JP2003009850A - Method for degrading polycyclic aromatic compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means for promoting the degradation of a polycyclic aromatic compound with a microorganism. SOLUTION: BPCI strain isolated from a microorganism consortium for degrading the polycyclic aromatic compound.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for decomposing polycyclic aromatic compounds such as benzopyrene, and more specifically, it utilizes a microorganism that produces a substance that promotes decomposition of polycyclic aromatic compounds by microorganisms. The present invention relates to a method for decomposing a ring aromatic compound.

[0002]

BACKGROUND OF THE INVENTION Polycyclic aromatic compounds are a by-product of petroleum spills, incomplete fossil fuel combustion and chemical industrial processes.
Released into the environment (Cerniglia, CE 1992. Biodegr
adation 3: 351-364.). It is known that many polycyclic aromatic compounds adversely affect the human body, and among them, benzopyrene is known to be particularly mutagenic and carcinogenic. Since it is impossible to selectively remove low concentrations of polycyclic aromatic compounds in the environment by physicochemical treatment, very expensive treatment such as transporting contaminated soil to other places is not possible. Be taken. On the other hand, microorganisms that decompose polycyclic aromatic compounds such as benzopyrene are also known (Cerniglia, CE 1992 Biodegradation 3: 351-36.
4.), and environmental restoration studies using them have also been conducted (Boonchan, B., ML Britz, and GA Stanley. 20
00. Appl. Environ. Microbiol. 66: 1007-1019). However, due to the slow degradation rate of these microorganisms (Kanaly, R.
A., and S. Harayama. 2000. J. Bacteriol. 182: 2059-
2067.), restoration of biological environment has not been put to practical use.

The present inventors have so far developed a soil microbial consortium (Kanaly, RA, R. Bartha, K. Watanabe) that decomposes polycyclic aromatic compounds such as benzopyrene relatively quickly.
andS. Harayama. 2000. Appl. Environ. Microbiol. 6
6: 4205-4211.). This consortium can rapidly decompose benzopyrene in the presence of diesel oil. However, the microorganisms present in them and their roles have not been fully understood, which has been an obstacle to the development of more efficient decomposition methods.

[0004]

One of the reasons why polycyclic aromatic compounds such as benzopyrene are difficult to biodegrade is
The reason is that they are hydrophobic and hardly dissolve in the aqueous solution. In other words, if there is a substance that solubilizes the polycyclic aromatic compound or a microorganism that produces the solubilized substance, biodegradation of the polycyclic aromatic compound is promoted, and rapid environmental purification can be achieved.

An object of the present invention is to provide a means for promoting the decomposition of polycyclic aromatic compounds by microorganisms.

[0006]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventor has found that (1) a novel bacterium BPC1 strain from a soil microbial consortium that decomposes polycyclic aromatic compounds such as benzopyrene. The present invention has been completed by finding that (2) the culture supernatant of the bacterium was added to the soil microbial consortia to promote the decomposition of benzopyrene.

That is, the first aspect of the present invention is that 16S rRNA represented by a nucleotide sequence having 95% or more homology with the nucleotide sequence of SEQ ID NO: 1.
It is a strain belonging to the genus Rodanobacter with a gene.

The second aspect of the present invention is to add the culture supernatant of the above strains to
A method for decomposing a polycyclic aromatic compound, which comprises adding the polycyclic aromatic compound to an environment contaminated with the polycyclic aromatic compound.

A third aspect of the present invention is a culture supernatant of the above strain,
And a microorganism decomposing a polycyclic aromatic compound or a consortium of such microorganisms is added to an environment polluted with the polycyclic aromatic compound.

A fourth aspect of the present invention is a polycyclic aromatic compound degradation accelerator containing the culture supernatant of the above strain as an active ingredient.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

The strain of the present invention belongs to the genus Rodanobacter having the 16S rRNA gene represented by the same or similar base sequence as the base sequence of SEQ ID NO: 1. The base sequence described in SEQ ID NO: 1 is the base sequence of the 16S rRNA gene of the BPC1 strain, and the strain having the 16S rRNA gene represented by the same or similar base sequence as this base sequence is a strain closely related to the BPC1 strain. Is considered to be. The term “similar base sequence” as used herein usually means a base sequence that is 95% or more homologous to the base sequence described in SEQ ID NO: 1, preferably a 98% or more homologous base sequence, and more preferably 99% or more. It means a nucleotide sequence homologous to at least%.

