WO2009125462A1

WO2009125462A1 - Microorganism capable of decomposing aromatic compounds and method of decomposing aromatic compounds using the same

Info

Publication number: WO2009125462A1
Application number: PCT/JP2008/056861
Authority: WO
Inventors: 祐子向井; 八千代川本; 豊一横田; 学佐見
Original assignee: アサヒビール株式会社
Priority date: 2008-04-07
Filing date: 2008-04-07
Publication date: 2009-10-15

Abstract

[PROBLEMS] To provide a microorganism capable of inducing an enzyme by which aromatic compounds including dioxin can be decomposed without using carbazole possibly posing a risk of environmental pollution. [MEANS FOR SOLVING PROBLEMS] A method of decomposing aromatic compounds characterized by comprising using a microorganism which has a promoter not needing induction and carAaI or carAaII originating in Sphingomonas sp. KAI strain and being under control thereby.

Description

Aromatic compound degrading microorganism and method for decomposing aromatic compound using the same

The present invention is characterized by cleaving a heterocycle when a substrate is an aromatic heterocycle by introducing two hydroxyl groups into the aromatic ring, and cleaving an ether bond when the substrate is phenol ether. The present invention relates to a microorganism having a gene encoding a characteristic enzyme and a novel method for decomposing aromatic compounds including dioxins using the microorganism.

One method of purifying soil and riverbed mud contaminated with dioxins is bioaugmentation. This is a method for purifying dioxins by spraying microorganisms that decompose dioxins on contaminated soil, and the microorganisms to be sprayed are white-rot fungi (Patent Document 1 “Method for Degrading Chlorinated Dioxins”), Patent Document 2 The microorganism must have a dioxin-degrading ability, such as the dioxin-degrading bacterium Sphingomonas sp. KA1 strain (FERM P-19744).

Such a decomposition method is cheaper in processing cost than a generally known physicochemical purification method. However, the period required for the purification is generally long, and in order to maintain the decomposition ability of microorganisms. , Soil aeration, moisturizing, and application of nutrients. Furthermore, since the degrading enzyme is almost always an inducing enzyme, it is necessary to realize a state in which the microbial enzyme is induced in the environment.

In the case of the above-mentioned white rot fungus, degrading enzymes are induced under carbon / nitrogen deficiency, so that it is difficult to apply to contaminated soil rich in nitrogen.

Also, in the case of KA1 strain, dioxin degrading enzyme is induced in the presence of carbazole, so that the bacterium is cultured with carbazole, and dioxin is effectively decomposed by adding carbazole to contaminated soil. However, culturing and spraying with carbazole to increase the degradation effect not only increases the treatment cost, but may cause new environmental contamination by carbazole, which is a major problem in practical use.

Here, an outline of the dioxin degrading bacterium Sphingomonas sp. KA1 strain (FERM P-19744) will be described. Two types of carbazole 1,9a-dioxygenase (CARDO) produced by the KA1 strain have been found and are called CARDOI and CARDOII, respectively. Since these enzymes can oxidize not only the original substrate carbazole but also various aromatic compounds including dioxin, they are expected to be applied to environmental purification as dioxin degrading enzymes.
CARDOI and CARDOII are multi-component enzymes composed of ferredoxin and ferredoxin reductase in addition to the main body (terminal oxygenase), and the genes encoding them are all found on the plasmid pCAR3 held by the KA1 strain. The terminal oxygenase genes are called carAaI and carAaII, respectively, and the homology between them is about 75% at the amino acid level. As a result of the examinations so far, it has been revealed that they show almost the same activity.
The operon formed by these gene groups is called a carbazole degrading operon. The operon promoter site is normally suppressed by the repressor protein CarRI produced by the transcriptional control gene carRI. However, in the presence of carbazole, the promoter is activated by removal of the repressor, and the downstream terminal oxygenase gene carAaI, carAaII and the like are expressed.
JP 2001-136957 A JP 2005-333874 A

The problem to be solved by the present invention is to provide a microorganism that produces a dioxin-degrading enzyme without requiring an inducer such as carbazole that may cause environmental pollution.

In the present invention, a dioxin-decomposing bacterium, Sphingomonas sp. We thought that bioaugmentation would be realized.

