Journal of Environmental Biotechnology
Vol. 15, No. 2, 105–108, 2016

Original paper (regular paper)

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) degradationby the thermophilic Geobacillus sp. UZO 3
Yuzo Suzuki1, Masaya Nakamura2*, Yuichiro Otsuka2, Nao Suzuki3, Keisuke Ohyama3,
Takeshi Kawakami1, Yusuke Umeka3, John Solomon Maninang4, Kanna Izawa-Sato3, Shojiro
Hishiyama2, Kouya Inoue5, Toshiji Kameyama5, Atsushi Takahashi1, Yoshihiro Katayama6
2

6

1
Takasago Thermal Engineering Co. Ltd., Shinjyuku, Tokyo 160–0022, Japan
Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305–8687, Japan
3
Graduate School of Bio-Applications and Systems Engineering,Tokyo University of Agriculture & Technology, Koganei, Tokyo 184–8588, Japan
4
Department of International Environmental and Agricultural Science,
Tokyo University of Agriculture & Technology, Fuchu, Tokyo 183–8538, Japan
5
Kantteku Co. Ltd., Bunkyo, Tokyo, 112–0004, Japan

College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252–0880, Japan* TEL: +81–29–829–8282 Fax: +81–29–873–3797* E-mail: nmasaya@ffpri.affrc.go.jp(Received: 24 February, 2016/Accepted: 28 March, 2016)

2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is the most toxic among the dioxin isomers. Despite previous breakthroughs, the global quest to discover bioremediation agents for this pollutant remains unabated. In this study, we characterize the 2,3,7,8-TCDD-degrading activity of the Geobacillus sp. UZO 3. Cell-free extracts of Geobacillus sp. UZO 3 wereincubated for 18 h at 65°C in a reaction milieu that contained 2,3,7,8-TCDD as substrate. Reaction products in the milieu
were dissolved in ethyl acetate and directly analyzed by GC-MS. GC-MS data revealed peaks that corresponded to3’,4’,4,5-tetrachloro-2-hydroxydiphenyl ether (TCDE). Moreover, performing a similar assay that instead contained TCDE
as substrate showed that the cell-free extract may also play a catalytic role in the subsequent conversion of TCDE todichlorophenol (DCP). Our present results suggest that this thermophilic specie catalyzes the reductive cleavage of the diaryl
ether bonds of 2,3,7,8-TCDD to TCDE as intermediate by-product which is then converted to DCP. We hereby demonstrate
the nouveau 2,3,7,8-TCDD-degrading activity by a bacterial cell-free extract.
Key words: Dioxin, 2,3,7,8-TCDD, Microbial degradation, Geobacillus sp. UZO 3

1. Introduction
Dioxins continue to pose serious threat to the environment.
Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), consisted of 75 and 135
isomers, respectively, are stable hydrophobic contaminants
which persist in the environment. PCDD/Fs are introduced
into the biosphere on a large scale not only as by-products
from the manufacture of chlorinated phenols as pesticides
in the 1930s, but also by incineration of wastes6,7,12). They
induce a broad spectrum of biochemical and toxic effects
to mammals and other organisms, such as teratogenesis,
immunosuppression and tumor promotion. Rapid advancesin the field of molecular toxicology revealed that these effects
by PCDD/Fs are mediated by aryl hydrocarbon receptors(AhR). Among the PCDD/F isomers, 2,3,7,8-TCDD has
been found highly toxic because it has the highest binding
affinity to AhR4,13,14).
From the mid-1980s, attention has been directed towards

the development of technology for the clean-up of PCDD/
Fs by bioremediation, marking the beginning of such kind ofresearch3,19). The aerobic bacterium Sphingomonas wittichii
RW1 is one of the most well studied dioxin-degrading
bacteria17,18). Armengaud et al.1) succeeded in cloning a
dioxin dioxygenase gene dxnA1A2 from Sphingomonas
wittichii RW1 and demonstrated that DxnA1A2 introduces
two atoms of oxygen at angular positions neighboring thediaryl ether bonds for subsequent degradation through an
unstable intermediate acetal structure. Nam et al.5,15) revealed
that resting cells from Sphingomonas wittichii RW1 degradated 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), 1,2,3-tric
hlorodibenzo-p-dioxin, 1,2,3,4-tetrachlorodibenzo-p-dioxin,
and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin to their corresponding chlorocatechols. On the other hand, in an anaerobic
bacterium, Bunge et al.2) reported that Dehalococcoides
sp. strain CBDB1 is capable of converting 1,2,3,4-tetrachlorodibenzo-p-dioxin to 2-monochlorodibenzo-p-dioxin,
and 1,2,3,7,8-pentachlorodibenzo-p-dioxin to 2,7- or 2,8-DCDD

Suzuki et al.

