Reductive dechlorination of cis-1,2-dichloroethene and vinyl chloride by Dehalococcoides ethenogenes

Article abstract of DOI:10.1021/es001285i

cis-Dichloroethene (DCE) and vinyl chloride (VC) often accumulate in contaminated aquifers in which tetrachloroethene (PCE) or trichloroethene (TCE) undergo reductive dechlorination. Dehalococcoides ethenogenes strain 195 is the first isolate capable of dechlorinating chloroethenes past cis-DCE. Strain 195 could utilize commercially synthesized cis-DCE as an electron acceptor, but doses greater than 0.2 mmol/L were inhibitory, especially to PCE utilization. To test whether the cis-DCE itself was toxic, or whether the toxicity was due to impurities in the commercial preparation (97% nominal purity), we produced cis-DCE biologically from PCE using a Desulfitobacterium sp. culture. The biogenic cis-DCE was readily utilized at high concentrations by strain 195 indicating that cis-DCE was not intrinsically inhibitory. Analysis of the commercially synthesized cis-DCE by GC/mass spectrometry indicated the presence of approximately 0.4% mol/mol chloroform. Chloroform was found to be inhibitory to chloroethene utilization by strain 195 and at least partially accounts for the inhibitory activity of the synthetic cis-DCE. VC, a human carcinogen that accumulates to a large extent in cultures of strain 195, was not utilized as a growth substrate, and cultures inoculated into medium with VC required a growth substrate, such as PCE, for substantial VC dechlorination. However, high concentrations of PCE or TCE inhibited VC dechlorination. Use of a hexadecane phase to keep the aqueous PCE concentration low in cultures allowed simultaneous utilization of PCE and VC. At contaminated sites in which D. ethenogenes or similar organisms are present, biogenic cis-DCE should be readily dechlorinated, chloroform as a co-contaminant may be inhibitory, and concentrations of PCE and TCE, except perhaps those near the source zone, should allow substantial VC dechlorination. cis-Dichloroethene (DCE) and vinyl chloride (VC) often accumulate in contaminated aquifers in which tetrachloroethene (PCE) or trichloroethene (TCE) undergo reductive dechlorination. `Dehalococcoides ethenogenes' strain 195 is the first isolate capable of dechlorinating chloroethenes past cis-DCE. Strain 195 could utilize commercially synthesized cis-DCE as an electron acceptor, but doses greater than 0.2 mmol/L were inhibitory, especially to PCE utilization. To test whether the cis-DCE itself was toxic, or whether the toxicity was due to impurities in the commercial preparation (97% nominal purity), we produced cis-DCE biologically from PCE using a Desulfitobacterium sp. culture. The biogenic cis-DCE was readily utilized at high concentrations by strain 195 indicating that cis-DCE was not intrinsically inhibitory. Analysis of the commercially synthesized cis-DCE by GC/mass spectrometry indicated the presence of approximately 0.4% mol/mol chloroform. Chloroform was found to be inhibitory to chloroethene utilization by strain 195 and at least partially accounts for the inhibitory activity of the synthetic cis-DCE. VC, a human carcinogen that accumulates to a large extent in cultures of strain 195, was not utilized as a growth substrate, and cultures inoculated into medium with VC required a growth substrate, such as PCE, for substantial VC dechlorination. However, high concentrations of PCE or TCE inhibited VC dechlorination. Use of a hexadecane phase to keep the aqueous PCE concentration low in cultures allowed simultaneous utilization of PCE and VC. At contaminated sites in which `D. ethenogenes' or similar organisms are present, biogenic cis-DCE should be readily dechlorinated, chloroform as a co-contaminant may be inhibitory, and concentrations of PCE and TCE, except perhaps those near the source zone, should allow substantial VC dechlorination.