The strain of the present invention can be isolated from soil or the like contaminated with a polycyclic aromatic compound using the nucleotide sequence of the 16S rRNA gene as an index.

An example of the strain of the present invention is BPC1 strain. This strain was deposited under the reference number FERM P-18 at the Patent Biological Depository Center, National Institute of Advanced Industrial Science and Technology.
Deposited as 387 (Deposit date: June 21, 2001)
Day). The strain of the present invention includes the BPC1 strain and a mutant strain of this strain. Here, the mutant strain of BPC1 strain produces a culture supernatant having the same function as the culture supernatant of BPC1 strain (function of promoting decomposition of polycyclic aromatic compounds by microorganisms), and the other one or In the above properties, it means a strain different from the BPC1 strain.

The culture supernatant of the strain of the present invention has a function of promoting the decomposition of polycyclic aromatic compounds by microorganisms. Therefore, by adding this culture supernatant to a polycyclic aromatic compound-contaminated environment together with a microorganism that decomposes the polycyclic aromatic compound, the polycyclic aromatic compound is decomposed and removed from the environment. You can Further, in the environment polluted with the polycyclic aromatic compound, usually, there is a microorganism that decomposes the polycyclic aromatic compound, so without adding a microorganism that decomposes the polycyclic aromatic compound from the outside, Only the culture supernatant of the strain of the present invention may be added.

[0016] The microorganism that decomposes the polycyclic aromatic compound is not particularly limited, and the microorganism may be not a single microorganism but a microbial consortia. RU.MBI.1997 can be mentioned as a preferable microbial consortia. This microbial consortia has been deposited at the National Institute of Advanced Industrial Science and Technology, Patent and Bio-organism Depositary, with deposit number FERM P-17934 (deposit date: June 30, 2000).

Examples of polycyclic aromatic compounds include benzo [a] pyrene, benzo [e] pyrene, dioxin, pyrene and PCB. The environment to which the culture supernatant is added usually means soil, but does not exclude other environments such as rivers and oceans.

Any culture supernatant may be used as long as it has a function of promoting the decomposition of polycyclic aromatic compounds by microorganisms, and its preparation means is not limited. Usually, the culture supernatant is obtained by culturing the strain of the present invention in an appropriate medium,
It can be obtained by subjecting the resulting culture to centrifugation or the like.

The strain of the present invention can be cultured in the same manner as the known strain of the genus Rodanobacter. As the medium, various media containing assimilable carbon sources, nitrogen sources, and inorganic substances can be used. As the carbon source, for example,
As the glucose, xylose, acetic acid, and nitrogen sources, yeast extract, ammonia, nitric acid, and other inorganic substances such as potassium phosphate, magnesium chloride, iron chloride can be used. As a preferable medium, SB medium, dCGY
A medium and the like can be exemplified. The temperature during culture is
The temperature is preferably 20 to 35 ° C, more preferably 25 to 30 ° C. The pH in the medium is preferably maintained in the range of 6-8. The culture period is not particularly limited, but it is usually preferably 1 to 5 days, more preferably 2 to 3 days. The condition of centrifugation is not particularly limited, but it is preferably 8000 rpm-10000 rpm for 5-10 minutes.
The culture supernatant obtained by centrifugation may be filtered through a suitable filtration membrane, if necessary. As the filtration membrane used at this time, a microfiltration membrane made of cellulose acetate, polyvinylidene fluoride, polycarbonate or the like can be used.

The amount of culture supernatant to be added to the environment may be determined according to the amount and type of polycyclic aromatic compound present in the environment and its degrading bacterium, for example, Microbial Consortia RU.MBI. 1997 is present in 1 L in only 1 ml, benzo [a]
Assuming an environment where only 1 mg of pyrene is present,
The amount of culture supernatant added to it is about 50 to 200 ml.