106

Fig. 1. Proposed degradation pathways for 2,3,7,8-TCDD by the Geobacillus sp. UZO 3 cell-free extract.
Compound definitions: 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD); 3’,4’,4,5-tetrachloro-2-hydroxydiphenyl ether (TCDE); anddichlorophenol (DCP).

by dechlorination. Despite numerous studies on the microbial
degradation of PCDD/Fs, no report has demonstrated a
2,3,7,8-TCDD-degrading activity of bacterial origin.
We have investigated the 2,3,7,8-TCDD-degrading activityof the Geobacillus sp. UZO 3 for several years. As
preliminary step, we have previously reported 2,7-DCDDdegrading activity of the cell-free extract from Geobacillus
sp. UZO 3 and demonstrated that it reductively cleaves
diaryl ether bonds of 2,7-DCDD in a sequential fashion
producing 4',5-dichloro-2-hydroxydiphenyl ether (DCDE) as
the intermediate, and 4-chlorophenol as the final reaction
product16). The structure of intermediate DCDE was identified by comparing it to the chemically-synthesized authentic
compound in GC-MS analysis. Moreover, the detectionof DCDE implicated the discovery of an unprecedented
dioxin degradation enzyme that reductively cleaves the diaryl
ether bonds similar to glutathione-S-transferase, a reduction
cleavage enzyme8–11).
To investigate the degradation activity of the cell-free extractfrom Geobacillus sp. UZO3 on 2,3,7,8-TCDD in vitro, we
chemically synthesized the authentic compound of putativeintermediate 3’,4’,4,5-tetrachloro-2-hydroxydiphenyl ether(TCDE) (Fig. 1). It appears difficult to demonstrate degradation of persistent organic pollutants like 2,3,7,8-TCDD
by existing analytical methods which are based on substratequantitative assay. To overcome this problem, structural
identification of the intermediate that demonstrates degradative reaction was employed in this study. We monitored
the production of the reaction intermediate TCDE in the
degradation of 2,3,7,8-TCDD mediated by Geobacillus sp.
UZO 3 cell-free extract.

2. Materials and Methods
2.1. Chemicals
2,3,7,8-TCDD was purchased from Cerilliant (Round
Rock, Texas, USA). DCP, Cu(OAc)2, CH2Cl2, CDCl3, tetramethylsilane and N,O-Bis(trimethyl silyl)trifluoroacetamide(BSTFA) were purchased from Wako Pure Chemical (Osaka,
Japan). 4,5-dichlorocatechol and 3,4-dichlorophenylboronic acid was purchased from Sigma-Aldrich (Steinheim,
Germany). Purities of these chemicals range from 96.6 to100%. All other chemicals used were of analytical grade andof the highest purity available.
2.2. Synthesis of TCDE
TCDE synthesis from 4,5-dichlorocatechol and 3,4-

dichlorophenylboronic acid was adapted from the method
for DCDE synthesis described previously16). Analysis by
GC-MS showed that the synthesized TCDE has a molecularion peak of m/z=396 (Fig. 2d) that corresponds to theestimated molecular weight of TCDE. Further analyses by
1
H-NMR and 13C-NMR have consistently confirmed theidentity of TCDE. The 1H-NMR spectrum δ (ppm, CDCl3)of TCDE is as follows: 5.51 (H, s, OH), 6.89 (1H, dd,
J=2.4 Hz), 6.94 (1H, d, J=8.5 Hz), 7.17 (1H, d, J=8.5 Hz),7.26 (1H, d, J=8.5 Hz), 7.44 (1H, dd, J=8.5 Hz). The
13
C-NMR spectrum δ (ppm, CDCl3) of TCDE is as follows:
154.7, 146.4, 142.0, 133.8, 131.4, 128.6, 128.2, 123.6, 120.2,
120.0, 118.1, 117.6.
2.3. Preparation for cell-free extracts, enzymatic reaction
assays and analytical methods
The preparation for cell-free extract of Geobacillus sp.
UZO 3 using french pressure cell press and the enzymatic
reaction assays were performed as described previously16).
Geobacillus sp. UZO 3 was maintained in tryptic soy agar
plates (Difco). Geobacillus sp. UZO 3 and routinely cultivated in liquid cultures of tryptic soy broth medium (Difco)
at 65°C with vigorous shaking. When the optical density
at 600 nm reached 1.2, cells were harvested by centrifugation, washed twice with 100 mM phosphate buffer (pH 7.0),
and resuspended in a reduced volume of the same buffer.
Cell free extracts was prepared from the disrupted bacterial cells using the french pressure cell press (Ohtake Co.
Ltd., Tokyo, Japan). The disrupted cells were centrifuged
at 25,000 g for 30 min (25°C) to remove intact cells andthe supernatant was collected as cell free extracts. Briefly,
the 1 mL reaction mixture contained 7.5 μM 2,3,7,8-TCDD
dissolved in dimethyl sulfoxide (final concentration 5%) andthe cell-free extract (about 2 mg of protein). The enzymatic
reaction was performed at 65°C for 18 h. Ethyl acetate
extracts from 2,3,7,8-TCDD reaction mixture were dried overanhydrous sodium sulfate and the solvent was volatilized bynitrogen gas. The dried enzyme reactant was derivatized by
N,O-Bis(trimethyl silyl)trifluoroacetamid prior to GC-MS
analysis. GC-MS and NMR analyses were performed as
described previously16).

