these phenol-resistant microorganisms are the same as those of the original KF-1 and KF-2 strains.

Each of the microorganisms of the present invention has the property of being well established in activated sludge and becoming a preferential species of activated sludge by acclimation. Therefore, the microorganism of the present invention can be used for various types of activated sludge. It is particularly useful in wastewater treatment methods. Then, the present inventors made the second invention of the present invention.
That is, Pseudom that decomposes polycyclic aromatic compounds
Activated soils of Onas and Rhodococcus
And when the fixing mud, SRT their acclimatization the (sludge retention time / sludge age), 5 days or more, a method preferably takes more than 10 days. As a result, the microorganism of the present invention can be satisfactorily and stably fixed to the activated sludge. Book
The third invention of the invention is obtained by the method of the present invention described above.
Activated soil used alone or in combination with microorganisms (strains)
This is a mud-type wastewater treatment method. And the above polycyclic aromatics
The present invention is particularly effective for treating wastewater containing a compound. The wastewater treatment method using the activated sludge method using microorganisms is well known, and the microorganism of the present invention can be applied to any known treatment method using activated sludge, and the method itself is particularly limited. It is not something to be done. still,
It goes without saying that the use of the above-mentioned microorganism includes the use of the culture or the use of the cultured product.

[0017]

Next, the present invention will be described more specifically with reference to examples. In the following examples, any one of phenanthrene, fluorene and dibenzothiophene was added to a medium having the composition shown in Table 1 to which the KF-1 strain or the KF-2 strain had been added. The concentration of the aromatic ring compound and the amount of bacterial cells were measured. More specifically, 100 ml of the medium was placed in a 500 ml Erlenmeyer flask, 100 mg of the polycyclic aromatic compound was added, 1 ml of the pre-cultured cells were added, and the cells were cultured at 30 ° C. and 180 rpm for a predetermined time. The culture was acidified with hydrochloric acid, and ethyl acetate 100
Then, the organic solvent layer was separated and the amount of the polycyclic aromatic compound was determined by gas chromatography.
The amount of the cells was measured by an absorption spectrophotometry.

[0018]

[Table 1] (*) Tween 80 manufactured by Atlas Powder

Example 1 In this example, phenanthrene was decomposed by the KF-1 strain. Table 2 shows the obtained results.

[0020]

[Table 2]

Example 2 Fluorene was decomposed by the KF-2 strain. Table 3 shows the results
Shown in

[0022]

[Table 3]

Example 3 Dibenzothiophene was decomposed by the KF-2 strain. Table 4 shows the results.

[0024]

[Table 4]

Example 4 Fluorene was decomposed by the KF-1 strain. Table 5 shows the results
Shown in

[0026]

[Table 5]

Example 5 Dibenzothiophene was decomposed by the KF-1 strain. Table 6 shows the results.

[0028]

[Table 6]

Example 6 In this example, an example of decomposing and removing phenanthrene by activated sludge to which the KF-1 strain was added will be described.

(1) Preparation of Activated Sludge KF-1 strain was added to activated sludge in a sewage treatment plant, and used after fixing and acclimatization. 2,000 initial MLSS of activated sludge
mg / l, and the KF-1 strain was adjusted to an initial cell concentration of 100 m.
g / l (dry concentration). (2) Synthetic wastewater and loading conditions SGP synthetic wastewater having the composition shown in Table 7 below was used.
0.1kg-BOD / kg-MLSS of SGP synthetic wastewater
・ Day, phenanthrene 0.05 kg / kg-ML
Both were added to the activated sludge at the same time so as to satisfy the loading condition of SS day.

(3) SRT (sludge residence time / sludge age) SRT was performed for 5, 10, 20, 30, and 50 days. (4) Treatment method The wastewater prepared in (2) was placed in a 1-liter Erlenmeyer flask with 50
0 ml and shake culture at room temperature for a predetermined time (10, 1
(5, 20 and 25 hours), and then allowed to stand for 4 hours to settle and separate sludge. An appropriate amount of the treated water was withdrawn, and phenanthrene in the treated water was analyzed. In addition, phenanthrene in MLSS was analyzed. The results are shown in Table 8 and FIG.
(Relation between processing time and phenanthrene concentration in MLSS)
Shown in When the SRT is 10 days or more, the phenanthrene concentration in the treated water is 1 mg /
1 (50 mg / l before treatment).

[0032]

[Table 7]

[0033]

[Table 8]

According to Table 8 and FIG. 1, if the SRT is taken for 5 days or more, preferably for 10 days or more, phenanthrene will be MLSS.
It is understood that stable processing is being performed without being accumulated in the inside. Therefore, it seems that the KF-1 strain stably colonizes the MLSS when the SRT is taken for 5 days or more, preferably 10 days or more.