[0021]

[Examples] [Example 1] Isolation and identification of BPC1 strain Soil microbial consortia (FERMP-17934) that decomposes polycyclic aromatic compounds such as benzopyrene was diluted with sterilized water to prepare dCG.
The Y medium (containing 0.5 g of casamino acid, 0.5 g of glycerol, 0.1 g of yeast extract per 1 L) was spread on an agar plate and cultured at 25 ° C. The colonies that had formed one week later were picked up, and again applied to the dCGY medium plate to purify the strains, and the BPC1 strain was isolated.

A small amount of colonies formed on the plate was collected and the method of Watanabe et al. (Watanabe, K., M. Teramoto, an
d S. Harayama. 1999. Appl. Environ. Microbiol. 65:
2813-2819.), The 16S rRNA gene was amplified by PCR and the nucleotide sequence was determined. This sequence is SEQ ID NO: 1
Shown in. BLAST database search based on the obtained sequence revealed that the BPC1 strain is closely related (98% homologous) to Rhodanobacter lindanoclasticus. From the sequence comparison, the possibility of a new bacterium of the genus Rhodobacter was considered.

The method of Kanaly et al. (Kanaly, RA, R. Bart
ha, K. Watanabe and S. Harayama. 2000. Appl. Enviro
n. Microbiol. 66: 4205-4211.), the ability of BPC1 strain to decompose benzo [a] pyrene was examined, but this strain did not decompose benz [a] pyrene. [Example 2] Promotion of benz [a] pyrene decomposition by culture supernatant of BPC1 strain BPC1 strain was treated with SB medium (Stanier's basal medium, Table 1 containing 500 mg / L yeast extract and 500 mg / L diesel oil). ).

[0024]

[Table 1] Centrifuge (8,000 rpm, 10 minutes) and filter filtration (acetate acetate membrane, 0.22 micron pores) from culture after 7 days.
The bacterial cells were removed by. During this process, diesel oil was removed from the culture supernatant.

[0025] Mix 15 ml of the culture supernatant with an equal amount of SB medium and
l of soil microbial consortia (FERMP-17934) suspension was added. To this, [ ¹⁴ C] benzo [a] pyrene was added to 1 mg / L, and the generated [ ¹⁴ C] CO ₂ was added to Oxosol C14 (National
(Diagnostics, Atlanta, USA) and measured with a scintillation counter. Also, as a control, BP
Instead of the culture supernatant of the C1 strain, a filter filtrate of SB medium containing yeast extract and diesel oil was added, and the same operation was performed. The result is shown in FIG.

The vertical axis in the figure represents the generation ratio of [ ¹⁴ C] CO ₂ with respect to the added [ ¹⁴ C] benzo [a] pyrene. Further, ■ in the figure represents the case where the culture supernatant was added, and ○ represents the case where the filtered product of the SB medium containing the yeast extract and diesel oil was added (control). As shown in this figure, when the culture supernatant was added, the decomposition of benz [a] pyrene was started immediately without lag time.

[0027] Kanaly et al.
It has been reported that the degradation of benz [a] pyrene by soil microbial consortia is accelerated (Kanaly, RA, R. Bart.
ha, K. Watanabe and S. Harayama. 2000. Appl. Envir
on. Microbiol. 66: 4205-4211.), the decomposition is accelerated about 5 days after the addition of diesel oil, which is obviously different from the case where the decomposition starts quickly without lag time. is there.

[0028]

INDUSTRIAL APPLICABILITY The present invention provides a microorganism which produces a substance having a function of promoting the decomposition of polycyclic aromatic compounds by the microorganism. By utilizing this microorganism, the environment polluted with the polycyclic aromatic compound can be efficiently purified.

