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Applied and Environmental Microbiology, May 2001, p. 2371-2374, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2371-2374.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
2-Bromoethanesulfonate Affects Bacteria in a
Trichloroethene-Dechlorinating Culture
Pei C.
Chiu* and
Minho
Lee
Department of Civil and Environmental
Engineering, University of Delaware, Newark, Delaware 19716
Received 3 August 2000/Accepted 28 February 2001
 |
ABSTRACT |
Long-term exposure to 2-bromoethanesulfonate (BES), an agent known
to inhibit methanogenesis, altered the bacterial community structure of
an anaerobic enrichment culture that reductively dechlorinated
trichloroethene (TCE). BES did not hinder the dechlorination of TCE or
other chlorinated ethenes as previously reported, although different
intermediates and end products were observed.
| |
TEXT |
Coenzyme M (CoM;
HSCH2CH2SO3
) is a
cofactor involved in the terminal step of methane biosynthesis,
where the methyl group carried by CoM is reduced to methane by
methyl-CoM reductase (18). 2-Bromoethanesulfonate
(BES; BrCH2CH2SO3) is
a structural analogue of CoM and a potent inhibitor of methanogenesis (9). Because this terminal step is involved in all methane biosynthesis and CoM is present only in methanogens (6),
BES has been used and regarded as a methanogen-specific inhibitor (14) in microbiological studies (1-3, 14,
17). It is often assumed implicitly that other organisms in
anaerobic mixed cultures are not affected by BES, and data are
interpreted accordingly to infer the roles of methanogens and other
microorganisms in the consortium.
However, it was found in recent years that BES can inhibit
nonmethanogens that reductively dechlorinate polychlorinated biphenyls (PCBs) (19) and chlorinated ethenes (12). Ye
et al. (19) reported that BES may compete with PCBs for
electrons, since the sulfonate moiety of BES can serve as an
alternative electron acceptor for sulfate-reducing bacteria, which were
presumably responsible for PCB dechlorination. A study by Löffler
et al. (12) showed that, in several nonmethanogenic
cultures, complete dechlorination of tetrachloroethene was inhibited by
BES, resulting in accumulation of intermediates, such as
dichloroethenes (DCEs) and vinyl chloride (VC). These authors therefore
concluded that inhibition of dechlorination by BES should not be taken
as evidence for the involvement of methanogens in the dechlorination
reaction. The mechanism through which BES inhibits microbial reduction
of chlorinated ethenes, however, remains unclear (12).
In this paper we present additional evidence, based on denaturing
gradient gel electrophoresis (DGGE) analysis, that BES affected bacteria in a trichloroethene (TCE)-dechlorinating enrichment culture.
In contrast to the previous observations (12), however, BES treatment did not hinder complete TCE dechlorination to ethene.
Effect of BES on bacteria (DGGE).
A TCE-dechlorinating culture
was isolated from a landfill site at Dover Air Force Base (Dover,
Del.). This culture was maintained in 120-ml serum bottles (Supelco,
Bellefonte, Pa.) containing lactate, acetate, HEPES buffer, vitamins,
and TCE, as described in detail elsewhere (11). The stock
culture was fed once a month by replacing half of the liquid culture
with fresh nutrient medium. The bottles were sealed with Teflon-lined
silicone septa and aluminum crimp caps, wrapped in foil, and incubated
at room temperature (22 ± 2°C) in an anaerobic glove bag
(I2R, Cheltenham, Pa.) under nitrogen. A nonmethanogenic
subculture derived from the stock culture was established by including
3 mM BES (Aldrich, Milwaukee, Wis.) in the culture medium for 18 consecutive feeding cycles.
To obtain DNA for PCR and DGGE analysis, cells were collected from the
stock culture and the BES culture at the end of the feeding cycle by
centrifuging (12,000 × g) 40 ml of each culture for 10 min.
