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Previous Article | Next Article 
Appl Environ Microbiol, March 1998, p. 940-947, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Complete Reductive Dehalogenation of Brominated
Biphenyls by Anaerobic Microorganisms in Sediment
Donna L.
Bedard* and
Heidi M.
Van Dort
GE Corporate Research and Development,
Schenectady, New York 12301
Received 9 October 1997/Accepted 7 January 1998
 |
ABSTRACT |
We sought to determine whether microorganisms from the
polychlorinated biphenyl (PCB)-contaminated sediment in Woods Pond (Lenox, Mass.) could dehalogenate brominated biphenyls. The PCB dechlorination specificities for the microorganisms in this sediment have been well characterized. This allowed us to compare the
dehalogenation specificities for brominated biphenyls
and chlorinated biphenyls within a single sediment. Anaerobic sediment
microcosms were incubated separately at 25°C with 16 different mono-
to tetrabrominated biphenyls (350 µM) and disodium malate (10 mM).
Samples were extracted and analyzed by gas chromatography with an
electron capture detector and a mass spectrometer detector at various
times for up to 54 weeks. All of the tested brominated biphenyls were
dehalogenated. For most congeners, including 2,6-dibromobiphenyl
(26-BB) and 24-25-BB, the dehalogenation began within 1 to 2 weeks. However, for 246-BB and 2-2-BB, debromination was first
observed at 7 and 14 weeks, respectively. Most intermediate products
did not persist, but when 2-2-BB was produced as a
dehalogenation product, it persisted for at least 15 weeks before it was dehalogenated to 2-BB and then to biphenyl. The
dehalogenation specificities for brominated and
chlorinated biphenyls were similar: meta and
para substituents were generally removed first, and
ortho substituents were more recalcitrant. However, the
brominated biphenyls were better dehalogenation substrates than the chlorinated biphenyls. All of the tested
bromobiphenyls, including those with ortho and unflanked
meta and para substituents, were ultimately
dehalogenated to biphenyl, whereas their chlorinated counterparts
either were not dehalogenation substrates or were only
partially dehalogenated. Our data suggest that PCB-dechlorinating microorganisms may be able to dehalogenate brominated biphenyls and may
exhibit a relaxed specificity for these substrates.
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INTRODUCTION |
There have been many studies of the
microbial dehalogenation of polychlorinated biphenyls
(PCBs) (1-3, 5-7, 9-11, 17-19, 22-24, 26-29), but only
two previous studies of dehalogenation of polybrominated biphenyls (PBBs) (15, 16).
Microorganisms from a PBB-contaminated site (Pine River, St.
Louis, Mich.) (16) and two PCB-contaminated sediments
(Silver Lake, Pittsfield, Mass., and Hudson River, river mile 193, Fort
Edward, N.Y.) (9, 10) partially dehalogenated the commercial
PBB mixture Firemaster BP6 (15). A single hexabromobiphenyl,
2,4,5,2',4',5'-hexabromobiphenyl (245-245-BB), which
constitutes more than 50% of Firemaster (21), was the
primary dehalogenation substrate. Microorganisms from the Pine River site removed 32% of the meta and
para bromines from Firemaster in 32 weeks
(15). The dehalogenation products were
predominantly 24-25-BB and 25-2-BB, with lesser amounts of 25-25-BB and
24-24-BB and a trace of 2-2-BB. In contrast, microorganisms collected
from the Pine River upstream of the PBB contamination did not
dehalogenate Firemaster. Hudson River and Silver Lake microorganisms
removed 12 and 3%, respectively, of the meta and para bromines from Firemaster in 32 weeks, but the main
product was 2-2-BB (15). No ortho debromination
was observed in any of these experiments. However, small amounts of
biphenyl and 2-BB were detected when Firemaster was incubated with
a pyruvate enrichment culture of PCB-dechlorinating microorganisms from
the Hudson River (15). The latter data suggested the
possibility of ortho debromination, since all PBB congeners
in Firemaster have at least one ortho bromine.