Full text of DOI:10.1021/es001285i

Environ. Sci. Technol. 2001, 35, 516-521
dechlorination is incomplete (2). Large accumulations of cis-
Reductive Dechlorination of
cis-1,2-Dichloroethene and Vinyl
Chloride by “Dehalococcoides
ethenogenes”
X A V I E R M A YM OÄ - G A T E L L , ‡
I V O N N E N I J E N H U I S , A N D
S T E P H E N H . Z I N D E R *
dichloroethene (DCE) are often found at contaminated sites
undergoing reductive dechlorination, and at some sites there
is little or no dechlorination past cis-DCE.
The reasons for cis-DCE accumulation are partially
understood. It appears that organisms that can dechlorinate
PCE to cis-DCE are diverse and cosmopolitan. Several
different organisms have been isolated from diverse sites
that reductively dechlorinate PCE and TCE no further than
cis-DCE (3-9). Organisms that dechlorinate past cis-DCE
are not always present at contaminated sites and seem to be
more nutritionally fastidious and slower growing than the
above organisms (10-14). Presently, the only isolated organ-
ism known to reductively dechlorinate chloroethenes further
than DCE is provisionally named “Dehalococcoides etheno-
genes”. Growth of this organism on cis-DCE was considerably
slower than with the other electron acceptors (PCE, TCE,
Department of Microbiology, Wing Hall, Cornell University,
Ithaca, New York 14853
cis-Dichloroethene (DCE) and vinyl chloride (VC) often
accumulate in contaminated aquifers in which tetrachloro-
ethene (PCE) or trichloroethene (TCE) undergo reductive
dechlorination. “Dehalococcoides ethenogenes” strain 195
is the first isolate capable of dechlorinating chloroethenes
past cis-DCE. Strain 195 could utilize commercially
synthesized cis-DCE as an electron acceptor, but doses
greater than 0.2 mmol/L were inhibitory, especially to PCE
utilization. To test whether the cis-DCE itself was toxic,
or whether the toxicity was due to impurities in the commercial
preparation (97% nominal purity), we produced cis-DCE
biologically from PCE using a Desulfitobacterium sp. culture.
The biogenic cis-DCE was readily utilized at high
concentrations by strain 195 indicating that cis-DCE was
not intrinsically inhibitory. Analysis of the commercially
synthesized cis-DCE by GC/mass spectrometry indicated the
presence of approximately 0.4% mol/mol chloroform.
Chloroform was found to be inhibitory to chloroethene
utilization by strain 195 and at least partially accounts for
the inhibitory activity of the synthetic cis-DCE. VC, a
human carcinogen that accumulates to a large extent in
cultures of strain 195, was not utilized as a growth substrate,
and cultures inoculated into medium with VC required a
growth substrate, such as PCE, for substantial VC
dechlorination. However, high concentrations of PCE or
TCE inhibited VC dechlorination. Use of a hexadecane phase
to keep the aqueous PCE concentration low in cultures
allowed simultaneous utilization of PCE and VC. At
contaminated sites in which “D. ethenogenes” or similar
organisms are present, biogenic cis-DCE should be readily
dechlorinated, chloroform as a co-contaminant may be
inhibitory, and concentrations of PCE and TCE, except perhaps
those near the source zone, should allow substantial VC
dechlorination.
1
,1-DCE, and 1,2-dichloroethane (DCA)). In this com-
munication, we examine more extensively the utilization of
this environmentally significant intermediate and demon-
strate that slower growth on cis-DCE is due to contaminants
in the commercial preparation.
When dechlorination proceeds past cis-DCE at a site,
accumulations of vinyl chloride (VC) are also undesirable
since VC is considered a human carcinogen (15). VC can be
utilized aerobically as a growth substrate by various micro-
organisms (16-18), and evidence for its oxidation under
anaerobic conditions in the presence of Fe3 (19) or humic
acids (20) has been obtained. VC can be reduced to ethene
and ethane under anaerobic conditions by a variety of mixed
cultures (14, 21-25) and in microcosms and field sites (10,
+
2
6, 27). When chloroethenes are utilized by strain 195 or
cultures containing strain 195, VC conversion to ethene (ETH)
was rate limiting and showed first-order kinetics (13, 28).
Moreover, its reductive dechlorination did not support growth
of strain 195 (29) so that VC utilization can be considered
cometabolic.