In the present invention, as a result of intensive studies, the present inventors succeeded in breeding a mutant strain of KA1 strain that is always produced by dioxin degrading enzyme. That is, the KA1 strain was subcultured in a culture system containing carbazole, which is considered to have been released from suppression of CARDO expression. As a result, mutant strains KA1G1 and KA1G2 carrying plasmids having a size different from that of pCAR3 were obtained at the 63rd passage. In these mutants, carAaI was deleted from the two cardo terminal oxygenase genes carAaI and carAaII encoded on pCAR3. When the presence or absence of transcription of carAaII into mRNA was examined by RT-PCR, transcription was not seen in the parent strain in the absence of carbazole, but transcription was seen in the KA1G1 and KA1G2 strains. It was estimated that it was a constitutive expression strain. When KA1G1 and KA1G2 strains were cultured using LB medium and the cells and dibenzo-p-dioxin were shaken in a buffer solution, significant reduction of dibenzo-p-dioxin was observed, which is an inducer. It was confirmed that the degrading enzyme was expressed, that is, constitutively expressed without adding carbazole.
In the present invention, the present inventors have also succeeded in breeding mutant strains that are more active than the KA1G1 and KA1G2 strains while constantly producing dioxin degrading enzymes. That is, the KA1G2 strain was subcultured in a culture system containing carbazole, and as a result, a mutant strain KA1G2-13012 having a plasmid of a size different from KA1G2 was obtained at the number of passages 67 times. While this mutant strain maintained the property of constitutive expression of CARDO, the CARDO activity was about twice that of the KA1 strain at the time of induction.
KA1G1 strain, KA1G2 strain and KA1G2-13012 strain were deposited internationally at the National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary, respectively, with accession numbers FERM BP-10861 (deposit date: July 19, 2006), FERM BP -10862 (Deposit date: July 19, 2006) and FERM BP-10954 (Deposit date: March 4, 2008).

That is, the present invention has the following gist.
(1)
A method for decomposing an aromatic compound, comprising using a microorganism having a carAaI or carAaII derived from a Sphingomonas sp. Strain KA1 and a promoter whose expression suppression mechanism is canceled by mutation or deletion of the transcriptional control gene carRI.
(2)
The degradation method according to item (1), wherein the microorganism is a genus Sphingomonas.
(3)
The degradation method according to item (1), wherein the microorganism is a Sphingomonas sp. KA1 strain or a strain derived therefrom.
(4)
Item 6. The method for decomposing an aromatic compound according to any one of items (1) to (3), wherein the heterocycle of the aromatic heterocyclic compound is cleaved.
(5)
Item 6. The method for decomposing an aromatic compound according to any one of Items (1) to (3), wherein the ether bond of phenol ether is cleaved.
(6)
6. The method for decomposing an aromatic compound according to any one of items (1) to (3), wherein a hydroxyl group is introduced into an aromatic ring of the aromatic compound to produce a dihydrodiol.
(7)
The decomposition method according to any one of items (1) to (6), wherein the aromatic compound is chlorinated dioxin.
(8)
Any of the items (1) to (6), wherein the aromatic compound is at least one selected from benzene, toluene, xylene, naphthalene, biphenyl, anthracene, dibenzofuran, dibenzothiophene, fluorene, fluoranthene, xanthene, phenoxathiin and diphenyl ether The decomposition method according to claim 1.
(9)
Sphingomonas sp. KA1G1 strain (FERM BP-10861), an aromatic compound-degrading microorganism.
(10)
An aromatic compound-degrading microorganism which is Sphingomonas sp. KA1G2 strain (FERM BP-10862).
(11)
An aromatic compound-degrading microorganism which is Sphingomonas sp. KA1G2-113012 strain (FERM BP-10954).
(12)
(9) A method for decomposing an aromatic compound, comprising using the microorganism according to any one of items (11) to (11).
(13)
(9) A method for decomposing an aromatic compound, comprising cleaving a heterocycle of an aromatic heterocyclic compound using the microorganism according to any one of (11) to (11).
(14)
(9) A method for decomposing an aromatic compound, which comprises cleaving an ether bond of phenol ether using the microorganism according to any one of items (11) to (11).
(15)
(9) A method for decomposing an aromatic compound, wherein a hydroxyl group is introduced into the aromatic ring of the aromatic compound using the microorganism according to any one of (11) to (11) to produce a dihydrodiol.
(16)
The decomposition method according to any one of (12) to (15), wherein the aromatic compound is chlorinated dioxin.
(17)
The aromatic compound is at least one selected from benzene, toluene, xylene, naphthalene, biphenyl, anthracene, dibenzofuran, dibenzothiophene, fluorene, fluoranthene, xanthene, phenoxathiin, and diphenyl ether, any of (12) to (15) Decomposition method according to the above.