The PCBs in Hudson River and Silver Lake sediments have undergone
extensive microbial dechlorination in situ (9, 10). In
addition, many laboratory experiments have confirmed that PCB dechlorinators are present in these sediments and can be eluted from
them and transferred to other sediments (1, 17-19). Neither sediment shows any evidence of PBB contamination. Hence, the
observation that microorganisms from two PCB-contaminated sites can
remove the meta and para bromines from
245-245-BB suggests that PCB dechlorinators might be able to
dehalogenate brominated biphenyls. We sought to test this
possibility by assessing the ability of the anaerobic microorganisms from the PCB-contaminated sediments of Woods Pond (Lenox, Mass.) to dehalogenate a variety of mono- through
tetrabrominated biphenyls.
The PCB dechlorination specificity for the microorganisms in Woods Pond
has been well characterized (3, 5, 6, 23, 24, 26-29). This
information provided us with the opportunity to directly compare the
dehalogenation specificities for PCBs and brominated
biphenyls within a single sediment. The microorganisms in Woods
Pond sediment can dechlorinate PCBs by removal of flanked meta or para chlorines from 3,4- (34-), 234-, 235-, 236-, 245-, 345-, 2345-, 2346-, 2356-, and 23456-chlorophenyl
rings and unflanked para chlorines from 24- and
246-chlorophenyl rings (3, 6, 24, 28, 29). However, the
unflanked meta chlorines on 3- and 25-chlorophenyl rings are
not dechlorinated by the microorganisms in this sediment and neither is
the unflanked para chlorine on 4-chlorophenyl rings
(6). Only three PCB congeners, 246-CB, 24-CB, and 2356-CB,
are known to be substrates for ortho dechlorination by the
microorganisms in Woods Pond (23, 26, 28). Unfortunately, 2-CB, 2-2-CB, 26-CB, and other ortho-chlorinated congeners
do not appear to be substrates (23, 26-29). We postulated
that brominated biphenyls might be favorable substrates for PCB
dechlorinators because they are PCB analogs. Furthermore, their
chemistry indicates that brominated biphenyls should be more
easily dehalogenated than PCBs. The aryl-bromine bond is weaker
than the aryl-chlorine bond (dissociation energies for the
C6H5-Br and C6H5-Cl
bonds are 80 and 95 kcal/mol, respectively (see Table 5, p. F-243, in reference 25). In addition, chemical
dehalogenations carried out by various reaction
mechanisms have consistently shown that aryl bromines are more
easily removed than aryl chlorines (8, 13, 20).
We investigated the dehalogenation of all commercially
available mono-, di-, and tribrominated biphenyls and
that of several tetrabrominated biphenyls. These were
2-BB, 3-BB, 4-BB, 24-BB, 25-BB, 26-BB, 2-2-BB, 4-4-BB, 245-BB,
246-BB, 345-BB, 25-2-BB, 25-3-BB, 25-4-BB, 24-25-BB, and 25-25-BB. We
expected to observe selective dehalogenation from
ortho, meta, and para positions as has
been observed for PCB dechlorination by microorganisms from Woods Pond
sediment (see above). Furthermore, because bromines should be
easier to dehalogenate, we reasoned that the unflanked bromines on
3-, 4-, 25-, and possibly 2- and 26-bromophenyl rings might also be
dehalogenated by these microorganisms even though the chlorines on
their PCB counterparts are not.
The results confirmed our expectations. All of the brominated
biphenyls were completely dehalogenated to biphenyl in
live samples, but no dehalogenation occurred in
autoclaved controls. Debromination occurred first from the
meta and para positions and then from the
ortho positions. Most of the congeners were dehalogenated
after a lag time of 1 to 2 weeks, but 2-2-BB required an acclimation
period of 14 weeks before dehalogenation commenced. Most dehalogenation intermediates did not accumulate,
but when 2-2-BB was produced as an intermediate it persisted for at
least 15 weeks.
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MATERIALS AND METHODS |
Chemicals.
Brominated biphenyls (97 to 99% purity)
were purchased from AccuStandard (New Haven, Conn.) or Ultra Scientific
(North Kingstown, R.I.). L-Malic acid (cell culture reagent
quality, catalog no. M-7387) was purchased from Sigma Chemical
Corporation (St. Louis, Mo.) and adjusted to pH 7 with sodium
hydroxide.
Slurry preparation and incubation.