In this publication, we describe the utilization of these
two important substrates by “D. ethenogenes” strain 195 in
greater detail. We demonstrate that difficulties utilizing cis-
DCE by strain 195 were due to inhibitory impurities in the
commercially synthesized product, one of which is chloro-
form. We also demonstrate that VC utilization by strain 195
requires another chloroethene to serve as an electron
acceptor for growth; however, high concentrations of these
chloroethenes also inhibit VC utilization during the period
of their utilization.
Materials and Methods
Chem icals and Analyses of Chloroethenes. Chloroethenes
and other chemicals were purchased as described previously
(
24). The commercially produced cis-DCE, with a nominal
purity of 97%, was purchased from Aldrich Chemical Co.,
Milwaukee, WI. We were unable to find cis-DCE from another
supplier.
For quantitative analysis of chloroethenes and ETH,
headspace gas samples (100 µl) were analyzed using a
temperature programmed Perkin-Elmer 8500 gas chromato-
graph (GC) equipped with a flame ionization detector (FID).
The GC contained a 60m × 0.53 mm RTX-502.2 capillary
column operated in splitless injection mode (3 µm film
thickness) (Restek Corp., Bellefonte, PA). The carrier gas
utilized was helium at a flow of 10 mL/ min. Peak areas were
calculated using the software supplied with the GC and were
compared to standard curves. In some experiments, chlo-
roethenes were quantified using a Perkin-Elmer Autosystem
Introduction
The solvents tetrachloroethene (PCE) and trichloroethene
(
TCE) are major groundwater pollutants (1). At anaerobic
sites, these solvents can undergo reductive dechlorination
to less-chlorinated ethenes. At some sites reductive dechlo-
rination proceeds completely to ethene, while at others
*
Corresponding author phone: (607)255-2415; fax: (607)255-3904;
e-mail: shz1@cornell.edu.
‡
Present address: McKinsey & Company, Barcelona, Spain.
5
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 3, 2001
10.1021/es001285i CCC: $20.00

2001 Am erican Chem ical Society
Published on Web 12/23/2000

XL FID gas chromatograph fitted with a 2 m × 3 mm stainless
steel column packed with 60/ 80 mesh Carbopak B/ 1% SP-
1
2
000 (Supelco, Bellefonte, PA) and operated isothermally at
00 °C as described previously (13). For analyses of con-
taminants in cis-DCE, the column was maintained at 45 °C
for 8 min, increased at 8 °C per minute to 220 °C, and then
held at that temperature for 15 min.
For gas chromatographic/ mass spectrometric analyses
of contaminants in cis-DCE, a Hewlett-Packard Model 5890
Series II gas chromatograph equipped with a 30 m × 0.25
mm × 0.23 µm film thickness, HP-5 (5%phenylmethyl
silicone, Hewlett-Packard) fused silica capillary column
connected to a Hewlett-Packard 5971 quadrupole mass
selective detector was operated under the conditions previ-
ously described (30). The column oven was maintained at 40
°
C for 8 min after injection of a 0.1 mL headspace sample,
then increased by 8 °C per minute to 220 °C, at which it was
held for 15 min. Under these conditions, both chloroform
and cis-DCE had a retention time of 2.7 min.
Growth Medium and Culture Conditions. Cultures of
strain 195 were grown as previously described (29). Unless
otherwise stated, inoculum sizes were 2% v/ v, all incubations
were done in duplicate, and each experiment presented was
performed at least twice with similar results.
