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Enzyme systems for biodegradation of polychlorinated dibenzo- p-dioxins.


Appl Microbiol Biotechnol (2010) 88:23–30 DOI 10.1007/s00253-010-2765-2

MINI-REVIEW

Enzyme systems for biodegradation of polychlorinated dibenzo-p-dioxins
Toshiyuki Sakaki & Eiji Munetsuna

Received: 21 April 2010 / Revised: 5 July 2010 / Accepted: 5 July 2010 / Published online: 22 July 2010 # Springer-Verlag 2010

Abstract The angular dioxygenase, cytochrome P450, lignin peroxidase, and dehalogenase are known as dioxinmetabolizing enzymes. All of these enzymes have metal ions in their active centers, and the enzyme systems except for peroxidase have each distinct electron transport chain. Although the enzymatic properties of the angular dioxygenase, lignin peroxidase, and cytochrome P450 have been studied well, the information about dehalogenase is much less than other enzyme systems due to its instability under the aerobic conditions. However, this enzyme system appears to be quite promising from the viewpoint of practical use for bioremediation, because dehalogenases are capable of degradation of polychlorinated dibenzo-pdioxins (PCDDs) with more than four chlorine substituents, whereas the other three enzyme systems prefer lowchlorinated PCDDs. On the other hand, protein engineering of angular dioxygenase, lignin peroxidase, and cytochrome P450 based on their tertiary structures has great potential to generate highly efficient dioxin-metabolizing enzymes. Actually, we successfully generated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-metabolizing enzyme by sitedirected mutagenesis of cytochrome P450. We hope that recombinant microorganisms harboring genetically engineered dioxin-metabolizing enzymes will be used for bioremediation of soil contaminated with PCDDs and polychlorinated dibenzofurans in the near future. Keywords Dioxin . Enzyme . Metabolism . Bioremediation . Microorganism
T. Sakaki (*) : E. Munetsuna Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan e-mail: tsakaki@pu-toyama.ac.jp

Introduction Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are known as environmental contaminants (Fig. 1). Although many physicochemical methods including incineration at a high temperature have been developed to destroy dioxins completely, from the economical point of view, thermal treatment of vast masses of contaminated soils and sediments is not feasible. Therefore, it is necessary to take biological alternatives into consideration. The destruction of organic compounds by microbial biocatalysts including bacteria and fungi plays an essential role in the global carbon cycle. Thus, bioremediation technologies using activated sludge microorganisms for industrial chemicals have been developed. Many reports have been published on the microbial degradation of PCDDs and PCDFs (Bunge et al. 2003; Habe et al. 2001; Harms et al. 1991; Kearny et al. 1972; Keim et al. 1999; Matsumura and Benezet 1973; Monna et al. 1993; Parsons et al. 1998; Philippi et al. 1981, 1982; Schreiner et al. 1997; Ward and Matsumura 1978; Wilkes et al. 1996; Wittich 1998; Wittich et al. 1992, 1999). More than two decades ago, Bumpus et al. (1985) reported that white-rot fungus Phanerochaete chrysosporium degraded 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). More recently, Bunge et al. (2003) reported that an anaerobic bacterium Dehalococcoides sp. strain CBDB1 is capable of conversion of 1,2,3,7,8-pentaCDD to 2,7- or 2,8-DCDD. These results strongly suggest that such bacteria and white-rot fungi have enzyme systems for degradation of PCDDs and PCDFs. An aerobic bacterium, Sphingomonas sp. RW1, which is the dibenzo-p-dioxin (DD)- and dibenzofuran (DF)mineralization strain, has a dioxin dioxygenase system (Armengaud et al. 1998, 2000; Armengaud and Timmis 1997, 1998). White rot fungi has both lignin peroxidase

24 Fig. 1 Structures of polychlorinated dibenzo-p-dioxins (PCDDs) (a), polychlorinated dibenzofurans (PCDFs) (b), and carbazole (c)