Multiple core samples (45 cm) of sediment were collected from the west side of Woods Pond
(5), a shallow impoundment on the Housatonic River. The core
samples were pooled in glass jars, topped with site water, and stored
at 4°C until used. On a dry-weight basis, each gram of sediment
contained 45.1 µg of partially dechlorinated Aroclor 1260 and 7,100 µg of weathered hydrocarbon oil. Slurries were prepared under an
atmosphere of 95 to 97% nitrogen-3 to 5% hydrogen in an anaerobic
chamber by mixing wet sediment (2 volumes) with glass-distilled water
(3 volumes). The slurries were dispensed into serum bottles, and a
bromobiphenyl congener was added from a concentrated stock
solution (70 mM in acetone) to give a final concentration of 350 µmol
per liter of slurry. (This corresponds to 560, 750, 940, and 1,130 µg
of brominated biphenyl per g [dry weight] of sediment for mono-
through tetrabrominated biphenyls, respectively.) Except where
indicated, disodium malate was also added to give a final concentration
of 10 mM. No other nutrients were added. The bottles were crimp sealed
with Teflon-lined butyl rubber septa. Sterile controls were prepared by
pasteurization (at 75°C for 20 min), followed by incubation (at 23 to
25°C for 24 h) and autoclaving (at 121°C for 3 h).
Duplicate samples and controls were incubated in the dark at 23 to
25°C. Although no bicarbonate or CO2 was added, all
incubations became methanogenic within 1 to 2 weeks.
The data for dehalogenation of 246-BB were obtained
from an experiment set up under different conditions. For this
experiment, microorganisms were eluted from a fresh slurry of Woods
Pond sediment by two consecutive gravity filtrations of the sediment
slurry through several layers of glass wool and then used to inoculate a pasteurized slurry prepared from decomposed freshwater marsh peat
collected from a beaver meadow in the Adirondack Mountains (N.Y.). The
marsh peat slurry was prepared in the anaerobic chamber, dispensed into
serum bottles as described above, and then pasteurized by heating twice
to 80°C for 10 min with a 24-h interval at 24°C between heatings.
Following pasteurization, 10 ml of the supernatant was removed from
each 30-ml sample of marsh slurry and replaced with the microbial
inoculum from Woods Pond. 26-BB or 246-BB, disodium malate, and Aroclor
1260 were then added to the resulting inoculated sediment to give final
concentrations per liter of 350 µmol, 10 mmol, and 10 mg,
respectively. The slurries were incubated at 22 to 25°C and became
methanogenic within a week or two.
Extraction and bromobiphenyl analysis.
Aliquots (1 ml each) of the slurries were sampled periodically and extracted by
vigorous shaking (24 h) with anhydrous ether (5 volumes) and elemental
mercury (1/4 volume, to remove sulfur) in vials with Teflon-lined
foam-backed screw caps. Samples were analyzed by gas chromatography
(GC) on a 5880A GC (Hewlett-Packard Co., Palo Alto, Calif.) equipped
with a Ni63 electron capture detector operated at 300°C
and a DB-1 (polydimethylsiloxane) capillary column (30 m long by 0.25 mm [inner diameter; 0.25-µm phase thckness], J & W Scientific,
Inc., Folsom, Calif.). We used a two-stage GC temperature program as
follows: 2 min at 40°C, increase at 20°C/min to 160°C, hold 3 min, increase at 2°C/min to 260°C, hold 20 min. The carrier and
makeup gas was nitrogen.
Bromobiphenyls formed as dehalogenation
products were initially identified by matching GC retention times with
those of authentic standards. Reference standards were not available
for 2-3-BB, 2-4-BB, 34-BB, 35-BB, and 24-2-BB. These intermediates were
identified from the possible debromination products based on
comparisons of their elution positions, relative to those of the other
brominated biphenyl congeners, with the relative elution
positions of PCBs with the same substitution patterns (12).
All intermediates were subsequently analyzed by selected ion monitoring
(electron impact ionization at 70 eV) with a Hewlett-Packard 5890/5971A GC-mass spectrometer (MS) equipped with a DB-1 capillary column as
described above and were confirmed to be brominated biphenyls. We used a multistage GC temperature program as follows: 2 min at
50°C, increase at 20°C/min to 150°C, increase at 4°C/min to 210°C, increase at 20°C/min to 270°C, and hold for 10 min.
Several isomers of each homolog class were scanned by GC-MS to
determine the fragmentation pattern and to identify the most abundant
ions. Scan windows were set to include the earliest and latest eluting
isomers of each homolog class. The masses of the most abundant ions of
the molecular ion cluster and its characteristic fragments were
monitored for each homolog class. These were, in order of relative
abundance, m/z 153 and 154 for biphenyl;
m/z 232, 234, and 152 for monobromobiphenyls;
m/z 312, 314, and 152 for dibromobiphenyls;
m/z 390, 392, 230, and 232 for tribromobiphenyls; and m/z 310, 389, 391, and 470 for 24-25-BB and 25-25-BB.