Solutions of PCE in hexadecane (HD) were prepared as
follows. HD was dispensed into 18 × 150 mm crimp-top
culture tubes (Bellco Glass, Vineland, NJ) and was purged
with a mixture of 70% N
were then sealed with Teflon-coated butyl rubber stoppers
Wheaton, Millville, NJ) and aluminum crimps and were
autoclaved at 121 °C for 30 min. PCE was purged with 70%
/ 30% CO for 5 min and was then added by filter
sterilization through a sterile Swinnex filter assembly fitted
with a 0.2 µm cellulose acetate filter (Millipore Inc., Woburn,
MA) as described by Holliger et al. (31). Cultures in 27 mL
tubes containing 9.25 mL of medium received 0.75 mL of
either a 2% v/ v (called “low”) or 5% v/ v (called “high”) PCE
dissolved in HD, equivalent to 14.7 or 37.5 mmol added per
liter of medium, respectively. Our initial studies indicated
that upon equilibration the aqueous concentration of PCE
was the equivalent of ca. 0.3 and 0.65 mmol PCE added per
liter and that VC was only sparingly soluble and ETH
practically insoluble in the HD phase. To accommodate the
2 2
/ 30% CO for 15 min. The tubes
(
FIGURE 1. Effects of different dosing schedules on product formation
from cis-DCE by strain 195. The doses of cis-DCE added to the
cultures were (a) 0.3, 0.5, and 0.7 mmol added per liter of culture
medium and (b) 0.15, 0.25, 0.35, and 0.50 mmol added per liter.
N
2
2
hexadecane than is PCE, was quantified in the culture
headspaces. Samples of the headspace were removed with
a sterile N -flushed syringe and were added to cultures of
2
strain 195 in 27 mL tubes containing 10 mL of medium to
achieve appropriate cis-DCE concentrations. Tubes not
inoculated with strain 195 and receiving PCE showed no
signs of dechlorination.
Results and Discussion
greatly increased amounts of PCE in cultures containing HD,
Utilization of Chem ically Synthesized cis-DCE by Strain
195. Cultures of strain 195, when transferred from medium
containing PCE as electron acceptor to medium containing
cis-DCE obtained from a commercial manufacturer, initially
utilized cis-DCE with increasing rates, indicative of growth,
but at much slower rates than other chloroethene electron
acceptors such as PCE, TCE, 1,1-DCE, or 1,2-dichloroethane
(29). When the dosing schedule typically used for PCE (24)
of ca. 0.3, 0.5, and 0.7 mmol cis-DCE added per liter of culture
medium was used (Figure 1a), the final dose was not
consumed, even after incubation for 8.5 days. When the doses
of cis-DCE added were reduced by 50% to ca. 0.15, 0.25, 0.35,
and 0.50 mmol added per liter (Figure 1b), the initial
performance of the culture was better. After 8.5 days from
the start of the experiments, the culture in Figure 1b produced
a total of 0.92 mmol VC per liter, nearly double the 0.52
mmol VC per liter produced by the culture in Figure 1a. Using
the dosing schedule in Figure 1b, cultures could be suc-
cessfully transferred in medium with cis-DCE at least three
times (data not presented).
-
increased amounts of H
2
and HCO
3
were added to respec-
tively serve as electron donor and increase buffer capacity.
Cultures were shaken at 150 rpm on a gyrotory shaker to
ensure adequate transfer of H and PCE between the three
2
phases. The amounts of VC and ETH in these cultures were
corrected for the absorption in the HD phase through use
of calibration curves prepared with the appropriate amounts
of HD and medium present.
Production of Biogenic cis-DCE. Biogenic cis-DCE was
prepared using a culture isolated in this laboratory, called
Desulfitobacterium strain DCE, which was isolated from a
contaminated site at Plattsburgh Air Force Base, NY. This
culture reduced PCE to cis-DCE (>99% of the products
detected). We determined ca. 1000 bases of its 16S rDNA
sequence (data not presented) as previously described (13),
and sequence analysis showed that it is a member of the
genus Desulfitobacterium. Its cultural characteristics re-
semble those of the recently described Desulfitobacterium
frappieri sp. strain PCE (7). Cultures of Desulfitobacterium
DCE were grown in 120 mL serum vials containing 60 mL of
basal mineral medium used for strain 195 without any of the
other nutrient supplements and were provided with 7 g/ L
sodium lactate as the electron donor and 1 mL 10% v/ v PCE
dissolved in hexadecane as previously described (31). After
consumption of the PCE by the culture (lactate was present
in excess), cis-DCE, which is considerably less soluble in
Utilization of PCE and Other Chloroethenes by Cultures
Grown on Chem ically Synthetic cis-DCE. We have previously
shown that cultures of strain 195 grown on TCE, 1,1-DCE,
and 1,2-dichloroethane are capable of immediately using
PCE (29), indicating that PCE dechlorination is constitutive
when grown on those substrates. We examined the ability of
cis-DCE-grown cells of strain 195 to dechlorinate chloro-
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FIGURE 2. Utilization of PCE by cells grown on cis-DCE.