Appl Microbiol Biotechnol (2010) 88:23–30

a
8 7

9

10

O

1 2 3

Clm

6

O 5

4

Cln

b
Clm
7 6 8

9

1

2

Cln
3

O
5

4

c
6 7

5

4 3

8

N 9a 1 H

9

2

and Mn-dependent peroxidase. Recent genome analysis revealed that P. chrysosporium has 148 cytochrome P450 genes (Larrondo et al. 2007; Matsuzaki and Wariishi 2004; Shary et al. 2008; Yadav et al. 2006). In contrast, the anaerobic bacterium, Dehalococcoides has multiple nonidentical reductive-dehalogenase-homologous (RDH) genes in its genome (Holscher et al. 2004; Waller et al. 2005). Judging from the fact that the microorganisms are capable of dioxin degradation, some enzyme systems consisting of dioxygenase, peroxidase, P450 monooxygenase, and dehalogenase may show degradation activity toward PCDDs and PCDFs including the most toxic 2,3,7,8-TCDD (Kearny et al. 1972; Matsumura and Benezet 1973; Philippi et al. 1981, 1982; Quensen and Matsumura 1983; Ward and Matsumura 1978). Although there are high expectations for the bacterial degradation of dioxins for the bioremediation of soil polluted with them, the ability of natural microorganisms appears to be insufficient for practical applications in the degradation of the most toxic dioxin, 2,3,7,8-TCDD. Thus, the discovery or generation of a 2,3,7,8-tetraCDD-metabolizing enzyme and its overexpression in microorganisms would be promising for bioremediation uses. In this report, the enzyme systems involved in the degradation of PCDDs and PCDFs are reviewed. We have studied metabolism of PCDDs by mammalian cytochrome P450s in addition to microbial cytochrome P450s, and we also describe their enzymatic properties.

molecular oxygen into the substrate (Fig. 2). Some terminal dioxygenases are homomultimers (α), while others are heteromultimers consisting of a large subunit (α), which belongs to a large family named Rieske non-heme iron oxygenases (Gibson and Parales 2000) and a small subunit (β). Recently, the crystal structures of the terminal oxygenase component, naphthalene 1,2-dioxygenase (NDO-O) of Pseudomonas resinovorans (Kauppi et al. 1998), carbazole 19a-dioxygenase (CARDO-O) of Janthinobacterium sp. strain 13 (Nojiri et al. 2005), and cumene dioxygenase (CumDO-O) of Pseudomonas fluorescens IP01 (Dong et al. 2005) have been determined. Docking simulation study of CARDO-O strongly suggests that carbazole binds to the substrate-binding pocket in a manner suitable for catalysis of angular dioxygenation (Nojiri et al. 2005). Among the carbon atoms of carbazole, those at the angular position (C9a) and its adjacent position (C1) are closed to both oxygen atoms (Fig. 1c). The entrance of the substrate, the size of substrate-binding pocket of CARDOO, and the docking model revealed why CARDO-O shows no detectable activity towards highly chlorinated dioxins. On the other hand, Sphingomonas sp. RW1, which is capable of using DD and DF as sole carbon sources, has a dioxin dioxygenase system. The electron transfer chain of dioxin dioxygenase consists of NADH-ferredoxin reductase

a
NAD+ NADH FAD

2Fe-2S β

Fe β
2F e-2 S

e2Fe-2S

e-

Fe
2Fe -2S

RedA2

Fdx1

β

Fe

DxnA1A2

b

NADP

+

c
CPR P450
X

e-

4Fe-4S 3Fe-4S Co

NADPH

FAD FMN

eheme

Cpr A

Cpr B

Angular dioxygenase The aromatic-ring hydroxylating dioxygenase system generally consists of a terminal dioxygenase and a reductase chain. Electrons from NAD(P)H are transferred to the dioxygenase via NAD(P)H-ferredoxin reductase (FDR) and ferredoxin (FDX) to catalyze the direct insertion of