The ratios of the areas of these ions were checked for each sample to
verify that they matched those of the standards. 26-BB and its
products, 2-BB and biphenyl, were quantified by GC-MS with a
linear three-point calibration curve.
| |
RESULTS |
Stoichiometric dehalogenation of 26-BB to
biphenyl.
We examined the dehalogenation
of 26-BB in our first experiments because we were particularly
interested in determining whether the microorganisms in Woods Pond
sediment could dehalogenate ortho-brominated biphenyls. As shown in Fig. 1A,
dehalogenation of 26-BB was first detected at 2 weeks
and the congener was completely dehalogenated to biphenyl
within 3 months. 2-BB accumulated as an intermediate while the 26-BB
was being dehalogenated, but some of it was further dehalogenated to
biphenyl even though significant amounts of 26-BB remained.
This result suggests that the rate of dehalogenation of
26-BB is higher than that for 2-BB. No dehalogenation
occurred in autoclaved controls.
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FIG. 1.
Dehalogenation of 26-BB to biphenyl. 26-BB (350 µmol per liter of slurry) was incubated in anaerobic microcosms of
Woods Pond sediment with or without malate (10 mM) as described in the
text. The time course of dehalogenation was monitored
by GC-MS as described in Materials and Methods. (A) No malate. (B)
Malate was added at the beginning of the incubation.  , 26-BB;  ,
2-BB,  , biphenyl.
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Effect of malate on dehalogenation of 26-BB.
We had previously determined that disodium malate (10 mM) accelerated
the dechlorination of 25-34-CB to 25-3-CB (4). Malate also
accelerated the dehalogenation of 26-BB (Fig. 1B), but
the degree of acceleration differed depending on the time of year at
which the sediment was collected and the length of time it was stored
before use. Subsequent experiments showed that the malate was depleted
within the first few days of incubation, prior to the onset of
dehalogenation. Furthermore, replenishing the malate
during the incubation had no effect on the
dehalogenation. Hence, it is unlikely that malate was
an electron donor for the dehalogenation reaction.
Malate also accelerated the dehalogenation at
concentrations of 2.5 and 5 mM, but these concentrations were slightly less effective than 10 mM (data not shown). Malate had no
significant effect at concentrations of 0.1 or 0.5 mM.
Dehalogenation of mono- and dibrominated biphenyls.
All but one of the mono- and dibromobiphenyls were
dehalogenated after only 1 to 2 weeks of acclimation. Following
acclimation, more than 90% of each of these congeners was
dehalogenated to biphenyl in 1 to 7 weeks (Fig.
2). The meta and
para bromines were removed first, and then the
ortho bromines were removed (Fig. 2). 4-BB was detected
as a transient intermediate of 4-4-BB but was further dehalogenated to
biphenyl without accumulating. The 2-BB produced from the
dehalogenation of 24- and 25-BB accumulated briefly
prior to dehalogenation as seen previously for 26-BB
(Fig. 1B). Unlike the other congeners, 2-2-BB required a long
acclimation period (14 weeks) before dehalogenation
commenced. However, although 2-BB was detected as an intermediate of
2-2-BB, it did not accumulate as in the other slurries but was rapidly
dehalogenated to biphenyl.
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FIG. 2.
Dehalogenation of mono- and dibrominated
biphenyls. a, length of incubation before
dehalogenation was observed, as evidenced by the
appearance of the first traces of dehalogenation
product(s). As little as 1 to 2% dehalogenation of the
substrate could be detected. b, length of time after acclimation for
dehalogenation of ~90% of the bromobiphenyl
to biphenyl.
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Dehalogenation of tribrominated biphenyls.
All
tribromobiphenyls except 246-BB were also dehalogenated
after only 1 to 2 weeks of acclimation, but complete
dehalogenation to biphenyl took much longer
than for mono- and dibromobiphenyls (Fig.