ethenes containing an equal or higher number of chlorines
after having consumed several doses of cis-DCE. Strain 195
was able to dechlorinate cis-DCE, 1,1-DCE, and TCE without
any lag as has been previously demonstrated for other
substrates (29). PCE, in contrast to the other chloroethenes
tested, was not dechlorinated, even 32 days after it was added
to the culture (Figure 2). Cultures grown on cis-DCE would
not utilize a dose of PCE in several repetitions of this
experiment, nor could they be transferred to fresh growth
medium containing PCE as the electron acceptor. However,
cultures transferred one time to a medium containing either
FIGURE 3. Utilization of synthetic or biogenic cis-DCE by strain 195.
Vinyl chloride was the primary product of reductive dechlorination
in these cultures. Data points represent the average of duplicate
tubes.
1
,1-DCE or TCE regained the ability to utilize PCE after
receiving two doses of the alternate substrate.
Magnuson et al. (32) have shown that mixed cultures
containing strain 195 contain a PCE reductive dehalogenase
which dechlorinates PCE to TCE, and a TCE reductive
dehalogenase, which dechlorinates TCE, cis-DCE, 1,1-DCE
to VC, and VC slowly to ETH. Preliminary results indicate
that these two enzyme are present in strain 195. These results
are consistent with greater inhibition of the PCE dehalogenase
by the inhibitory factor(s) in cis-DCE than TCE dehalogenase.
Utilization of Biogenic cis-DCE by Strain 195. The slow
utilization of commercially prepared cis-DCE, especially at
high concentrations, indicated toxicity of this preparation to
strain 195. However, it was not clear whether it was the cis-
DCE itself or an impurity in the preparation we used that
was responsible for the toxicity. Initial studies indicated that
gaseous cis-DCE derived from the headspace of a vial
containing the neat liquid was as inhibitory to growth as was
the liquid itself (data not presented), indicating that the toxic
agent was either cis-DCE or a volatile contaminant.
We decided to examine the toxicity of cis-DCE further by
producing cis-DCE biologically using a culture, called De-
sulfitobacterium DCE, which reductively dechlorinated PCE
stoichiometrically to cis-DCE (see Materials and Methods).
As shown in Figure 3, a culture of strain 195 transferred to
medium containing 0.14 or 0.65 mmol/ L biogenic cis-DCE
converted it nearly stoichiometrically to VC within a few days,
whereas cultures supplied with synthetic cis-DCE did not
convert it in that time. Cultures grown on biogenic cis-DCE
retained the ability to utilize PCE (data not presented).
Thus, cis-DCE is not intrinsically inhibitory, since biogenic
cis-DCE was readily utilized by strain 195. There is other
evidence of toxicity of synthetic cis-DCE to chloroethene-
dechlorinating cultures. There is evidence for cis-DCE toxicity
to an anaerobic enrichment culture that reductively dechlo-
rinated cis-DCE and VC (14), as demonstrated lack of toxicity
of a biogenic cis-DCE preparation (P. L. McCarty, personal
communication). This culture had properties different from
those of strain 195 in that it could not use PCE or TCE and
used VC more rapidly than does strain 195 (14). Neumann
et al. (33) noted a 50% inhibition of a PCE reductive
dehalogenase from Dehalospirillum multivorans by 14 mM
cis-DCE. Since cis-DCE has not found widespread industrial
FIGURE 4. Analyses of synthetic and biogenic cis-DCE. Gas
chromatograms of biogenic cis-DCE (a), synthetic cis-DCE (b), and
chloroform (c). Analyses were performed as described in the
Materials and Methods. Mass spectral analysis (d) of the gas
chromatographic peak eluting at 2.7 min (see Materials and Methods)
obtained from a sample containing synthetic cis-DCE. Both cis-DCE
and chloroform elute at this time.
use, it is generally considered to be biogenic rather than
anthropogenic at contaminated sites. Therefore, it is unlikely
that this inhibition phenomenon is relevant to those sites.