Fig. 2 Electron transport chains of angular dioxygenase (a), microsomal CYP (b), and dehalogenase derived from Desulfitobacterium dehalogenans (c). Angular dioxygenases belonging to class IB, II, and III of Rieske non-heme iron oxygenase systems (ROSs) form α3β3 configuration, whereas those belonging to class IA of ROSs form α3 configuration (Nojiri et al. 2005). RedA2, Fdx1, and DxnA1A2 mean an NADH-ferredoxin reductase, a [2Fe–2S] ferredoxin, and a terminal dioxygenase, respectively. Angular dioxygenase systems are localized in the cytoplasm of aerobic bacteria, and microsomal P450 system is localized on endoplasmic reticulum membrane. CPR means NADPHP450 reductase (b). Dehalogenase system of D. dehalogenans contains CprA, CprB, and unknown electron donor of CprA designated as X. CprA is localized in the periplasmic space, and CprB appears to play a role in membrane anchor of CprA (van de Pas et al. 1999)

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named RedA2 (Armengaud and Timmis 1998), a [2Fe–2S] ferredoxin named Fdx1 (Armengaud and Timmis 1997), and a terminal dioxygenase named DxnA1A2 (Fig. 2a). Among the four dioxygenase systems, only the dioxin dioxygenase system contains a putidaredoxin-type [2Fe– 2S] ferredoxin, Fdx1, while other systems contain a Riesketype [2Fe–2S] ferredoxin. Recently, Chang's group revealed that Sphingomonas sp. RW1 transformed 2,7-DCDD, 1,2,3-TriCDD, 1,2,3,4TCDD, and 1,2,3,4,7,8-HxCDD into corresponding chlorocatechols (Chang 2008; Hong et al. 2002; Nam et al. 2006). However, it was unable to transform 2,3,7-TriCDD and 1,2,3,7,8-PeCDD. These results suggest that 1,2,3TriCDD, 1,2,3,4-TCDD, and 1,2,3,4,7,8-HxCDD are good substrates for DxnA1A2, but 2,3,7-TriCDD and 1,2,3,7,8PeCDD are bad substrates for DxnA1A2. Based on these results, the substrate-binding pocket of DxnA1A2 appears to be much larger than that of CARDO-O. Crystal structure of DxnA1A2 would help us understand its enzymatic properties. In addition, site-directed mutagenesis of DxnA1A2 might make it possible to construct an active dioxygenase for the most toxic dioxin, 2,3,7,8-TCDD.

Cytochrome P450 Manmalian cytochrome P450s Cytochrome P450s (CYPs) are widely distributed in the biological kingdom and constitute a superfamily of hemoproteins, which catalyze the incorporation of a single oxygen atom from molecular oxygen into a large variety of endogenous and exogenous lipophilic compounds. While bacterial CYPs are water-soluble, mammalian CYPs are bound to either endoplasmic reticulum (Fig. 2b) or mitochondrial membranes. The metabolism of PCDDs has been studied in vivo using experimental animals (Hu and Bunce 1999a; Van Leeuwen et al. 2000; Petroske et al. 1997; Poiger et al. 1982; Tai et al. 1993; Tulp and Hutzinger 1978). The insertion of a single oxygen atom into the PCDD molecule to form an epoxide by CYP is considered to be the initial reaction for metabolism of PCDDs (Fig. 3b, c). Hu and Bunce (1999b) suggested that CYP1A1 and CYP1A2 play an important role in the metabolism of PCDDs. Thus, CYPs appear to be key enzymes for the metabolism of PCDDs in mammals. In vivo studies suggest that the CYP-dependent metabolism includes multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and opening the dioxin ring. All of these reactions appear to be detoxification reactions of PCDDs.