3). For most tribromobiphenyls,
small to moderate amounts of the parent congener persisted for long
times, even though most of the congener had already been dehalogenated
to biphenyl. Most of the tribromobiphenyls were
dehalogenated by two different routes, but it was generally not
possible to determine whether one route was favored, because most
dibrominated intermediates did not accumulate to substantial levels
before further dehalogenation. However, meta
and para bromines were generally removed before
ortho bromines. When formed, 26-BB and 2-BB briefly
accumulated to substantial levels.
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FIG. 3.
Dehalogenation of tribrominated biphenyls. a,
length of incubation before dehalogenation was
observed, as evidenced by the appearance of the first traces of
dehalogenation products. b, length of time after
acclimation for dehalogenation of ~90% of the
bromobiphenyl to biphenyl. c, the first time point in
these incubations was at 14 days; by that time, significant
dehalogenation to di- and monobrominated products had
already occurred. d, a moderate amount of 245-BB was still present at
this time. e, small amounts of 246-BB and 25-4-BB, respectively,
persisted in these samples. f, this congener was rapidly dehalogenated
to 2-2-BB, which persisted at this time; biphenyl was first observed at
136 days. g, a large amount of 345-BB and a moderate amount of 3-BB
persisted. h, this dehalogenation reaction was observed
only when 246-BB was the initial substrate.
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There were two instances in which ortho debromination
occurred prior to complete meta and para
dehalogenation (Fig. 3). (i) The 246-BB was
dehalogenated to both 24-BB and 26-BB. Subsequently, 2-BB and 4-BB were
detected prior to dehalogenation to biphenyl. (ii) The 2-3-BB that was formed as an intermediate from 25-3-BB was
dehalogenated to both 2-BB and 3-BB before
dehalogenation to biphenyl.
The 345-BB was dehalogenated to 34-BB and 35-BB and then to 3-BB, 4-BB,
and biphenyl. The 35-BB and 3-BB both accumulated to high
levels for a short time before dehalogenation to
biphenyl.
The 25-2-BB was exclusively dehalogenated by the pathway 25-2-BB 
2-2-BB 2-BB biphenyl. The initial
dehalogenation product, 2-2-BB, was first detected at 6 days and persisted without further dehalogenation for
at least 18 weeks. No 2-BB or biphenyl was detected until 136 days.
Dehalogenation of 24-25-BB and 25-25-BB.
Figure
4 shows the pathways for
dehalogenation of 24-25-BB and 25-25-BB. The
meta and para dehalogenation of
these tetrabromobiphenyls to 2-2-BB commenced within 2 weeks,
but the 2-2-BB accumulated and persisted for at least 15 weeks. When
the slurries were sampled again at 54 weeks, some of the
tetrabromobiphenyl and 2-2-BB was still present, but most had
been dehalogenated to biphenyl. Figure 5 shows the product distribution at
various times for one of duplicate incubations of 24-25-BB.
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FIG. 4.
Dehalogenation pathway for 24-25-BB and 25-25-BB. Both
congeners were dehalogenated to 2-2-BB, which began to accumulate at 14 days and persisted for at least 16 weeks. Dehalogenation of 2-2-BB
began sometime after 17 weeks and prior to 54 weeks. In both cases,
small amounts of 2-2-BB and tetrabromobiphenyl still persisted
at 54 weeks.
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FIG. 5.
Total ion chromatogram of 24-25-BB and its
dehalogenation products at various times. The
dehalogenation of 24-25-BB was monitored by GC-MS as
described in Materials and Methods. The areas plotted for each compound
represent the sum of selected ions for that compound and therefore are
not directly comparable to concentration. However, the plots do
indicate the progress of the dehalogenation. (A)
Autoclaved control at 120 days. (B through E) Live samples showing the
dehalogenation products present at 21, 29, 43, and 377 days. The retention times have shifted slightly for the sample at 377 days because this sample was analyzed many months later than the other
samples.
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DISCUSSION |
Dehalogenation specificity: brominated versus chlorinated
biphenyls.
Despite no history of prior exposure to
brominated biphenyls, the microorganisms in Woods Pond sediment
dehalogenated all of the tested brominated congeners to
biphenyl. As expected, brominated biphenyls were better
substrates for microbial dehalogenation than
chlorinated biphenyls. Perhaps as a result, the specificity for
brominated biphenyl dehalogenation was less
stringent than that observed for PCBs. Table 1 compares the
dehalogenation of various brominated biphenyls
and their chlorinated counterparts in microcosms of Woods Pond
sediment. Although all of these
brominated biphenyls were dehalogenated, most of their
chlorinated analogs were not dehalogenated, despite prolonged
incubation. Furthermore, the brominated biphenyls were totally
dehalogenated to biphenyl, but the chlorinated
biphenyls that were substrates were only partially dehalogenated.