The microorganism that constitutively expresses the dioxin-degrading enzyme of the present invention has the following advantages compared to the microorganism that is induced and expressed by conventional carbazole.
(1) Any medium may be used for the growth as long as the microorganism can grow, and the bacterial cells can be produced at low cost.
(2) There is no worry of causing new environmental pollution by the inducer.

Therefore, when bioaugmentation is performed using the microorganism of the present invention, various pollutants in the soil, for example, aromatic compounds such as dioxins, benzene, toluene, naphthalene, anthracene, and fluoranthene can be efficiently purified. .

The graph which shows the decomposition rate of dioxins.

The aromatic compound to be decomposed according to the present invention is selected from chlorinated dioxins or benzene, toluene, xylene, naphthalene, biphenyl, anthracene, dibenzofuran, dibenzothiophene, fluorene, fluoranthene, xanthene, phenoxathiin and diphenyl ether. At least one.

The microorganism used may be a promoter that does not require induction and that has carAaI or carAaII derived from the Sphingomonas sp. KA1 strain under its control.

A promoter that does not require induction is a promoter in which transcription of mRNA is not repressed even if the strain is cultured under any culture conditions. In addition to the gfp promoter, the nptII promoter, and the like, It includes a promoter that is in a state in which induction suppression is not caused by mutation or deletion.

The gene expressed under the control of such a promoter may be any gene as long as it contains the enzyme genes carAaI or carAaII that are the main components of dioxin degradation. Preferably, these enzymes are combined with ferredoxin that conducts electrons and ferredoxin reductase. It is better to have it. Examples of the gene encoding ferredoxin include carAcI and carAcII possessed by the KA1 strain. Examples of genes encoding ferredoxin reductase include fdrI and fdrII possessed by the KA1 strain.

Sphingomonas sp. KA1G1, KA1G2 and KA1G2-13012 are mutant strains obtained by mutation from the dioxin-degrading bacterium Sphingomonas sp. KA1 in order to satisfy these conditions.

In KA1G1 and KA1G2 strains, mutations occurred in pCAR3, and carAaI was deleted from the two cardo terminal oxygenases carAaI and carAaII. Is released and the remaining carAaII is constantly expressed.
Furthermore, the ferredoxin gene carAcII, which conducts electron transfer, is also encoded downstream of carAaII and is simultaneously transcribed as a single mRNA, so that CARDO activity is expressed regardless of the conditions under which these strains grow. To do. In addition, in the KA1G2-13012 strain, carAaII was detected at least twice that of the KA1 strain, and it is assumed that the increase in the number of genes contributed to the enhancement of CARDO activity.

CARDO thus expressed promotes the spontaneous cleavage of the heterocycle by introducing two hydroxyl groups into the benzene ring of carbazole and produces 2′-aminobiphenyl-2,3-diol.
This enzyme has a relatively wide substrate specificity and can introduce two hydroxyl groups into an aromatic compound having a similar reaction site structure. When the heteroatom is an oxygen atom instead of a nitrogen atom as in dibenzofuran, the heterocycle is cleaved in the same manner as carbazole. Similarly, in the case of dibenzo-p-dioxin and its chlorinated compound, the corresponding cleavage compound is produced.
When the aromatic compound to be decomposed does not have a heterocycle like diphenyl ether and belongs to phenol ether, the reaction occurs in exactly the same manner, and the ether bond is cleaved to generate two molecules.
When the aromatic compound to be decomposed has neither a heterocycle nor a phenol bond, such as benzene and naphthalene, CARDO introduces a hydroxyl group into the aromatic ring to form a dihydrodiol. That is, the benzene ring on which CARDO has acted is converted to a 6-membered ring having two double bonds.
Thus, since CARDO acts on various aromatic compounds, these Sphingomonas sp. Strains KA1G1, KA1G2 and KA1G2-13012 can be said to be highly practical degrading bacteria.