Most supernatant was removed, and the cells were vortexed with the
remaining liquid (approximately 3 ml). The suspension was then
transferred to a 1.5-ml microtube and centrifuged for 2 min
(10,000 × g), and the supernatant was discarded. This step was repeated to remove all liquid. One hundred microliters of Tween 20 (0.1%; Bio-Rad, Hercules, Calif.) was added to the microtube and
vortexed to promote cell lysis, and the mixture was heated to 100°C
for 10 min in an iCycler (Bio-Rad). The cells were resuspended by being
vortexed for 10 s and centrifuged for 1 min. Eighty microliters of
InstaGene Matrix (Bio-Rad) was used to remove the cell lysis products,
and the mixture was vortexed. The sample was incubated at 56°C for 20 min, vortexed, incubated again at 100°C for 8 min, and vortexed again
for 10 s. The sample was then centrifuged for 2 min, and the
supernatant containing DNA was stored at 
20°C before PCR.
The forward and reverse primer sets used for bacteria and methanogens
were P63f (5' CAG GCC TAA CAC ATG CAA GTC 3') and P518r
(5' ATT ACC GCG GCT GCT GG 3') and M23f (5' TGG TTG ATC
CTG CCA
GAG G 3') and M440r (5' CGG CTG GCA CCG GTC TTG C
3'), respectively.
P63f had been shown to be applicable to a wide
range of bacteria
(
13). P518r was based on a universally
conserved region of bacteria
(
16). M23f and M440r were
designed based on the conserved regions
of the methanogenic 16S
ribosomal RNA sequence (
10). A GC clamp
of 40 bases was
added to both forward primers. These primer sets
were expected to give
PCR products of approximately 495 bp (P63f
and P518r) and 493 bp (M23f
and
M440r).
The PCR mixture consisted of 3.5 µl of 10×
Taq buffer,
0.28 µl of 100 mM deoxynucleoside triphosphate mix (0.25 mM/base),
0.18 µl of
Taq polymerase (5 U/µl), and 7 µl of Q
solution (PCR
Core Kit; Qiagen, Valencia, Calif.). Primers P63f and
P518r (Operon,
Alameda, Calif.) were added in the amounts equivalent to
50 pmol
per 100 µl of reaction mixture. One microliter of extracted
DNA
was used as template. Sterile deionized (DI) water was added to
make up the mixture volume to 35 µl. PCR was performed using a
Bio-Rad iCycler. The temperature program for the bacterial DNA
was (i)
95°C for 5 min; (ii) 30 cycles of 92°C for 1 min, 55°C
for 1 min,
and 72°C for 1 min; and (iii) 72°C for 10 min. The
program for
methanogen DNA was (i) 94°C for 2 min; (ii) 30 cycles
of 94°C for 1 min, 50°C for 1 min, and 72°C for 1.5 min; and (iii)
72°C for 2 min (
10).
DGGE was performed using a Bio-Rad DCode System. Thirty microliters of
PCR products and 5 µl of 6× gel loading dye (6% bromophenol
blue,
6% xylene cyanol, 100% glycerol, and DI water) were loaded
onto an
8% (wt/vol) polyacrylamide gel in 1× Tris-acetate-EDTA
running
buffer. The linear 40 to 70% denaturing gradient was created
by mixing
7 M urea and 40% formamide (J. T. Baker, Phillipsburg,
N.J.). The
electrophoresis was performed at 60°C at 75 V for 12
h. After
electrophoresis, the gels were soaked for 30 min in a
SYBR green I
solution (Molecular Probes, Eugene, Oreg.). The stained
gel was viewed
using a UV transilluminator (VWR, Baltimore, Md.)
and photographed with
a Polaroid camera (Cambridge, Mass.). The
major bacteria bands (Fig.