Example 7 In this example, a method for imparting phenol resistance to the KF-1 strain and the KF-2 strain and the results thereof will be described. (1) Determination of logarithmic growth phase 1 ml of the pre-culture solution of the above strain, which had been pre-cultured in Nutrient Broth (NB) medium, was inoculated into 100 ml of NB medium and incubated at 30 ° C.
Shaking culture was performed. Changes in the turbidity, viable cell count, and ATP amount of this culture over time were measured, and the logarithmic growth phase was determined.
The logarithmic growth phase of the KF-1 and KF-2 strains was 4
-10 hours and 6-14 hours. (2) Determination of UV Irradiation Conditions (Irradiation Distance and Irradiation Time) Mutants by ultraviolet irradiation are usually obtained using a logarithmically growing bacterial cell under conditions that result in a high mortality (about 99.99%). Done. The cells in the logarithmic growth phase are diluted to an appropriate concentration (10 ⁻² to 10 ⁻³ ), and ultraviolet rays are irradiated for a predetermined distance (10 to 10 ⁻³ ).
Irradiation was carried out for a predetermined time (10 to 180 seconds), and the number of viable cells was measured to determine the optimal conditions. The ultraviolet irradiation was performed with a Toshiba lamp GL150 (manufactured by Toshiba Corporation).

The above results are shown in Tables 9 and 10. KF
The optimal irradiation conditions for the -1 strain (the killing rate of the bacterial cells is 99.99%) are as follows.
At 0 cm, irradiation is for 30 seconds. The optimum irradiation conditions for the KF-2 strain are 10 seconds irradiation at an irradiation distance of 10 to 30 cm, 5 seconds irradiation,
At 0 cm, irradiation is for 60 seconds.

[0037]

[Table 9]

[0038]

[Table 10]

(3) Obtaining Ultraviolet Irradiated Mutants 0.1 ml of KF-1 strain and KF-2 strain in logarithmic growth phase
The plate medium was smeared and irradiated with ultraviolet light under the respective optimum conditions determined in (2). (4) Improvement of phenol resistance A colony irradiated with ultraviolet rays was subjected to a phenol concentration of 500 ppm.
Was added to a phenanthrene medium (test tube). A dilution plate was prepared from the test tube in which growth was observed, and irradiated with ultraviolet light under the above-mentioned optimum conditions. The appeared colonies were cultured in a phenanthrene medium having a phenol concentration of 600 ppm. This operation was repeated until the phenol concentration reached 1
It was increased by 00 ppm.

The KF-1 strain had a phenol resistance of 700 ppm in the phenanthrene medium, but had a phenol resistance of 14 ppm due to the improvement of repeated irradiation and adaptation.
Improved to 00 ppm. Similarly, the phenol resistance was remarkably improved in the KF-2 strain.

[0041]

EFFECT OF THE INVENTION Pseud which decomposes polycyclic aromatic compounds
omonas spp. and Rhodococcus spp.
And a phenol-resistant bacterium obtained by the method of the present invention.
And by the use of acclimating microorganisms, it is possible to decompose and remove the polycyclic aromatic compounds persistent in the wastewater
Was .

[Brief description of the drawings]

FIG. 1 is a diagram showing test results of Example 6.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:38) (C12N 1/20 C12R 1:01) Examiner Toshio Uchida (56) References JP-A-63-291575 (JP) JP-A-63-207378 (JP, A) JP-A-5-276933 (JP, A) JP-A-7-155174 (JP, A) JP-A-7-23773 (JP, A) 63-202374 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 1/20 C12N 1/26 C02F 3/34 BIOSIS (DIALOG) WPI (DIALOG)

Claims (7)

(57) [Claims] 1. Pseud for decomposing a polycyclic aromatic compound
Omonas spp. and Rhodococcus spp.
Mutations are caused, and the resulting mutants are
Decomposes polycyclic aromatic compounds characterized by habituation
A method for imparting phenol resistance to bacteria. 2. Pseudomonas spp.
domonas paucimobilis, R
Rhodococcus spp.
The method for imparting phenol resistance to a microorganism that degrades a polycyclic aromatic compound according to claim 1, which is a rubber. 3. Pseudomonas paucim
obilis is Pseudomonas paucim
obilis 421Y strain (FERM BP-512)
2), and the Rhodococcus rubber is R
hodococcus ruber TA 0902 strain confers resistance to phenolic in bacteria degrade polycyclic aromatic compound according to claim 2 which is (FERM BP-5121)
how to. 4. Pseudo for decomposing a polycyclic aromatic compound
Omonas and Rhodococcus sp.
Specially added to municipal sludge and acclimatized in SRT 10 days or more.
Pseudomo that decomposes polycyclic aromatic compounds
How to acclimate nas and Rhodococcus
Law. 5. The Pseudomonas spp.
domonas paucimobilis and Rh
Odococcus sp. is Rhodococcus r
5. The polycyclic aromatic compound according to claim 4, which is
Pseudomonas spp. And Rhodoco
How to acclimatize ccus spp. 6. Pseudomonas paucim
obilis is Ps eudomonas paucim
obilis 421Y strain (FERM BP-512)
2), and the Rhodococcus rubber is R
hodococcus rubber TA0902 strain
(FERM BP-5121).
Pseudomonas sp. That decomposes polycyclic aromatic compounds
A method of acclimating bacteria and Rhodococcus spp. 7. The method according to claim 1, 2 or 3,
Of polycyclic aromatic compounds with phenol resistance.
Pseudomonas spp. And Rhodoco
a phenol-resistant bacterium of the genus ccus;
Yoshiaki Takan, acclimated by the method described in 4, 5, or 6
Pseudomonas spp. That degrades aromatic compounds and
Use of an acclimatized bacterium of the genus Rhodococcus
A method for treating polycyclic aromatic compound-containing wastewater.