In order to purify soil contaminated with these decomposing bacteria, the decomposed bacteria and the pollutant may be contacted by spraying the decomposing bacteria over the contaminated sites. The spraying method may be any form such as spraying the culture solution as it is if the degrading bacteria are alive, or immobilizing on activated carbon, diatomaceous earth, or the like. The treatment temperature may be in a range called normal temperature, but is preferably 10 ° C. to 35 ° C., more preferably 25 ° C. to 30 ° C. However, even if sprayed in this way, the degrading bacteria do not always survive in the contaminated area for a long time and do not always continue to decompose the pollutants. It is possible to further purify by repeatedly spraying the decomposed bacteria cultured on the surface.
(Example)
EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not limited to a following example.

Method for obtaining mutant strains that express dioxin degrading enzyme CFMM medium (Na ₂ HPO ₄ 2.2 g, KH ₂ PO ₄ 0.8 g, NH ₄ NO ₃ 3.0 g, MgSO ₄ per liter) _{· 7H 2 O 0.2 g, FeCl} 3 · 6H 2 O 50 mg, CaCl 2 · 2H 2 O 10 mg) 5ml and was added carbazole solution (100 mg / ml DMSO solution) 50 [mu] l was inoculated with Sphingomonas sp. KA1 strain. This was cultured with shaking at 30 ° C., and subculture was repeated every 3 days under the same culture conditions. During the 63rd culture, the white or pale yellow color of the culture broth changed to brown, so the culture broth was extracted with ethyl acetate under neutral conditions and the extracted components were converted to GC-MS. In comparison with the culture solution of KA1 strain before subculture, significant accumulation of catechol and anthranilic acid was observed in the culture solution after subculture, and some mutations occurred in KA1 strain by subculture It has been suggested.

Two mutant strains KA1G1 and KA1G2 were isolated from the main culture. When plasmids were extracted from each of them and subjected to DNA sequencing, it was found that both mutants had a large portion of plasmid pCAR3 possessed by KA1 strain deleted at least about 60 kb. Among the two gene groups that degrade dioxins, the deletion site included an operon containing carAaI and a catechol-degrading operon responsible for metabolism downstream of carbazole. In the KA1G2 strain, a portion not related to the carbazole degradation gene was further deleted from the KA1G1 strain by about 300b.

Even when these gene groups were deleted, the mutant strain was able to grow using carbazole as the sole carbon source because the operon containing another carbazole-degrading gene carAaII on the plasmid and on the chromosome downstream of catechol. This is because a gene group exists. Furthermore, as a result of intensive studies, it was found that the deletion site contained carRI that regulates the transcription of operons including carAaI and carAaII. It was inferred that this gene was lost because carAaII was constitutively expressed.

Method for Obtaining Mutant Strain KA1G2-13012 Constitutively Expressing Dioxin Degrading Enzyme Strain KA1G2 was subcultured in the same manner as in Example 1. In the 67th culture, the brown color of the culture broth changed to pale yellow, suggesting that subculturing caused some mutation in the KA1G2 strain.
A mutant strain KA1G2-13012 was isolated from the main culture. Total DNA was extracted from each, and the abundance ratio of carAaII and 16SrDNA was analyzed by real-time PCR. As a result, the value of this mutant strain was more than twice that of the KA1G2 strain. From this, it was inferred that this mutant strain was a strain in which the region containing carAaII was duplicated on the genome by homologous recombination or the like.

Dioxin-degrading gene expression level (activity)
When CARDO acts on naphthalene, it produces cis-1,2-dihydroxy-1,2-dihydronaphthalene. By using this reaction and quantifying the product by liquid chromatography, the KA1 strain, KA1G1 strain, KA1G2 strain, and KA1G2-13012 strain were compared in CARDO activity.

Suspend 2 ml of phosphate buffer solution, 5 mg of naphthalene, and cultured cells of KA1, KA1G1, KA1G2, and KA1G2-113012 in an Ø10 mm glass test tube so that OD ₆₀₀ = 0.5 did. In order to culture the KA1 strain, a CFMM medium supplemented with 0.01% carbazole was used. In order to culture the KA1G1 strain, the KA1G2 strain, and the KA1G2-113012 strain, a CFMM medium supplemented with 0.01% sodium succinate was used. After the reaction, the culture broth was filtered, cis-1,2-dihydroxy-1,2-dihydronaphthalene in the filtrate was quantified by liquid chromatography, and the initial production rate was measured.

