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Applied and Environmental Microbiology, January 1999, p. 327-329, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
2-Bromoethanesulfonate, Sulfate, Molybdate, and Ethanesulfonate
Inhibit Anaerobic Dechlorination of Polychlorobiphenyls by
Pasteurized Microorganisms
Dingyi
Ye,1,2,*
John F.
Quensen III,1
James M.
Tiedje,1,3 and
Stephen A.
Boyd1
Department of Crop and Soil
Sciences1 and
Center for Microbial
Ecology,3
Michigan State University,
East Lansing, Michigan 48824, and Department of Biology, Hong Kong
Baptist University, Hong Kong, People's Republic of
China2
Received 17 June 1998/Accepted 15 October 1998
 |
ABSTRACT |
Dechlorination of Aroclor 1242 by pasteurized microorganisms was
inhibited by 2-bromoethanesulfonate (BES), sulfate, molybdate, and
ethanesulfonate. Consumption of these anions and production of sulfide
from BES were detected. The inhibition could not be relieved by
hydrogen. Taken together these results suggest that pattern M
dechlorination is mediated by spore-forming sulfidogenic bacteria.
These results also suggest that BES may inhibit anaerobic dechlorination by nonmethanogens by more than one mechanism.
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TEXT |
Effects of 2-bromoethanesulfonate
(BES), sulfate, and molybdate on dechlorination of polychlorinated
biphenyls (PCBs) have been reported (for a review, see reference
2) and were also investigated in our previous
studies with nonpasteurized microorganisms (21). However, in
all these studies, the microbial communities contained methanogens. Due
to the complicated relationships between methanogens and sulfidogens
(9, 10, 17, 20), it is usually difficult to interpret the
results. In this study pasteurization eliminated methanogens and still
retained a partial dechlorination activity (pattern M [2]), thus
simplifying the dechlorinating community. Therefore, we investigated
the effects of the same anions on PCB dechlorination by microorganisms
that withstood repeated pasteurization. Information from such
inhibition study should provide some information about the composition
of the dechlorinating community and consequently facilitate isolation
of the PCB-dechlorinating microorganisms.
Preliminary inhibition experiment.
The inoculum was collected
from site H7 sediments, upper Hudson River, N.Y. (3).
Inoculum preparation and pasteurization were as described elsewhere
(22). Each 60-ml serum bottle contained 10 g of
PCB-free Hudson River sediments and was prepared as previously described (22). The final volumes of the revised anaerobic
mineral medium (RAMM) (16) and inoculum in each bottle were
20 and 10 ml, respectively, and the final concentration of PCBs
(Aroclor 1242; Monsanto Co., St. Louis, Mo.) was 800 µg/g of dry
sediment. Stock solutions of BES, sulfate, and molybdate (all were
sodium salts) were bubbled with N2, autoclaved, and then
introduced. The controls were autoclaved twice, 1 h each time with
an interval of incubation at 37°C for 5 h before PCBs were
added. After addition of PCBs the samples were shaken for 1 h and
then incubated at 25°C in the dark. Methane production was determined
by gas chromatography with a flame ionization detector (23).
The headspace gas was analyzed to measure methane production after a
culture was shaken and before the slurry was sampled for PCB analysis
(23). To analyze PCBs, 2 ml of the sediment slurry was
shaken, extracted, and analyzed by capillary gas chromatography with an
electron capture detector as previously described (14).
No methane production was detected in any pasteurized slurries as
previously reported (22), indicating no growth of
methanogens (5). Elimination of methanogens was also
established by the following: (i) we previously reported that the
Hudson River pasteurized microorganisms survived not only
pasteurization but also ethanol treatment, which should eliminate
thermophilic methanogens (22), and (ii) no methane was
detected in triplicate pasteurized slurries containing no PCBs after 4 months of incubation (data not shown), ruling out the unlikely
possibility that some thermophilic methanogens happened to survive the
pasteurization and that methane was not detected due to a shift of
electron flow to dechlorination.
In this preliminary inhibition experiment (Fig.
1), the initial concentrations of BES and
molybdate were 50 and 5 mM, respectively.
To replenish the inhibitors,
the same amounts of molybdate and
half as much BES were refed at 2, 4, 6, and 8 weeks. The initial
concentration of sulfate was 20 mM, and the
same amount was added
at 4 and 8 weeks.
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FIG. 1.
Inhibition by BES, sulfate, and molybdate of anaerobic
dechlorination of Aroclor 1242 by pasteurized microorganisms. The error
bars indicate standard deviations of triplicate samples. The
concentrations of the anions are given in the text.
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The dechlorination pattern observed was the typical
meta-preferential dechlorination pattern M (
2).
Dechlorination was
completely inhibited in bottles with all three
anions throughout
12 weeks of incubation (Fig.
1). The slurries amended
with either
sulfate or BES turned dark black after 2 weeks of
incubation.
