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Applied and Environmental Microbiology, November 1998, p. 4603-4606, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Spectrum of the Reductive Dehalogenation Activity
of Desulfitobacterium frappieri PCP-1
D.
Dennie,
I.
Gladu,
F.
Lépine,
R.
Villemur,
J.-G.
Bisaillon, and
R.
Beaudet*
Centre de Recherche en Microbiologie
Appliquée, Institut Armand-Frappier, Université du
Québec, Ville de Laval, Quebec, Canada H7N 4Z3
Received 21 April 1998/Accepted 6 August 1998
 |
ABSTRACT |
Desulfitobacterium frappieri PCP-1 was induced for
ortho- and para-dechlorinating activities by
different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol
tested and the position of the chlorine removed. D. frappieri shows a broad substrate specificity; in addition to
tetrachloroethylene and pentachloropyridine, strain PCP-1 can
dehalogenate at ortho, meta, and
para positions a large variety of aromatic molecules with
substituted hydroxyl or amino groups. Reactions of O demethylation and
reduction of nitro to amino substituents on aromatic molecules were
also observed.
| |
TEXT |
Reductive dehalogenation is the most
important mechanism involved in the transformation of halogenated
pollutants by anaerobic microorganisms. To date, few organisms which
can reductively dehalogenate chlorinated aromatic and aliphatic
compounds have been isolated. Desulfomonile tiedjei DCB-1, a
strictly anaerobic, gram-negative, sulfate-reducing bacterium can
meta dehalogenate a variety of substituted halobenzoates
(7) and chlorophenols (9). An anaerobic
microorganism phylogenetically related to myxobacteria which can grow
anaerobically via reductive dehalogenation of 2-chlorophenol has been
isolated (3). Different anaerobic gram-positive
microorganisms belonging to the genus Desulfitobacterium and
capable of reductive dehalogenation were isolated.
Desulfitobacterium dehalogenans (15, 16),
Desulfitobacterium chlororespirans (10),
Desulfitobacterium hafniense (2), and
Desulfitobacterium sp. strain PCE1 (4) can
ortho dehalogenate polychlorinated phenols. Strain PCE1 can also reductively dechlorinate tetrachloroethylene (PCE) to
trichloroethylene and, to a smaller extent, to dichloroethylene
(4).
From a methanogenic consortium (6, 7), Bouchard et al.
(1) have isolated Desulfitobacterium frappieri
PCP-1, a strictly anaerobic bacterium which can dechlorinate
pentachlorophenol (PCP) to 3-chlorophenol and different chlorophenols
at the ortho, meta, and para
positions. Two dehalogenation systems exist in D. frappieri. One system is inducible for ortho dechlorination, and the
second system is inducible for meta and para
dechlorinations. However, strain PCP-1 cannot dechlorinate
2,3-dichlorophenol, 2,5-dichlorophenol, 3,4-dichlorophenol, and
the monochlorophenols (1).
In this paper, we present a study on the induction of
ortho-, meta-, and para-dechlorinating
activities by different chlorophenols and on the substrate specificity
of D. frappieri PCP-1.
Cultures and chemical analysis.
D. frappieri PCP-1 (ATCC
700357) was cultivated anaerobically at 37°C in 70-ml serum bottles
containing 35 ml of mineral salt medium supplemented with 55 mM
pyruvate and 0.1% (wt/vol) yeast extract (1).
2,4,6-Trichlorophenol (5 mg/liter) was added to induce the
ortho-dechlorinating activity, whereas 3,5-dichlorophenol (5 mg/liter) was added to induce the meta- and
para-dechlorinating activity. Each bottle was inoculated
with 1.5 ml (4% [vol/vol]) of strain PCP-1 from an exponentially
growing culture. After 24 h of incubation, the xenobiotic to be
tested was added to the culture, generally at a final concentration of
5 mg/liter. Autoclaved cultures were used as abiotic controls.
Experiments were performed in triplicate.