We have examined metabolism of PCDDs by using recombinant yeast cells expressing human or rat CYPs (Sakaki et al. 2002; Shinkyo et al. 2003a, b). When each of DD, mono-, di-, and tri-CDDs was added to the cell culture of the recombinant yeast expressing CYP1A1 or CYP1A2, a remarkable metabolism was observed. The metabolism contained multiple reactions seen in vivo animal studies. Kinetic analysis using microsomal fractions prepared from the recombinant yeast cells revealed that 2,7-DCDD and 2,3,7-triCDD were good substrates for both CYP1A1 and CYP1A2. When 2,3,7-triCDD was added to the yeast cells expressing each of rat CYP1A1 and CYP1A2, most of 2,3,7-tri-CDD was first converted to 8-hydroxy-2,3,7-triCDD, and further metabolized to more hydrophilic compounds whose ethereal bridges were cleaved (Sakaki et al. 2002). These results clearly demonstrated that mammalian CYPs are capable for degradation of mono-, di-, and triCDD, although many of microbiologists might have an idea that mammals have no enzyme systems to degrade PCDDs and PCDFs. It is noted that low-chlorinated dioxins are good substrates for mammalian CYPs, whereas rat or human CYPs expressed in yeast cells showed no detectable activity towards 2,3,7,8-TCDD. On the contrary, Poiger et al. (1982) detected the metabolites of 2,3,7,8-TCDD in dogs, suggesting that dogs have some CYPs catalyzing hydroxylation of 2,3,7,8-TCDD with or without NIH shift which undergoes an intramolecular migration of chloride atom during a hydroxylation reaction (Fig. 3c). Based on these results, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-tetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by sitedirected mutagenesis. Among the mutants examined, F240A showed a conversion of 2,3,7,8-TCDD to 8hydroxy-2,3,7-triCDD (Shinkyo et al. 2003b). To our best knowledge, the F240A mutant of rat CYP1A1 is the first enzyme to be verified as a 2,3,7,8-TCDD-metabolizing enzyme. Several years ago, we successfully expressed rat CYP1A1 in white-rot basidiomycete fungus cells (Orihara et al. 2005). In addition, we successfully expressed Nterminal truncated F240A mutant (ΔF240A) in Escherichia coli cells (Shinkyo et al. 2006). These results suggest possible application of fungal or prokaryotic cells expressing F240A or ΔF240A to the bioremediation of PCDDcontaminated soil. Fungal cytochrome P450 Previous reports demonstrated that the white-rot fungus P. chrysosporium has been shown to possess biodegradative capabilities of DD (Joshi and Gold 1994), 2,7-DCDD (Valli et al. 1992), and 2,3,7,8-TCDD (Bumpus et al. 1985). The

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Appl Microbiol Biotechnol (2010) 88:23–30

a
O O O O

OH O OH OH

OH

H OH

b
O O Cl O O Cl O O OH Cl

O

HO HO

Cl

OH

O

Cl

+
OH OH OH

c
Cl Cl O O Cl Cl
Cl Cl O O H Cl

Cl Cl

O O

Cl
OH

(III)
O Cl Cl Cl O O

Cl

(I)

Cl OH

(II)

(IV)

d
O Cl O Cl

eCl

O

Cl

H2O H+, HCl
Cl

O O

O

+
O

(I)

(II)

(III)

eH2O
O O O OH O Cl Cl O O OH

H+
O

+

(VI)

(V)

(IV)

e
Cl Cl Cl Cl O O Cl Cl Cl Cl O Cl O Cl Cl Cl O Cl Cl O O Cl O Cl O Cl O Cl

Fig. 3 Putative metabolic pathways of PCDDs by angular dioxygenase (a), rat CYP1A1 (b), dog CYP (Poiger et al. 1982), and the mutant F240A of rat CYP1A1 (c), lignin peroxidase (d), and dehalogenase derived from Dehalococcoides sp. strain CBDB1

(Bunge et al. 2003) (e). Compounds in square brackets mean putative intermediates. Rat CYP1A1 has more complicated metabolic pathways of 2-MCDD in addition to the pathway in this figure as described in our previous report (Shinkyo et al. 2003a, b)