1, bands a, b, c, and d) were excised
with a sterile blade from the polyacrylamide gel, and the DNA
was
extracted using a Qiagen gel extraction kit. (Several minor
bands were
also obtained; however, they were too faint to excise
precisely and/or
to yield reliable sequences.) The DNA was sequenced
using an ABI Prism
377 DNA Sequencer (Applied Biosystems, Foster
City, Calif.) at the
University of Delaware Center for Agricultural
Biotechnology. The same
PCR primers were used for sequencing.
The major bacteria were
identified by comparing the sequences
of their 16S ribosomal DNA (rDNA)
fragments to the sequences of
known organisms using BLAST 2.0 (Basic
Local Alignment Search
Tool) of the National Center for Biotechnology
Information (NCBI;
http://www.ncbi.nlm.nih.gov). The highest percent
match for each
of the bands ranged from 88 to 95%.
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FIG. 1.
DGGE profiles of the 16S rDNA fragments of the stock
culture amplified with universal bacterial PCR primers (lane B1) and
with methanogen-specific primers (lane M1) and of the BES culture
amplified with universal bacterial primers (lane B2). No PCR products
were obtained from the BES culture using methanogen-specific primers
(lane M2).
|
|
Figure
1 shows the DGGE profiles of the PCR-amplified 16S rDNA
fragments from the stock culture and the BES-amended culture.
Using the
bacterium-specific primers, the stock culture was found
to contain
three dominant bacteria (bands a, b, and c in lane
B1). Sequencing
results showed that bands a (93%) and b (88%)
corresponded to
uncultured bacteria in the
Cytophaga-Flavobacter-Bacteroides phylum (
5; E. Stackebrandt, submitted to Deutsche Sammlung
von Mikroorganismen und Zellkulturen, accession no.
AJ287664).
No
specific information was available for band a. Band b was closely
related to the genus
Porphyromonas (
5), which
belongs to the
family
Bacteroidaceae, a group of
gram-negative, obligate anaerobes.
Band c matched (95%) a bacterium
found at a site contaminated
with hydrocarbons and chlorinated
solvents, which was most likely
a gram-positive
Syntrophomonas (
7). For the culture receiving
BES, bands a and c essentially disappeared, whereas band b was
not
affected. Interestingly, a new band (band d in lane B2), which
was very
faint in lane B1, became dominant as a result of BES
treatment. This
new band matched (92%) an uncultured bacterium
found in an anaerobic
digester (
8). Three prominent bands were
obtained from the
stock culture with the methanogen-specific primers
(lane M1). These
methanogen bands, which matched
Methanosaeta concilii (82 to
98%), were eliminated completely by BES, as expected.
No PCR products
were obtained for DGGE analysis from the BES-treated
culture using the
methanogen-specific primers (lane
M2).
The data in Fig.
1 show that long-term exposure to BES not only
eliminated methanogenic archaea but also altered the bacterial
community structure. It is possible that BES affected some of
these
bacteria directly by acting as a competing substrate (
19)
or through other mechanisms (
12). Alternatively, bacteria
which
consume and/or produce substrates for methanogenesis, such as
H
2 and acetate, would be affected indirectly by BES through
the
elimination of methanogens. That is, one cannot expect to
specifically
remove one group of organisms without eventually affecting
the
metabolically associated members in the community. Therefore,
exposure time should be taken into account when using BES as a
specific
inhibitor for methanogens. For short-term experiments,
caution should
also be exercised in interpreting the data, as
BES has been shown to
directly inhibit certain bacteria. Further
studies are necessary to
fully understand the limitations of using
BES as an inhibitor in
enzymatic and microbial ecology
studies.
Effect of BES treatment on TCE dechlorination.