The different substrates and products were analyzed by high-pressure
liquid chromatography with a reverse-phase NovaPak C18 column (3.9 by 150 mm). A water-acetonitrile gradient containing 0.1%
(vol/vol) acetic acid was used to elute the samples. Chlorophenols were
analyzed as described by Juteau et al. (5, 6). Some substrates and degradation intermediates were analyzed by gas chromatography (Varian; model 3500) using a DB5 capillary column (5%
phenyl, 95% methyl silicon; 30 m long with a 0.2-mm internal diameter). The chromatograph was coupled to a mass spectrometer (Ion
trap 800; Finnigan Mat). Substrates and degradation intermediates were
extracted with ethyl acetate, concentrated, and injected into the gas
chromatograph/mass spectrometer. Pyrene (4.94 mM) was used as an
internal standard. The quantification was done by comparison with
commercially available authentic standards. 2,3,6-Trichloroaniline and
3,5,6-trichloroguaiacol were isolated from the culture media and
identified by nuclear magnetic resonance by comparison with the results
of Smith et al. (12).
Xenobiotics were from Aldrich (Milwaukee, Wis.) and Helix Biotech
(Richmond, British Columbia, Canada). Pentachloroaniline
was made by
reduction of pentachloronitrobenzene as described
by Vogel
(
17). 2,3,5,6-Tetrachloropyridine was synthesized by
the
method described by Sobieralski (
13).
3,5-Dichloro-4-hydroxybiphenyl
and 3,5-dichloro-2-hydroxybiphenyl were
synthesized by chlorination
of the corresponding hydroxybiphenyl with
chlorine. Typically,
a solution containing 100 mg of a hydroxybiphenyl
in 20 ml of
ethyl ether was bubbled with chlorine gas for 10 min. After
2
h, the solution was extracted with concentrated NaOH solution,
dried, and evaporated. The organic residue was purified by thick-layer
chromatography (diethyl ether-hexane; 1:3). The resulting
hydroxy-polychlorinated
biphenyls were identified by gas
chromatography/mass
spectrometry.
Dehalogenation of chlorophenols.
D. frappieri was grown
without chlorophenol for four successive transfers. The dehalogenating
activities were then tested with different chlorophenols. These
cultures dechlorinated PCP, 2,4,6-trichlorophenol,
2,3,4-trichlorophenol, 2,3,5 trichlorophenol, 2,6-dichlorophenol, and
2,4-dichlorophenol at the ortho position, suggesting
that these substances were also inducers of
ortho-dechlorinating activity. These compounds,
however, were not dehalogenated when the noninduced cultures were
supplemented with chloramphenicol (50 µg/ml) prior to the addition of
the chlorophenol, confirming the induction of the
ortho-dehalogenating activity. For D. dehalogenans, the ortho-dehalogenating activity
was induced by 2,4,6-trichlorophenol, 2,4-dichlorophenol,
2,3-dichlorophenol, and 3-Cl-4-hydroxyphenylacetate but was
not induced by PCP, 2,3,4-trichlorophenol, 2,3,6-trichlorophenol, and 2,6-dichlorophenol (15).
Cultures of strain PCP-1 were also able to dechlorinate
3,4,5-trichlorophenol and 3,5-dichlorophenol to 3-chlorophenol,
suggesting
that these two chlorophenols can induce
meta- and
para-dechlorinating
activity. No dechlorinating activity was
observed when chloramphenicol
was added to the culture before the
chlorophenol.
D. tiedjei DCB-1
can
meta
dechlorinate PCP and different chlorophenols in the presence
of
3-chlorobenzoate; however, neither PCP nor the other chlorophenols
induced dehalogenation (
9).
Different dehalogenation rates in induced cultures of strain PCP-1 were
observed according to the chlorophenols tested and
the position of the
chlorine removed (Table
1).
2,3,5-Trichlorophenol
was the most rapidly
ortho
dehalogenated, with a rate of 1,158
nmol/min/mg of cell protein, while
the fastest rate observed for
meta dehalogenation was for
3,5-dichlorophenol, with a rate of
667 nmol/min/mg of cell protein. The
maximum rate of PCP
meta dehalogenation observed for
D. tiedjei by Mohn and Kennedy (
9)
was 0.9 nmol
of Cl
/min/mg of protein. This rate is approximately 1,000 times lower
than the rate observed for the
ortho
dehalogenation of PCP by
D. frappieri.
Transformation of different halogenated compounds.
A list of
halogenated compounds transformed by D. frappieri PCP-1 is
presented in Table 2.
