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Dehalogenation of Polyhalogenated Dioxins.

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Bacterial Degradation of Dioxin
Dehalogenation of Polyhalogenated Dioxins
Karl-Heinz van Pe*
chlorine · dechlorination · dehalorespiration · dioxins ·
In her book “The Silent Spring”, which
was published in 1962, Rachel Carson
described very impressively the consequences of the use of chlorinated herbicides for the environment and their
hazards for mankind. This book prompted US president Kennedy to institute a
commitee for the investigation of this
problem. This commitee reached the
same conclusions as Rachel Carson.
However, it was also president Kennedy
who approved the use of “Agent Purple”
as a defoliant during the Vietnam War.
“Agent Purple”, like the later used
“Agent Orange” contained not only
the main components 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), but
also 2,3,7,8-tetrachlorobenzo-p-dioxin
(dioxin, 1) and other polychlorinated
dibenzo-p-dioxins as contaminants. After an accident during the production of
trichlorophenol in the village of Soveso
near Milan in 1976, 1 in particular
became tragically known as the “Seveso
poison”. It became evident during investigations following this industrial disaster that polychlorinated dioxins and
dibenzofurans are not only extremely
toxic, but that they can also be formed
[*] Prof. Dr. K.-H. van P*e
Institut f,r Biochemie
Technische Universit/t Dresden
01062 Dresden (Germany)
Fax: (+ 49) 351-463-35506
very easily during the incineration of
organic compounds in the presence of
chlorine. In addition, the natural production of polychlorinated dioxins (for
example spongiadioxin A, 2) was also
ditions.[4] Changes in the composition of
dioxins in sediments were the first hints
of anaerobic degradation of dioxins.[5]
The removal of halogen atoms from
aromatic compounds under anaerobic
conditions proceeds reductively. Reductive dehalogenation steps are also
known from the degradation of pentachlorophenol; however, here oxidative
as well as reductive dehalogenation
steps occur.[6] A process called “dehalorespiration” plays an important role in
the degradation of halogenated compounds in sediments and thus in the
absence of oxygen. In this process
reductive dehalogenation is coupled to
the conservation of energy and cell
growth. Such reactions have been
known for quite some time for the
dehalogenation of compounds such as
chlorophenols.[7, 8]
Until now, investigations on the
reductive dehalogenation of polychlorinated dioxins were performed with
mixed cultures, such as in the investigations of the degradation of 1,2,3,4-tetrachlorodibenzo-p-dioxin (3, Scheme 1)
by Bunge et al.[9] For this purpose,
The dioxin problem became especially important because of the fact that
polyhalogenated dioxins are extremely
lipophilic and thus strongly adsorb onto
hydrophobic material, such as humus, or
they can get enriched in fatty tissue.
During the 1980s the German federal
government considered the dioxin problem so urgent that it was prepared to
finance the building of special laboratories for the investigation of the biological degradation of polyhalogenated
dioxins. At that time, data on the
degradation of halogenated aromatic compounds by microorganisms and the direct
elemination of chlorine atoms from aromatic
were already available,[3] however, nothing was known about
the degradation of dioxin-like compounds.
on the degradation of
dioxins by microorganisms were conduct- Scheme 1. Proposed main pathway for the degradation of 1,2,3,4ed under aerobic con- tetrachloro-p-dioxin (3) according to Bunge et al.[10]
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200301662
Angew. Chem. Int. Ed. 2003, 42, 3718 – 3720
bacterial cultures were isolated from
sediments of the Spittelwasserbach, Bitterfeld region, Germany, since these
sediments contain high concentrations
of polyhalogenated dioxins. When 3 was
added to these mixed cultures it was
transformed into a mixture of 1,3- and
2,3-dichlorodibenzo-p-dioxin (5). During subsequent investigations,[10] these
mixed cultures were transferred six
times into a defined minimal medium
without loss of dehalogenating activity.
However, most of the transferred cultures now transformed 1,2,4-trichlorodibenzo-p-dioxin preferentialy to 5 and
only to a lesser extent into 1,3-dichlorodibenzo-p-dioxin.
The structural similarity of chlorobenzene with chlorinated dioxins and
the fact that Dehalococcoides sp. strain
CBDB1 is the only bacterium known
until now to dehalogenate chlorobenzene[11] meant that it did not seem far
fetched to screen for related strains in
the mixed cultures by using primers for
16S ribosomal DNA (rDNA). An additional Dehalococcoides strain, D. ethenogenes strain 195, dechlorinated tetrachloroethene to give ethene by dehalorespiration. Thus, 16S rDNA primers for
this strain were also used. The
16S rDNA sequences could be amplified
from the mixed cultures by employing
specific PCR primers for these Dehalococcoides strains. These sequences were
identical to the corresponding sequence
of Dehalococcoides sp. strain CBDB1
and showed an identity of 98.5 % to the
corresponding sequence of D. ethenogenes strain 195. The identity of the
same 16S rDNA sequence amplified
from the mixed cultures with that of
Dehalococcoides sp. strain CBDB1 implicates that a corresponding strain
should be present in the mixed culture.