Analysis of Synthetic and Biogenic cis-DCE and Chlo-
roform Inhibition of Dechlorination. Figure 4a-b shows gas
chromatograms obtained for synthetic cis-DCE and biogenic
cis-DCE at low attenuation so that the cis-DCE peak eluting
near 13.5 min was offscale and small peaks representing
impurities are emphasized. The chromatogram for biogenic
cis-DCE (Figure 4a) showed peaks that comigrated with 1,1-
DCE and with TCE, the former being a minor sideproduct
the latter an intermediate in PCE dechlorination to cis-DCE
by strain DCE. The chromatogram for synthetic cis-DCE
(Figure 4b) showed several small peaks, including ones
comigrating with 1,1-DCE and TCE, and more prominent
peaks eluting immediately before cis-DCE and after cis-DCE
as a shoulder. The peak eluting before cis-DCE as well as the
other small peaks did not comigrate with any compound
tested, including dichloromethane, 1,1-dichloroethane, 1,1,1-
5
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trichloroethane, or carbon tetrachloride. The peak that
migrated as a shoulder on the cis-DCE comigrated with
chloroform (Figure 4c).
We were unable to obtain better resolution between cis-
DCE and chloroform using a RTX-502.2 capillary column
(
see Materials and Methods) which is designed for use with
chlorinated solvents and provides excellent resolution be-
tween the three DCE isomers. A perusal of sample chro-
matograms for various GC columns designed to separate
aliphatic chlorinated organic compounds did not reveal any
columns in which chloroform did not elute almost im-
mediately after cis-DCE, thereby making it making it difficult
to quantify chloroform in the presence of large amounts of
cis-DCE. The boiling points of cis-DCE and chloroform are
similar (60.7 °C and 61.7 °C, respectively (34)) and their
Henry’s Law constants are 0.167 and 0.150 at 24.8 °C (35).
Thus, their similar boiling points and hydrophobicity may
partially explain the difficulty in resolving them. Analysis of
the synthetic cis-DCE preparation indicated that if the peak
was indeed chloroform, it would represent ca. 0.4% of the
amount of cis-DCE in the sample.
We also examined the synthetic cis-DCE preparation by
gas-chromatography/ mass spectroscopy. Chloroform and
cis-DCE coeluted at 2.7 min from the column available for
use with this instrument (see Materials and Methods), but
analysis of the peak at 2.7 min from synthetic cis-DCE showed
that ions with m/ z values of 83, 85, and 87 were present
(
Figure 4d) which were not found in biogenic cis-DCE (data
not presented) or the database mass spectrum for cis-DCE.
These three mass values represent the fragment produced
FIGURE 5. VC dechlorination to ETH by strain 195 in the absence
(a) and presence (b) of PCE at the time of inoculation. After 30 days
of incubation, three consecutive increasing doses of PCE (0.3, 0.5,
and 0.7 mmol/L) were added to the culture in (a).
by removal of a chlorine atom from chloroform to form
+
CHCl
2
. This fragment forms a triplet in mass spectrograms
3
5
37
because it can contain either Cl or Cl (natural ratio ≈ 3:1
3
5
+
2
(
34)) and therefore each fragment can consist of CH Cl ,
3
5
37
+
37
+
2
CH Cl Cl , or CH Cl
in order of abundance. The ratio
roethenes. Studies in J. M. Gossett’s laboratory (36) on a
mixed PCE-dechlorinating anaerobic culture showed that
ca. 1 mg/ L aqueous concentration (ca. 8.4 µM) of chloroform
led to complete inhibition of PCE dechlorination within a
day, an inhibition similar to that which we detected in our
culture (Figure 4). This similar inhibition is not surprising
since the culture studied was derived from the one from
which strain 195 was isolated. Chlorinated methanes were
inhibitory to a PCE reductive dehalogenase from D. multi-
vorans, with chloroform (50% inhibition at 0.25 µM) and
chloromethane (50% inhibition by 0.8 µM) being the most
inhibitory (33). This same enzyme was also inhibited by high
concentrations of cis-DCE, which was presumably com-
mercially synthesized. Chloroform is known to be a potent
inhibitor of methanogenesis (37, 38) and is believed to
interfere with corrinoid function. The presence of corrinoids
has been demonstrated or implicated in chloroethene
reductive dehalogenases (32, 39, 40).