The results are shown in Table 1. The unit unit used here represents the ng number of cis-1,2-dihydroxy-1,2-dihydronaphthalene produced in one hour in this reaction system. The KA1G1 and KA1G2 strains showed a production rate of about 40% of the KA1 strain induced with carbazole without adding carbazole. Moreover, the KA1G2-13012 strain showed about twice the production rate of the KA1 strain. From these results, it was confirmed that all these mutants expressed constitutive expression of CARDO. The KA1G1 and KA1G2 strains are less active than the KA1 strain in which CARDO was induced because one of the two enzyme genes was deleted in the mutant strain. it is conceivable that. On the other hand, the high activity of the KA1G2-13012 strain is considered to be due to duplication of carAaII in the genome. It is not clear whether the effect is only due to the large number of genes or whether the transcriptional activity is also increased.

Degradation ability of the aromatic compound of the obtained mutant strain 1 ml of 50 mM phosphate buffer (pH 7.2) was placed in a 20 ml glass vial and cultured in the same manner as described in Example 3, KA1 strain, KA1G1 strain, The KA1G2 strain and the KA1G2-113012 strain were suspended so that OD ₆₀₀ = 1.5. 10 μg each of benzene, toluene, xylene, naphthalene, biphenyl, anthracene, dibenzofuran, dibenzothiophene, fluorene, fluoranthene, xanthene, phenoxathiin, and diphenyl ether was added to these cell suspensions, and the mixture was sealed and reacted at 30 ° C. for 24 hours. In the case of benzene, toluene and xylene, the remaining material was extracted from the gas phase and the product was extracted from the liquid phase and subjected to GC-MS analysis. In the case of other test substances, residual substrates and products were extracted from the liquid phase and subjected to GC-MS analysis.
Table 2 shows the ratio (%) of the recovered remaining substrate. All strains degraded the test substance well. In particular, in the KA1G2-113012 strain, many substances were degraded at a high degradation rate.
On the other hand, the detected decomposition products were dibenzofuran, dibenzothiophene and diphenyl ether, except for compounds to dihydrodiol, dibenzofuran to trihydroxybiphenyl and dibenzofurandihydrodiol, dibenzothiophene to dibenzothiophene dihydrodiol and dibenzothiophene-5- To oxide, diphenyl ether was converted to catechol and phenol. As a result, it was confirmed that these mutant strains degraded the aromatic compound by the same mechanism as the parent strain KA1.

Decomposition ability of tetrachlorinated dioxin 5 ml of 50 mM phosphate buffer (pH 7.2) was placed in a 30 ml Erlenmeyer flask, and cultured in the same manner as in Example 3. KA1, KA1G1, KA1G2 and KA1G2-113012 were OD. _It was suspended to ₆₀₀ = 1.5. To this, 10 ng of 2,3,7,8-TeCDD was added and stirred at 30 ° C. for 24 hours. Residual substrates were extracted and quantified from these samples, and the decomposition rates as shown in the table below were obtained. As a control, cells obtained by autoclaving the KA1G1 strain at 121 ° C. for 5 minutes were used, and the degradation rate was calculated based on the ratio to the recovered amount from this control.

Thus, it was revealed that any of the obtained mutant strains decomposes 2,3,7,8-TeCDD even when a culture medium without carbazole is used for the culture. In particular, the KA1G2-113012 strain decomposed the added 2,3,7,8-TeCDD by 60% or more in 24 hours and was shown to have a very strong 2,3,7,8-TeCDD degradation ability.

Decomposition capacity of highly chlorinated dioxins 5 ml of 50 mM phosphate buffer (pH 7.2) was placed in a 30 ml Erlenmeyer flask and cultured in the same manner as in Example 3. KA1 strain, KA1G1 strain and KA1G2-13012 strain were OD ₆₀₀ = 1 Suspended to be .5. A dioxin mixed solution was added thereto and stirred at 30 ° C. for 24 hours. The amount of dioxin added is 10 ng for tetrachlorinated dioxins, pentachlorinated dioxins, tetrachlorinated dibenzofurans, pentachlorinated dibenzofurans, and coplanar PCBs, 50 ng for octachlorinated dioxins and octachlorinated dibenzofurans, and other dioxins for dioxins 20 ng. In these samples, the residual substrate was extracted and quantified, and the residual ratio as shown in FIG. 1 was obtained. As a control, microbial cells obtained by autoclaving the KA1G1 strain at 121 ° C. for 5 minutes were used, and the residual rate was calculated as a ratio to the recovered amount from this control.