Thus, the Dehalococcoides sp. strain
CBDB1 isolated by Adrian et al.[11] was
used to check whether this strain could
also dehalogenate dioxins. It was found
that this strain dechlorinates 3 to 2monochlorodibenzo-p-dioxin (6) with 5
as an intermediary product. This intermediate was also detected during the
degradation of 1,2,3-trichlorodibenzo-pdioxin (4, Scheme 1). However, 1,3dichlorodibenzo-p-dioxin was formed
as an intermediate in up to 50 mol %
during the degradation of 1,2,4-trichlorodibenzo-p-dioxin. In the course of the
transfer of the mixed cultures, the preferred position for the dechlorination
changed from the lateral to the periposition. This result suggests that the
original mixed culture contained additional organisms with the ability to
dechlorinate dioxins and that the transfer resulted in enrichment of the Dehalococcoides sp. strain CBDB1.
1,2,3,7,8 - Pentachlorodibenzo - p - dioxin (7) was used as a model substance
for polychlorinated dioxins chlorinated
on both benzene rings. This compound
was dehalogenated very slowly (over
104 days) and only to an extent of
2.8 mol % to 2,3,7,8-tetrachlorodibenzo-p-dioxin (8), 2,7-dichlorodibenzo-pdioxin (9) or 2,8-dichlorodibenzo-p-dioxin (10), and to a minor amount of
1,3,7,8-tetrachlorodibenzo-p-dioxin and
(Scheme 2).
The important novelties in the results of Bunge et al.[10] is the identification of a bacterial strain responsible for
the dehalogenation of polychlorinated
dioxins which originates from a sediment sample contaminated with dioxins,
and the demonstration of the dehaloge-
Scheme 2. Proposed main pathway for the degradation of 1,2,3,7,8-pentachlorodibenzo-p-dioxin
(7) according to Bunge et al.[10]
Angew. Chem. Int. Ed. 2003, 42, 3718 – 3720
nation of polyhalogenated dioxins by a
pure culture of a Dehalococcoides sp.
strain which is part of a mixed culture.
The fact that bacteria with the ability to
degrade polyhalogenated dioxins are
present in sediments contaminated with
these compounds shows nature's adaptability and strengthens the hypothesis
that compounds with structural similarity to compounds synthesized by living
organisms, such as the polyhalogenated
dioxin 2,[2] can be detoxified and decomposed.
The results presented by Bunge
et al.[10] give rise to the hope that detoxification of polychlorinated dioxins by
anaerobic reductive dehalogenation reactions and subsequent further degradation of dioxins is possible in nature
and that the decontamination of dioxincontaminated soil can be accomplished
by this means. This hope is especially
strengthened by the finding that Dehalococcoides sp. strain CBDB1 cannot
grow in a synthetic medium in the
absence of a chlorinated electron acceptor. Since, according to these finding,
Dehalococcoides sp. strain CBDB1
seems to be strictly dependent on dehalorespiration for energy conservation, it
could be possible that this strain even
dehalogenates polychlorinated dioxins
in the presence of nonhalogenated nutrients. This could be a crucial advantage
over the aerobic dehalogenation of
polychlorinated dioxins.[12] However,
the length of time required for the
degradation and the degradation rates
at about 60 mol % after 56 days for the
transformation of 4 into 6 are still a long
way from application. Molecular genetics to clone the dehalogenase genes and
overexpression of the genes could be
used to improve the degradation of
polychlorinated dioxins in contaminated
The repeated occurrence of contamination of feed stuff by polyhalogenated
dioxins is only one example showing the
necessity to find ways for the degradation and detoxification of polyhalogenated dioxins. However, in this respect it
has to be mentioned that highest priority
should be given to preventing the formation of polyhalogenated dioxins. The
dehalogenation of dioxins is also a big
challenge for basic research. Interesting
research projects could involve the isolation of the dehalogenating enzymes
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
and their characterization or to find an
answer to the question of why these
Dehalococcoides strains are strictly dependent on halogenated electron acceptors. The low degradation rates and
the long cultivation times required,
however, indicate that it will take some
time until we will see any progress
towards these goals.
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Angew. Chem. Int. Ed. 2003, 42, 3718 – 3720
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dioxin, dehalogenation, polyhalogenated
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