Thus, the inhibition of reductive dechlorination of chlo-
roethenes by chloroform may be a general phenomenon that
may be relevant to sites at which chloroform is a co-
contaminant with chloroethenes. These problems may be
obviated by the breakdown of chloroform at those sites. At
this time, the only process known for anaerobic breakdown
of chloroform is a slow cometabolic dehalogenation by
anaerobic respirers such as methanogens, acetogens, and
sulfate reducers (37, 41, 42). Indeed recent studies on a high-
rate PCE-dechlorinating enrichment culture (43) in which
molecular biological techniques indicated the presence of
an organism closely related to strain 195 showed that carbon
tetrachloride was reductively dechlorinated to chloroform,
which was subsequently transformed to dichloromethane.
There was considerable inhibition of reductive dechlorination
of chloroethenes in this culture.
between these peaks was 8.9/ 5.9/ 1.0 in the synthetic cis-
DCE preparation, whereas their ratios were 9.5/ 6.1/ 1.0 and
9
.0/ 5.9/ 1.0 in mass spectra from authentic chloroform and
the database values for chloroform, respectively.
A set of small peaks from chloroform centered near m/ z
+
1
18 representing the fragment CCl
3
were also visible in the
spectrum from the synthetic cis-DCE (Figure 4d) but not in
the biogenic material, while peaks from chloroform near m/ z
4
7 were obscured by the cis-DCE peaks in that region.
Although this analysis was not quantitative, it did verify the
presence of a component in the synthetic cis-DCE with a
mass spectrum essentially identical with chloroform. The
+
fragm ent CHCl
2
could also be derived from dichlo-
romethane, but previous gas chromatographic analysis
Figure 4b) demonstrated that this compound was not
present.
We examined whether adding chloroform to biogenic cis-
(
DCE in amounts comparable to the amount found in the
synthetic preparation was toxic to growth of strain 195. We
found that 1.6 µM, about double the amount of chloroform
as that estimated a completely inhibitory dose of synthetic
cis-DCE, was required for complete inhibition of cis-DCE
dechlorination (data not presented). Gas chromatographic
analysis (Figure 4b) of the synthetic cis-DCE preparation
indicates the presence of other compounds which could also
be toxic. Also unclear was the fate of small amounts of
chloroform in the cultures. Preliminary studies (not pre-
sented) indicate little or no chloroform absorption into the
stopper, but it is not clear whether chloroform is metabolized
by strain 195. No dichloromethane was found in gas
chromatographic traces of cultures exposed to chloroform.
Whether or not chloroform is completely responsible for
the inhibition of reductive dechlorination in strain 195 by
cis-DCE, it is a potent inhibitor. There is other evidence for
chloroform inhibition of reductive dechlorination of chlo-
ETH Production from VC in Inoculated Cultures. As
shown in Figure 5a, during 30 days of incubation with ca. 0.4
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FIGURE 7. Total ETH (a) and VC (b) produced and aqueous levels
of PCE (a) by strain 195 grown in media with a hexadecane (HD)
phase. PCE was added differently to cultures in a HD phase either
at “low” (total of 14.7 mmol/L PCE added) or “high” (total of 36.7
mmol/L PCE added) concentrations (see Materials and Methods).
Also included are cultures without HD (4 doses; total of 2.4 mmol/L
PCE added).