With a few exceptions mentioned in the table, the compounds tested were
generally not dehalogenated by noninduced cultures, suggesting that
they cannot induce their own dehalogenation. 2,4,6-Tribromophenol was
debrominated to 4-bromophenol without the addition of an inducer,
suggesting that this substrate can induce ortho
dehalogenation. However, fluorine compounds such as
2,4,6-trifluorophenol and 2,3,5-trifluorophenol were not dehalogenated. This result was also observed with D. dehalogenans
(15). Fluorine substituents have less of a tendency than
chlorine and bromine substituents to be removed through reductive
dehalogenation.
Tetrachlorocatechol was completely dechlorinated to catechol when the
bacteria were induced with 3,5-dichlorophenol and
2,4,6-trichlorophenol.
Without inducers, 3,4,5-trichlorocatechol,
3,4,6-trichlorocatechol,
and 4,5-dichlorocatechol were the only
products detected. O demethylation
of tetrachloroguaiacol,
tetrachloroveratrole, and pentachloroanisole
was observed after a first
step of dechlorination at the
meta position relative to the
methoxy group for the two first compounds
or at the
para
position for the last compound. After demethylation,
these compounds
were dechlorinated further. Tetrachloroguaiacol
was transformed to
2-chlorocatechol in the absence of
inducers.
3,5-Dichloro-4-hydroxybiphenyl was dechlorinated by strain PCP-1 to
3-chloro-4-hydroxybiphenyl. However,
3,5-dichloro-2-hydroxybiphenyl,
2-hydroxy-2',5'-trichlorobiphenyl, and
4-hydroxy-2,2',5'-trichlorobiphenyl
were not degraded,
suggesting that the presence and the position
of the hydroxyl group
were
important.
When the hydroxyl function was replaced by an amino group,
dechlorination was also possible, as observed with
pentachloroaniline
and 2,3,5,6-tetrachloroaniline. These substances
were both dechlorinated
to different tri- and dichloroanilines. No
dechlorination was
observed in the absence of inducers, suggesting that
they cannot
induce the dechlorinating activities.
Pentachloronitrobenzene
was first transformed by
D. frappieri to pentachloroaniline, which
was dechlorinated as shown
above. In the presence of both inducers,
pentachloropyridine was
dechlorinated to 2,3,5,6-tetrachloropyridine
and to an unidentified
trichloropyridine.
PCE was dechlorinated to trichloroethylene. Dechlorination of PCE to
trichloroethylene and dichloroethylene was observed with
Desulfitobacterium sp. strain PCE1 (
4), cell
extracts of
D. tiedjei (
14), and
Dehalospirillum multivorans (
11).
Unlike
D. dehalogenans (
15,
16), which can
dechlorinate 3-Cl-4-hydroxyphenylacetate, strain PCP-1 did not
dehalogenate
this substrate but transformed it to 2-chlorophenol. Also,
2,3,6-trichlorophenylacetate
(Fenac), 2,4,5-trichlorophenoxyacetate,
and 2,4,6- and 2,3,5-trichlorobenzoate
were not dechlorinated. These
results suggest that aromatic molecules
with substituted acetate or
carboxyl groups are not
dehalogenated.
In conclusion,
D. frappieri PCP-1 can dehalogenate at the
ortho,
meta, and
para positions a
large variety of aromatic molecules
with substituted hydroxyl, methoxy,
or amino groups. This substrate
specificity differs from those observed
for
D. tiedjei (
9)
and for other strains
belonging to the
Desulfitobacterium genus
(
2,
4,
10,
15). The capacity of
D. frappieri to demethylate,
deacetylate, and reduce nitro substituents on aromatic molecules
could
be useful in the bioremediation
process.
| |
ACKNOWLEDGMENTS |
This work was supported by the Natural Sciences and Engineering
Research Council of Canada and by Fonds pour la Formation de Chercheurs
et l'Aide à la Recherche.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre de
Recherche en Microbiologie Appliquée, Institut Armand-Frappier,
Université du Québec, Ville de Laval, C.P. 100, Quebec,
Canada H7N 4Z3. Fax: (450) 686-5501. E-mail:
Rejean_Beaudet{at}IAF.UQUEBEC.CA.
| |
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Applied and Environmental Microbiology, November 1998, p. 4603-4606, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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