As shown in FIG. 1, it was found that the KA1, KA1G1 and KA1G2-13012 strains can decompose various toxic chlorinated dioxins other than 2,3,7,8-TeCDD.
Particularly high in 2,3,7,8-TeCDF, 3,4,4 ′, 5-TeCB, 3,3 ′, 4,4′-TeCB, 3,3 ′, 4,4 ′, 5-PeCB, etc. The decomposition rate could be obtained.
Considering the test results described in Example 3 and Example 5, it is presumed that the KA1G2 strain not used in this test has the same degradation activity as the KA1G1 strain.

In addition, the abbreviation in FIG. 1 points out the following compounds.
TeCDF: Tetrachlorodibenzofuran
PeCDF: Pentachlorodibenzofuran
HxCDF: Hexachlorodibenzofuran
HpCDF: Heptachlorodibenzofuran
OCDF: Octachlorodibenzofuran
TeCDD: Tetrachlorodibenzo-p-dioxin
PeCDD: Pentachlorodibenzo-p-dioxin
HxCDD: Hexachlorodibenzo-p-dioxin
HpCDD: Heptachlorodibenzo-p-dioxin
OCDD: Octachlorodibenzo-p-dioxin
TeCB: Tetrachlorobiphenyl
PeCB: Pentachlorobiphenyl
HxCB: Hexachlorobiphenyl
HpCB: Heptachlorobiphenyl

Since the present invention can decompose aromatic compounds without causing environmental pollution, the present invention is extremely useful as an environmental purification technique.

Claims

A method for decomposing aromatic compounds, comprising using a microorganism having a carAaI or carAaII derived from a Sphingomonas sp. KA1 strain and a promoter whose expression suppression mechanism is released by mutation or deletion of the transcriptional control gene carRI.
The degradation method according to claim 1, wherein the microorganism is a genus Sphingomonas.
The degradation method according to claim 1, wherein the microorganism is Sphingomonas sp. KA1 strain or a strain derived therefrom.
The method for decomposing an aromatic compound according to any one of claims 1 to 3, wherein the heterocycle of the aromatic heterocyclic compound is cleaved.
The method for decomposing an aromatic compound according to any one of claims 1 to 3, wherein the ether bond of phenol ether is cleaved.
The method for decomposing an aromatic compound according to any one of claims 1 to 3, wherein a hydroxyl group is introduced into the aromatic ring of the aromatic compound to produce a dihydrodiol form.
The decomposition method according to any one of claims 1 to 6, wherein the aromatic compound is chlorinated dioxin.
The aromatic compound is at least one selected from benzene, toluene, xylene, naphthalene, biphenyl, anthracene, dibenzofuran, dibenzothiophene, fluorene, fluoranthene, xanthene, phenoxathiin and diphenyl ether. The disassembly method described in 1.
Aromatic compound-degrading microorganism, which is Sphingomonas sp.
Aromatic compound-degrading microorganisms, which are Sphingomonas sp.
Aromatic compound-degrading microorganism, Sphingomonas sp. KA1G2-113012 strain (FERM BP-10954).
A method for decomposing an aromatic compound, comprising using the microorganism according to any one of claims 9-11.
A method for decomposing an aromatic compound, comprising cleaving a heterocycle of an aromatic heterocyclic compound using the microorganism according to any one of claims 9-11.
A method for decomposing an aromatic compound, comprising cleaving an ether bond of phenol ether using the microorganism according to any one of claims 9-11.
A method for decomposing an aromatic compound, comprising using the microorganism according to any one of claims 9 to 11 to introduce a hydroxyl group into an aromatic ring of the aromatic compound to produce a dihydrodiol form.
The decomposition method according to any one of claims 12 to 15, wherein the aromatic compound is chlorinated dioxin.
The aromatic compound is at least one selected from benzene, toluene, xylene, naphthalene, biphenyl, anthracene, dibenzofuran, dibenzothiophene, fluorene, fluoranthene, xanthene, phenoxathiin and diphenyl ether. The disassembly method described in 1.