3. Results and Discussion
3.1. 2,3,7,8-TCDD-degrading activity producing TCDE
by Geobacillus sp. UZO 3 cell-free extract
We analyzed the 2,3,7,8-TCDD-degrading activity ofNew dioxin-degrading bacteria degrades 2,3,7,8-TCDD

107

Fig. 2. GC-MS analyses of the reaction mixtures for 2,3,7,8-TCDD and TCDE degradations mediated by Geobacillus sp. UZO 3 cell-free
extract.
The MS chromatograms of the detected intermediates TCDE at m/z=396 (b) from 2,3,7,8-TCDD degradation, and DCP at m/z=234 (e)from TCDE degradation, and their corresponding MS spectrums. The MS chromatograms at m/z=396 (c) and at m/z=234 (f) represent
the control chromatograms obtained when the cell-free extract was lacking from the enzyme assay. The MS chromatograms of the authentic compounds TCDE at m/z=396 (a) and DCP at m/z=234 (d), and their corresponding MS spectrums.

Geobacillus sp. UZO 3 cell-free extract after 18 h of incubating the reaction milieu at 65°C. GC-MS analysis of theethyl acetate extract of the complete reaction milieu inaddition to the substrate 2,3,7,8-TCDD showed peak for
TCDE at retention time 47.75 min (Fig. 2b). The peaksof the substrate and product corresponded well with thoseof their respective authentic compounds (Fig. 2a, b). Ethyl
acetate extract of a reaction mixture that did not contain thecell-free extract (control) was detected of 2,3,7,8-TCDD, butshowed no peak for TCDE (Fig. 2c). These results suggestthat the chlorinated dioxin degradation enzymes containedin Geobacillus sp. UZO 3 cell-free extract reductively cleaves
diaryl ether bonds of 2,3,7,8-TCDD to produce TCDE.

3.2. Geobacillus sp. UZO 3 cell-free extract degrades TCDE
to DCP
We further investigated the TCDE-degrading activityof Geobacillus sp. UZO 3 cell-free extract. We likewise
analyzed by GC-MS the ethyl acetate extract of a similar
reaction milieu that instead contained TCDE as substrate.
DCP was detected in the ethyl acetate extract (Fig. 2e)
implying that the Geobacillus sp. UZO 3 cell-free extract
catalyzes the reductive cleavage of the two diaryl ether bonds
in 2,3,7,8-TCDD in a sequential fashion. That is, TCDE
is first produced as a reaction intermediate which is thenconverted to DCP.

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The results of this study provide strong evidence that
Geobacillus sp. UZO 3 cell-free extract reductively cleaves
the diaryl ether bonds of 2,3,7,8-TCDD (Fig. 1). This is
the first report to demonstrate a 2,3,7,8-TCDD-degrading
activity of bacterial origin. Work is in progress to clone thegenes of the enzymes involved in the reductive cleavage ofthe diaryl ether bonds of 2,3,7,8-TCDD. Further enzymological characterization of the response mechanism towards
2,3,7,8-TCDD is envisioned to contribute in the remediationof PCDD/Fs-contaminated areas.

Acknowledgments
This work was supported in part by grant-in-aid for
Scientific Research (A), 21248037, from the Ministry of
Education, Culuture, Sports, Science and Technology ofJapan and a fund from Ministry of Environment of Japan.
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