For brominated biphenyls, there appeared to be no particular
preference for which meta or para
bromine was removed first, regardless of whether the bromine
was flanked or unflanked. Generally, all possible initial
meta and para dehalogenation
products were observed (Fig. 2 through 5). Usually all meta
and para bromines were removed prior to ortho
dehalogenation. The unflanked meta bromines appeared to be slightly more difficult to remove, as evidenced by the brief accumulation of 3-BB and 35-BB when these congeners were produced as intermediates.
In contrast, PCB dehalogenation primarily targets the
flanked meta and para chlorines in Woods Pond
sediment (Table 1 and references 3, 24, and
26). Furthermore, the substitution pattern of the
PCB substrate determines whether meta or para
chlorines will be removed. For example, only one product was seen for
the dechlorination of 345-CB, 235-CB, 25-34-CB, and 24-34-CB (3, 26). Both possible initial meta and para
dechlorination products were observed for 245-CB and 234-CB, but the
ratio of the two products was >99:1 (24). The substitution
pattern of the added PCB substrate also determines the specificity of
the dechlorination of Aroclor 1260 that will be primed in the sediment
(24). Unflanked para chlorines on 24- and
246-chlorophenyl groups can also be dehalogenated, but usually only
after long acclimation times (6, 26, 28, 29). Unflanked
meta dechlorination of PCBs has never been observed in Woods
Pond sediment.
For brominated biphenyls, all ortho bromines
were ultimately removed, but they were apparently more difficult to
remove than the meta and para bromines. This
was evidenced by the longer acclimation times for 2-BB, and especially
2-2-BB, and by the accumulation and persistence of these congeners when
they were produced as dehalogenation intermediates. For
chlorinated biphenyls, the ortho chlorines are not
dehalogenation substrates, except for those on 2356-CB,
246-CB, and 24-CB (23, 26, 28).
Despite the differences discussed above, there are similarities in the
relative reactivity preferences for brominated and chlorinated
biphenyls in Woods Pond sediment. For brominated
biphenyls, the observed order of reactivity was as follows:
flanked meta 
flanked para unflanked para > unflanked meta > ortho bromines. For PCBs, the order of reactivity is as
follows: flanked meta flanked para > unflanked para 
ortho > unflanked
meta chlorines.
Morris and colleagues reported that microorganisms collected upstream
of the PBB contamination in the Pine River could not dehalogenate
Firemaster, but microorganisms eluted from the PBB-contaminated section
of the Pine River and from two sites with a history of exposure to
PCBs, but not to PBBs, were able to dehalogenate Firemaster (15). These observations suggest that prior
acclimation to halogenated biphenyls is necessary for PBB
dehalogenation and that PCB dechlorinators might be
able to dehalogenate brominated biphenyls. Our data for microorganisms from Woods Pond, which also has no history of PBB contamination, are consistent with this interpretation. Furthermore, the observed similarities in the relative reactivity preferences for
chlorinated and brominated biphenyls suggest that the PCB dechlorinators in Woods Pond may exhibit cross-reactivity for brominated biphenyls. However, given that PCBs composed
of certain chlorophenyl groups (e.g., those substituted at positions
2-, 3-, 4-, 25-, and 26-) appear to totally resist
dehalogenation in this sediment, we were
surprised that all of the brominated biphenyls were completely
dehalogenated to biphenyl.
Clearly, the difference between PCB and brominated biphenyl
dehalogenation is not simply a matter of reduction
potential. The reduction potentials of 4-4-CB and 25-25-CB are higher
than that of 4-BB (20); yet these PCBs are not
dehalogenation substrates in Woods Pond (Table 1 and
references 3 and 24). If the same
microorganisms do indeed dehalogenate both brominated and chlorinated
biphenyls, it appears that the dehalogenases show a
relaxed specificity for bromophenyl groups. There is precedent for such
relaxed specificity; Desulfomonile tiedje can remove chlorine only from the meta positions of halogenated
benzoates, but it can remove iodine and bromine from
ortho, meta, and para positions
(reviewed in reference 14). A full understanding of our own results will require isolation of the responsible
dehalogenating microorganisms and their dehalogenases.