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degradation of more highly chlorinated DDs by P. chrysosporium has also been demonstrated (Takada et al. 1996). As mentioned above, we have revealed mammalian CYPdependent metabolism of PCDDs. Based on the fact that P. chrysosporium genome contains 148 CYP genes, it is possible to assume that some P. chrysosporium CYPs are involved in the metabolism of PCDDs. Recently, we have obtained 120 clones expressing individual CYPs of P. chrysosporium (Kasai et al. 2010). Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and the clone expressing CYP5145A3 showed much higher activity than any other clones towards 1-MCDD, 2-MCDD, and 2,3-DCDD. However, CYP5145A3 showed no detectable activity toward 2,7-DCDD and higher chlorinated dioxins. Unfotunately, we have not yet discovered 2,3,7,8TCDD-metabolizing enzyme in P. chrysosporium. Procaryotic cytochrome P450 Bacterial CYP102A1 (P450BM-3) is a water-soluble fusion protein between CYP and NADPH-P450 reductase containing FAD and FMN. Interestingly, CYP and the reductase domains of CYP102A1 are significantly homologous with animal CYP and its reductase, suggesting the eukaryotic origin of CYP102A1 (Omura 2010). Carmichael and Wong (2001) generated CYP102A1 mutants showing high activity for polycyclic aromatic hydrocarbons such as phenanthrene, fluoranthene, and pyrene by site-directed mutagenesis of amino acids at active site and the entrance of the substrate access channel. We also generated CYP102A1 mutants and examined metabolism of PCDDs. The triple mutant named AL4V (Ala74Gly, Phe87Val, Leu188Gln) showed significantly higher activities toward PCDDs than the wild type of CYP102A1 (Sulistyaningdyah et al. 2004). However, the hydroxylation activities of AL4V towards PCDDs are much lower than those of mammalian CYPs. The activities of AL4V towards 1MCDD and 2,3,7-TriCDD are approximately 0.5 and 0.001 (mole per minute per mole P450), respectively. On the other hand, the hydroxylation activities of rat CYP1A1 towards 1-MCDD and 2,3,7-TriCDD are approximately 16 and 23 (mole per minute per mole P450), respectively (Shinkyo et al. 2003b).

including PCDDs, while Mn-dependent peroxidase is involved in the oxidation after a dioxin ring cleavage (Valli et al. 1992). One of the major isozymes of Lip (Lip-2) of P. chrysosporium with 343 amino acid residues was purified, and its crystal structure was revealed (Edwards et al. 1993). Lip has 4 disulfide bonds, and 11 helical segments with the heme sandwiched between the distal and proximal helices. Valli et al. (1992) proposed the mechanism of Lipdependent degradation of 2,7-DCDD by using the purified sample of Lip (Fig. 3d). The first step of the reaction is oneelectron oxidation of 2,7-DCDD by the oxidized enzyme intermediate, compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), resulting in the formation of the aryl cation radical (Fig. 3d). The cation radical cannot be detected because of its short lifetime based on the fact that chloride is a much better leaving group than hydrogen. Then, the cation is attacked by H2O to lose chloride. The final products of 2,7-DCDD by Lip are benzoquinone compounds by cleavage of both C–O–C bonds (Fig. 3d V and VI) via quinone intermediate with one C–O–C bond (Fig. 3d IV). We expect that Lip is capable of degradation of 2,3,7,8-TCDD in a similar metabolism pathway based on the report by Bumpus et al. (1985). However, no reports showing evidence of Lip-dependent metabolism of 2,3,7,8-TCDD have been published.

Dehalogenase Several bacteria use chlorinated compounds such as chlorinated ethane (Neumann et al. 1996), chlorinated benzenes (Adrian et al. 2000), and PCDDs (Bunge et al. 2003) in their energy-generating process. Holscher et al. (2004) revealed that four members of Dehalococcoides group have multiple reductive-dehalogenase-homologous (RDH) genes with a common ancestry. For example, Dehalococcoides sp. CBDB1 has 14 RDH genes. Sequence analysis of RDH genes revealed the presence of two closely linked open reading frames designated as orfA encoding dehalogenase with 400–500 amino acid residues and orfB encoding a hydrophobic protein with about 100 amino acids. The dehalogenase derived from orfA contains twinarginine translocation (Tat) signal at the amino terminus including RRXFXK. Tat signal sequence is a characteristic for periplasmic enzymes containing complex redox cofactors, suggesting that the dehalogenase is located on the bacterial cell inner membrane. Actually, PCE reductive dehalogenase (Neumann et al. 1996) and orthochlorophenol dehalogenase CprA (van de Pas et al. 1999) are purified from cell membrane. In addition, the small hydrophobic protein CprB is assumed to act as a membrane anchor of CprA. EPR analysis of CprA indicated one [4Fe– 4S] cluster, one [3Fe–4S] cluster, and one cobalamin per