The effect of
BES treatment on the culture's ability to reductively dechlorinate TCE
was investigated by using batch reactors similar to those described
elsewhere (11). The experiment was conducted using 63-ml
amber reactors (Alltech, Deerfield, Ill.) set up in duplicate at room
temperature under anaerobic and light-excluded conditions. Immediately
after feeding, 31.5 ml of the stock or BES culture was placed into each
reactor, leaving 31.5 ml of N2 headspace. Each reactor was
capped with a Mininert valve (Precision, Baton Rouge, La.), sealed with
low-permeability vinyl tape (3M, St. Paul, Minn.), and incubated in an
inverted position on a shaker at 150 rpm. At different elapsed times,
100 µl of headspace samples were withdrawn with a gas-tight syringe
and analyzed using a Hewlett Packard 6890 gas chromatograph
(Wilmington, Del.) equipped with a flame-ionization detector and a 30-m
GS-GasPro capillary column (J&W, Folson, Calif.). The temperature
program used was 40°C for 2 min, 25°C/min to 115°C, 10°C/min to
200°C, and 200°C for 1 min. The analytes had the following
retention times: methane, 0.422 min; ethane, 0.625 min; ethene, 0.791 min; acetylene, 1.58 min; VC, 5.87 min; 1,1-DCE, 7.74 min;
trans-DCE, 8.60 min; cis-DCE, 9.83 min; and TCE,
11.0 min. Quantification of peak areas was based on external
calibration standards.
The stock culture dechlorinated TCE quantitatively to ethane via
cis-DCE, VC, and ethene as intermediates (Fig.
2a). No 1,1-DCE
and only trace amounts of
trans-DCE were formed. Upon complete
conversion of TCE to
ethane, additional ethene was spiked, which
was reduced to ethane
quantitatively, indicating that ethene was
the precursor of ethane
(data not shown). Large quantities of
methane were produced throughout
the experiment (Fig.
2c). In
comparison, the BES-treated culture
dechlorinated TCE to ethene
(but not ethane) via a mixture of
cis- and 1,1-DCE (approximately
3:1 ratio) and VC as
intermediates (Fig.
2b). Dechlorination of
cis-DCE and VC by
the BES culture was not hindered in the presence
of BES (3 mM), in
contrast to the nonmethanogenic, dechlorinating
cultures studied
previously (
12). The appearance of 1,1-DCE
suggests that
BES either selected for organisms that dechlorinated
TCE to 1,1-DCE or
inhibited organisms that dechlorinated 1,1-DCE
in the stock
culture. Methanogens were probably not involved in
the observed
dechlorination, as BES reduced methanogenic activities
by three orders
of magnitude and only minute amounts of methane
were formed (Fig.
2c,
insert).
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FIG. 2.
Reductive dechlorination of TCE by the stock culture (a)
and the BES culture (b) and methane production by the two
cultures (c).
|
|
The inhibitory effect of BES on ethene reduction to ethane has been
reported elsewhere (
4), but the organism(s) involved
and
the role of methanogens remain unknown. Interestingly, we
observed,
under various conditions, that ethane was formed only
during active
methanogenesis; however, the reverse was not true.
That is, high
methanogenic activities did not guarantee ethane
formation. For
example, when H
2 (instead of lactate) was used
as the
electron donor to stimulate methanogenic and dechlorination
activities,
ethene was the final product (data not
shown).
In summary, our results show that extended exposure to BES altered the
bacterial community structure of a TCE-dechlorinating
consortium but
did not affect the culture's ability to completely
dechlorinate TCE,
even in the presence of 3 mM BES. Different
reduction intermediates and
products of TCE were observed, which
suggests that dehalogenators (and
other organisms) were influenced
by BES. Therefore, data from
studies involving the use of BES
as a methanogen inhibitor should be
interpreted with
care.
| |
ACKNOWLEDGMENTS |
We thank Michael D. Lee for sharing the stock culture, Bruce
Kingham for sequencing the DNA, and Mark Radosevich for reviewing the manuscript.
This study was supported in part by the National Science Foundation
(award no. 9984669).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Civil and Environmental Engineering, University of Delaware, Newark, DE
19716. Phone: (302) 831-3104. Fax: (302) 831-3640. E-mail: pei{at}ce.udel.edu.
| |
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Applied and Environmental Microbiology, May 2001, p. 2371-2374, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2371-2374.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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