[0029]

[Sequence list]                                SEQUENCE LISTING        <110> MARINE BIOTECHNOLOGY INSTITUTE CO., LTD. <120> METHOD FOR DEGRATING POLYCYCLIC AROMATIC COMPOUND <130> P01-041 <160> 1 <170> PatentIn Ver. 2.1 <210> 1 <211> 1532 <212> DNA <213> Rhodanobacter sp. <400> 1 gatcctggct cagattgaac gctggcggca tgcctaacac atgcaagtcg aacggcagca 60 cagaggagct tgctccttgg gtggcgagtg gcggacgggt gagtaatgca tcgggatcta 120 cccagacgtg ggggataacc tcgggaaacc gggactaata ccgcatacgt cctacgggag 180 aaagcggggg accttttagg cctcgcgcgg ctggacgaac cgatgtgcga ttagctagtt 240 ggtggggtaa tggcctacca aggcgacgat cgctagctgg tctgagagga tgatcagcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga atattggaca 360 atgggcgcaa gcctgatcca gcaatgccgc gtgtgtgaag aaggccttcg ggttgtaaag 420 cacttttatc aggaacgaaa tctgcatgct taatacgtgt gcagtctgac ggtacctgag 480 gaataagcac cggctaactt cgtgccagca gccgcggtaa tacgaagggt gcaagcgtta 540 atcggaatta ctgggcgtaa agggtgcgta ggcggttgct taagtctgtc gtgaaatccc 600 cgggctcaac ctgggaatgg cgatggatac tgggcagcta gagtgtgtca gaggatggtg 660 gaattcccgg tgtagcggtg aaatgcgtag agatcgggag gaacatcagt ggcgaaggcg 720 gccatctggg acaacactga cgctgaagca cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccct aaacgatgcg aactggatgt tggtctcaac tcggagatca 840 gtgtcgaagc aaacgcgtta agttcgccgc ctggggagta cggtcgcaag actgaaactc 900 aaaggaattg acgggggccc gcacaagcgg tggagtatgt ggtttaattc gatgcaacgc 960 gaagaacctt acctggcctt gacatgtccg gaatcctgca gagatgcggg agtgccttcg 1020 ggaatcggaa cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 1080 agtcccgcaa cgagcgcaac ccttgtcctt agttgccagc acgtaaaggt gggaactcta 1140 aggagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaagtca tcatggccct 1200 tacggccagg gctacacacg tactacaatg gtcggtacag agggttgcaa taccgcgagg 1260 tggagccaat cccagaaagc cgatcccagt ccggattgga gtctgcaact cgactccatg 1320 aagtcggaat cgctagtaat cgcggatcag ctatgccgcg gtgaatacgt tcccgggcct 1380 tgtacacacc gcccgtcaca ccatgggagt gggttgctcc agaaggcgtt agtctaaccg 1440 caaggaggac gacgcccacg gagtggtcca tgactggggt gaagtcgtaa caaggtagcc 1500 gtatcggaag gtgcggctgg atcacctcct ta 1532

[Brief description of drawings]

FIG. 1 is a view showing a change with time in benzo [a] pyrene decomposition of a microbial consortia to which a culture supernatant of BPC1 strain was added.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C02F 3/34 C12N 1/20 A // (C12N 1/20 C12R 1:01 C12R 1:01) B09B 3 / 00 E (72) Inventor Kazuya Watanabe 75-1 Hirata No. 3 75-1 Hamata, Kamaishi-shi, Iwate Inside the Marine Biotechnology Research Institute Kamaishi Research Institute, Inc. No. 1 F-Term in Kamaishi Research Center, Marine Biotechnology Research Institute, Inc. (reference) 4B065 AA01X AC20 BA22 BB04 BB40 CA56 4D004 AA41 AB05 AC07 CA18 CC07 4D040 DD03

Claims (5)

[Claims] 1. A strain belonging to the genus Rodanobacter having a 16S rRNA gene represented by a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 1 by 95% or more. 2. The strain according to claim 1, wherein the strain belonging to the genus Rhodobacter is Rhodobacter sp. BPC1 strain or a mutant strain thereof. 3. A method for decomposing a polycyclic aromatic compound, which comprises adding the culture supernatant of the strain according to claim 1 or 2 to an environment contaminated with the polycyclic aromatic compound. 4. A culture supernatant of the strain according to claim 1 or 2,
And a method for decomposing a polycyclic aromatic compound, which comprises adding a microorganism decomposing a polycyclic aromatic compound or a consortium of such microorganisms to an environment contaminated with the polycyclic aromatic compound. 5. A polycyclic aromatic compound degradation accelerator comprising the culture supernatant of the strain according to claim 1 or 2 as an active ingredient.