Dehalogenation of 246-BB.
Unexpectedly, the acclimation time
for dehalogenation of 246-BB was 7 weeks versus 1 to 2 weeks for all other tribromobiphenyls. Furthermore, although
bromines were almost always more readily removed from
meta and para positions than from
ortho positions, this was not true for 246-BB. As previously
observed for 246-CB, the unflanked halogen in the para
position proved as difficult to remove as those in the ortho
positions. Since the unflanked para bromines on 4-BB and
24-BB and the ortho bromines on 2-BB and 26-BB were
removed after very short acclimation times, we do not understand the
relative recalcitrance of 246-BB. The incubation conditions for 246-BB
were different from those for the other congeners. However, it is
unlikely that this explains the difference, because the acclimation
time for the dehalogenation of 26-BB was the same under
both incubation conditions.
Ability of individual brominated biphenyls to trigger
dehalogenation.
Morris and coworkers
reported that the microorganisms from three different sediments
dehalogenated 245-245-BB only when it was incubated as a component of
Firemaster and not when it was incubated alone (15).
This was unexpected, because the total concentrations of 245-245-BB in
the Firemaster experiments and in the 245-245-BB experiments were
comparable. Morris and coworkers proposed that one or more of the other
components of Firemaster might be required to elicit a response (e.g.,
enzyme induction) that triggers dehalogenation. This
proposal is not consistent with our results with Woods Pond sediment.
Each of the 16 brominated biphenyls that we studied triggered
dehalogenation, regardless of the substitution pattern.
However, we did not study 245-245-BB.
The relative recalcitrance of 2-2-BB and its implications for the
dehalogenation of Firemaster BP6.
Very long
acclimation times were required for ortho
dehalogenation when ortho bromines were
juxtaposed on both rings, as in 2-2-BB, 25-2-BB, 24-25-BB, and
25-25-BB. Prior acclimation for meta and/or para
dehalogenation did not facilitate ortho
dehalogenation of 2-2-BB, suggesting that the
microorganisms that dehalogenate 2-2-BB are distinct from those that
dehalogenate the other congeners tested.
Microorganisms from the Pine River, the Hudson River, and Silver Lake
meta- and para-dehalogenated the 245-245-BB in
Firemaster to di-ortho-substituted products including
2-2-BB, but no ortho dehalogenation
was observed in the 32-week incubations (15). This is
consistent with the relative recalcitrance of 2-2-BB that we
observed in Woods Pond sediment. It is important to note that Morris
and coworkers terminated their experiments when fewer than one-third of
the meta and para bromines had been removed
from Firemaster (15). Given that we observed
ortho dehalogenation of congeners with
ortho bromines juxtaposed on both rings only after
nearly all of the meta and para bromines had
been removed, and then only after a long acclimation to 2-2-BB (see
Results and Fig. 5), it is possible that ortho
dehalogenation to biphenyl would have occurred
in the Firemaster experiments if the incubations had been extended
further. On the other hand, it is also possible that the microorganisms
from the sites studied in the Firemaster experiments are incapable of
ortho-dehalogenating brominated biphenyls. It is
well established that microorganisms from Woods Pond can ortho-dehalogenate certain PCB congeners, but no conclusive
evidence for ortho dechlorination of PCBs has been reported
for microorganisms from any of the locations studied by Morris and
coworkers (11, 16, 18, and 22 and
references 9 and 10 as
reevaluated in reference 2). Longer experiments with
microorganisms from the Pine River, the Hudson River, and Silver Lake
incubated with 245-245-BB, 24-24-BB, 24-25-BB, 25-25-BB, 25-2-BB, and
2-2-BB would determine whether these sediments harbor microorganisms capable of totally dehalogenating the key components of Firemaster BP6.
| |
ACKNOWLEDGMENTS |
We thank Ralph J. May for assistance in developing GC-MS methods
of analysis, Rosanna Stokes for malate analysis, Lynn Smullen for help
in preparing the figures, and Kim DeWeerd for helpful comments on the
manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: GE Corporate
Research & Development, Bldg. K-1, Room 3B12, P.O. Box 8, Schenectady, NY 12301. E-mail: bedardd{at}crd.ge.com.
Present address: Department of Chemistry, Purdue University,
Lafayette, IN 47907-1393.
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Appl Environ Microbiol, March 1998, p. 940-947, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