Lignin peroxidase White rot fungus P. chrysosporium produces a number of extracellular enzymes including lignin peroxidases (Lip) and Mn-dependent peroxidases (MnP) in response to nutrient nitrogen of carbon depletion. The physiological role of Lip is considered to be lignin biodegradation. Lip is capable of initial oxidation of environmental pollutants

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Appl Microbiol Biotechnol (2010) 88:23–30

monomer (Fig. 2c). These iron–sulfur clusters appear to be involved in the electron transfer to the active site containing cobalamin. Although two models for reaction mechanism have been proposed (Neumann et al. 1996; Schumacher et al. 1997), further studies are needed to be generally accepted. It is well-known that dehalogenases are quite unstable under aerobic conditions. In addition, they are membrane proteins. Overproduction of the functional dehalogenases in E. coli has not been succeeded although nonfunctional dehalogenase was overproduced (Neumann et al. 1998). These results indicate that structure–function analysis of the dehalogenases is quite difficult. However, the previous reports by Bunge et al. (2003) strongly suggest that some RDH are capable of dechlorination of PCDDs. Thus, we eagerly expect an identification of the dehalogenases for 2,3,7,8-TCDD and more chlorinated PCDDs in the near future.

Conclusions and prospects Many reports showing microbial potential for bioremediation of dioxin-polluted soils have been published. In particular, some reports have indicated the biodegradation of 2,3,7,8TCDD, the most toxic dioxin, suggesting the presence of the enzyme catalyzing 2,3,7,8-TCDD degradation. The potent candidates are angular dioxygenase, cytochrome CYP, lignin peroxidase, and dehalogenase as mentioned above. All of these enzymes have metal ions in their active centers, and three enzyme systems, angular dioxygenase, CYP, and dehalogenase have their original electron transport chains as shown in Fig. 2. Angular dioxygenase and prokaryotic CYP are cytosolic proteins, while lignin peroxidase is extracellular protein. On the other hand, mammalian and fungal microsomal CYPs and dehalogenase are membrane-bound proteins. The enzymatic properties including tertiary structure and reaction mechanism of the angular dioxygenases, lignin peroxidase, and prokaryotic CYP, all of which are watersoluble proteins, have been extensively studied. Although mammalian microsomal CYPs in the families one, two, and three are membrane-bound proteins, their enzymatic properties have been studied well because they are essential drugmetabolizing enzymes. Recently, most of their crystal structures including human CYP1A2 have been revealed. On the other hand, the information about dehalogenases is much less than other enzyme systems for the reasons described in the session of dehalogenases. It is noted that dehalogenases prefer highly chlorinated PCDDs with more than four chlorine substituents, whereas the other three enzyme systems prefer low-chlorinated PCDDs. Thus, dehalogenases appear to be quite promising from the viewpoint of practical use for bioremediation.

Combination of distinct enzyme systems appears to be useful. Actually, white-rot fungi have lignin peroxidase, Mndependent peroxidase, and CYPs. Expression of dehalogenases in white-rot fungi appears to be quite attractive. However, it is noted that CYP reactions require molecular oxygen while dehalogenases require strictly anaerobic conditions. Thus, the functional expression of both systems in the single cell appears to be impossible. On the three enzyme systems, angular dioxygenases, lignin peroxidase, and CYP, protein engineering based on their tertiary structures has great potential to generate highly efficient dioxin-metabolizing enzymes. On the other hand, in the case of the dehalogenase, identification of PCDD-dechlorinating enzymes is an urgent matter. Although genetically engineered microorganisms (GEMs) have the potential risks to affect biological environment, recent progress to generate suicidal genetically engineered microorganisms (Paul et al. 2005) will make it possible to apply GEMs expressing dioxin-metabolizing enzyme for bioremediation of soil contaminated with PCDDs and PCDFs in the near future.

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