Additionally, these slurries emitted a strong sulfide smell
when
they were sampled while being flushed with
N
2-CO
2. In contrast,
slurries amended with
molybdate were yellowish and did not emit
the sulfide smell when
sampled.
Inhibitor fate and concentration effect.
Since BES, sulfate,
and molybdate inhibited the dechlorination in the preliminary
experiment, further experiments were done to evaluate the dose-response
relationship and to determine whether the inhibitors were metabolized.
Two types of experiments were conducted; both were prepared in the same
way as the preliminary inhibition experiment except as follows: the
inocula were taken from a culture previously pasteurized at 90°C for
15 min and retained the pattern M dechlorination activity, and then
they were repasteurized at 90°C for 10 min before inoculation; the
experimental vessels were 28-ml serum tubes (Bellco Glass Inc.,
Vineland, N.J.); each tube received 1 g of sediment, 4.5 ml of
RAMM, and 0.5 ml of inoculum; the concentration of Aroclor 1242 was 500 µg/g of dry sediment; and the tubes were incubated at 30°C instead
of 25°C. Inhibitor concentrations were as plotted in Fig. 2 and 3.
In the experiment shown in Fig.
2,
sulfate and molybdate amended at 4 mM were analyzed by high-pressure
ion chromatography
using an HPIC AS4A column (20 cm) and a conductivity
detector.
The mobile phase was 1.7 mM NaHCO
3-1.8 mM
Na
2CO
3, and the flow
rate was 2.3 ml/min.
Before the cultures were sampled, the tubes
were vortexed for 10 min.
After the sediments had settled, the
headspace gas was analyzed for
methane production, and then 1
ml of the liquid portion was withdrawn
while the vessel was flushed
with N
2. The liquid samples
were acidified with 1 N HCl while
being bubbled with N
2 for
5 min to drive out H
2S. The samples
were then centrifuged,
filtered (0.22-µm-pore-size filters; Millipore
Co., Bedford, Mass.),
and analyzed for sulfate and molybdate.
In the experiment shown in Fig.
3, BES and ethanesulfonate were
analyzed
by high-pressure liquid chromatography according to the
method
described by Löffler et al (
8).
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FIG. 2.
Effects of BES, sulfate, and molybdate at different
concentrations ( 1 mM) on anaerobic dechlorination of Aroclor 1242 by
the pasteurized microorganisms. The error bars indicate standard
deviations of triplicate samples.
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FIG. 3.
Effects of BES, sulfate, molybdate, and ethanesulfonate
at different concentrations ( 1 mM) on anaerobic dechlorination of
Aroclor 1242 by the pasteurized microorganisms. The error bars indicate
standard deviations of triplicate samples.
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Both BES and sulfate completely inhibited the dechlorination at
concentrations of
1 mM (Fig.
2 and
3). Statistical analysis
(analysis
of variance; data not shown) showed that there was no
significant
difference in molybdate inhibition at 2, 4, and 8
mM. Ion
chromatography results showed that after 4 weeks of incubation,
sulfate and molybdate in the 4 mM-amended group decreased 23.1
and
27.7%, respectively, compared to their killed-cell controls.
Differences in the color of the slurries amended with sulfate,
BES, and
molybdate were also observed. Unlike the nonamended group,
the 8 and 16 mM sulfate-amended slurries and the 16 mM BES-amended
slurry
turned black, while the molybdate-amended slurry was yellowish.
This
observation was consistent with that in the preliminary
inhibition
experiment and has also been observed in similar
experiments with
nonpasteurized microorganisms (
21).
Molybdate partially inhibited the dechlorination at a lower
concentration (1 mM) and did not inhibit the dechlorination at
concentrations of
0.5 mM (Fig.
3). The inhibitory effects of
sulfate and BES also decreased with a decrease in concentrations,
and no inhibitory effects were observed when their concentrations
were
0.1 mM. In this experiment, ethanesulfonate, a structural
analog
of BES, also inhibited the dechlorination and no significant
difference
in the inhibitory effect between ethanesulfonate and
BES was observed.
Concentrations of BES and ethanesulfonate used
for amendment at
1 mM were assayed with high-pressure liquid chromatography
and
were found to have decreased by 47.1 and 42.3%, respectively,
compared to their killed-cell controls. In the BES-amended
samples,
no ethanesulfonate, the potential debromination product of
BES,
was detected. To examine whether the inhibition could be relieved
by hydrogen, an additional eight samples were prepared with 60-ml
serum
bottles, instead of 28-ml tubes, to increase the capacity
for headspace
gas, and were amended with (in duplicate) either
1 mM sulfate, 1 mM
BES, 1 mM ethanesulfonate, or 16 mM molybdate.
These samples were
flushed with H
2-CO
2 (80:20) twice a week to
completely displace the headspace gas. Inhibition of the dechlorination
could not be relieved by replenishment of
hydrogen.
Organosulfonates have been reported to be used as electron acceptors
(
6,
7,
15), and reduction of organosulfonate
to sulfide has
also been documented (
6). To determine whether
the sulfonic
moiety of BES may be reduced to sulfide, an experiment
similar to that
described by Häggblom and Young (
4) was performed
with
the following modifications: the experimental vessels were
28-ml serum
tubes containing 4.5 ml of medium (the freshwater
medium described in
reference
4) and 0.5 ml of inoculum; sulfide
was
quantified by the methylene blue method (
19);
H
2S was driven
off by nitrogen; and the incubation time was
3 weeks. The freshwater
growth medium was modified as follows:
Na
2SO
4 was replaced by
2 mM BES; 1.5 mM
Na
2S was reduced to 0.5 mM; ascorbic acid was
added at
concentration of 0.1 g/liter; a mixture of lactate, pyruvate,
and
acetate (1 g of each per liter; all were sodium salts) was
chosen as
the substrate; the headspace gas was H
2-CO
2
(80:20);
and the trace elements and vitamins were replaced by those
used
in RAMM (
16). A molybdate (20 mM)-inhibited
BES-containing culture
and a culture without BES served as controls.
The amount of sulfide
S recovered from the BES-amended culture was more
than doubled
the 72 µg of sulfide S from Na
2S (reductant
in the medium) (Fig.
4). This result
proved production of sulfide from BES.
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FIG. 4.
Amount of sulfide S recovered from different amendment
cultures. The error bars indicate standard deviations of triplicate
samples.
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Results of the inhibitor concentration experiments suggest that the
inhibition of the dechlorination by BES, sulfate, molybdate,
and
ethanesulfonate was probably not due to general toxic effects
of these
anions. Effects of BES on a wide variety of microorganisms,
including
the spore-formers genera
Clostridium and
Bacillus
and
different types of anaerobes, were investigated, and 25 mM BES
had
no significant side effect (
12,
18). Similarly, it has
also
been documented that ~2 mM molybdate is not toxic (
10).
In
our experiment, 1 mM BES completely inhibited dechlorination
and 1 mM
molybdate partially inhibited the dechlorination. The
effective
concentration of molybdate in the slurries should have
been even lower
because some molybdate should have adsorbed onto
the clay surfaces
(
13) present in the sediment slurries and
become
nonbioavailable. The dechlorination was also completely
suppressed by 1 mM sulfate, and general toxicity of sulfate at
this concentration has
never been
reported.
Both the bromide moiety and the sulfonic moiety of BES are potential
electron acceptors (
7,
11,
15) and may compete
with PCBs for
electrons (
11). In our experiment, (i) no debromination
product (ethanesulfonate) was detected in the BES-amended samples,
(ii)
reduction of the sulfonic moiety to sulfide was detected,
and (iii)
ethanesulfonate also inhibited dechlorination. Based
on these
observations, we suggest that the inhibition is mainly
due to the
sulfonic
moiety.
Apparently the tested anions inhibited dechlorination by selectively
affecting certain target microorganisms. Molybdate, a
specific
inhibitor of sulfate-reducing bacteria (SRB), is able
to cause the
death of SRB by rapidly depleting their ATP pools
(
12).
Sulfate is a electron acceptor of SRB, while both BES
and
ethanesulfonate are potential electron acceptors of SRB, and
production
of sulfide from BES was detected in this experiment.
Together, these
data suggest that pattern M dechlorination is
mediated by spore-forming
sulfidogens.
BES has long been regarded as a specific inhibitor of methanogens
(
1,
12). Recently, Löffler et al. reported that BES
inhibited dechlorination of chloroethanes in the absence of
methanogens,
but no change in BES was observed under their experimental
conditions
(
8). Our results provide evidence that BES also
inhibits anaerobic
aromatic dechlorination by nonmethanogens. In our
case, however,
production of sulfide from BES was detected; this is the
first
report confirming reduction of the sulfonic moiety of BES to
sulfide.
It appears that BES may inhibit anaerobic dechlorination by
nonmethanogens
by more than one
mechanism.
| |
ACKNOWLEDGMENTS |
This work was supported in part by the General Electric Co.,
Michigan State University Institute for Environmental Toxicology, the
Great Lakes and Mid-Atlantic Hazardous Substance Research Center of
EPA, the SERDP Bioconsortium, and Hong Kong Baptist University.
We thank Linda Schimmelpfennig for technical assistance. Investigations
of ethanesulfonate and hydrogen were suggested by reviewers, and we
appreciate this suggestion.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Hong Kong Baptist University, 224, Waterloo Rd., Kowloon Tong, Hong Kong, People's Republic of China. Phone: (852) 2339 7062. Fax:
(852) 2336 1400. E-mail: dingyiye{at}hkbu.edu.hk.
| |
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Applied and Environmental Microbiology, January 1999, p. 327-329, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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