FIGURE 6. ETH formation in the presence or absence of PCE during
growth. Cultures were either fed a new dose of PCE before the
previous one had been consumed (a) or were allowed 2 days to
dechlorinate VC after each PCE dose had been consumed (b).
mmol VC added per liter of growth medium, there was a
slow conversion of VC to ETH. The slightly more rapid VC
disappearance versus ETH appearance was attributed to
absorption into the stopper as indicated by uninoculated
controls (data not presented). Figure 5c shows this ETH
appearance with greater sensitivity and demonstrates that
there was a gradual decrease in the rate of ETH appearance
during the first 30 days. A dose of 0.3 mmol/ l PCE added at
Day 30 was utilized in 14 days, but with only a slight
accumulation of dechlorination products (mainly VC), with
the decrease in PCE concentration attributed mainly to its
absorption into the stopper. Two subsequent PCE doses were
consumed at increasing rates, following which there was
significant ETH accumulation.
As shown in Figure 5b, when a dose of PCE was added
together with VC at the time of inoculation, PCE was
metabolized to VC by Day 4, and, and once the PCE was
depleted, there was a much more rapid accumulation of ETH
than when VC was added in the absence of PCE. Thus VC
dechlorination by strain 195 does not support growth, follows
first-order kinetics (13, 28), and requires the addition of a
metabolically useful substrate, such as PCE, for sufficient
growth to occur to allow significant VC dechlorination. These
are hallmarks of a cometabolic process. It is not clear why
strain 195 cannot conserve energy from VC dechlorination,
since it is as thermodynamically favorable as dechlorination
of the other chloroethenes (44). There is evidence that other
organisms are capable of conserving energy from reductive
dechlorination of VC to ETH (14, 21), but the microorganisms
in these mixed cultures are as yet poorly characterized.
Inhibition of VC dechlorination to ETH by PCE. Figure
6
b), ETH was produced almost exclusively during the periods
when PCE was absent from the medium. Cultures in which
days were allowed for VC dechlorination after PCE was
4
consumed only led to about 20% more ETH produced from
VC than those shown in Figure 6b (data not presented).
The other chloroethenes that strain 195 could use as
electron acceptors, TCE, cis-DCE, and 1,1-DCE, had similar
inhibitory effects on VC dechlorination to ETH (data not
presented). These results are similar to those for the mixed
culture containing strain 195 (28) in which all other chlo-
roethenes except trans-DCE were inhibitory to VC dechlo-
rination. It cannot be determined whether this inhibition is
direct, i.e., binding to the same site on an enzyme or cofactor,
2
or perhaps a competition for electrons derived from H .
Recent evidence in our laboratory (Nijenhuis and Zinder,
unpublished) has shown that dechlorination rates in whole
cells were much higher when the artificial electron donor
reduced methyl viologen was used as than when H
2
was
utilized, suggesting that electron flow from H
limiting in these cells.
2
was rate
VC Dechlorination to ETH in Cultures Grown with PCE
Dissolved in Hexadecane. To examine further the interaction
between the presence of PCE and VC dechlorination to ETH,
we introduced a hexadecane (HD) phase into our cultures.
PCE is highly soluble in hexadecane, and such a method is
a useful way to add large amounts of PCE while maintaining
a low aqueous concentration (31). Preliminary studies showed
that similar to results with Dehalobacter restrictus (31), strain
195 grew well when PCE was dissolved in a HD phase.
Figure 7 shows a comparison for the total amounts of VC
and ETH produced by cultures to which two different
concentrations of PCE dissolved in HD had been added
(called “low” and “high” see Materials and Methods) as well
as a control culture to which 2.4 mmol/ L of PCE was added
in four doses, not allowing the PCE concentration to reach
6
a demonstrates that in a culture in which four doses of PCE
were added in rapid succession without allowing PCE to be
depleted to concentrations below 1 mmol per liter, ETH only
accumulated slightly until the final dose of PCE was
consumed, after which there was significant ETH accumula-
tion. In cultures in which an additional dose of PCE was
added 2 days after the previous dose was consumed (Figure
5
2 0
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zero, as in Figure 6a. Cultures receiving the “low” PCE/ HD
solution produced the same amount of VC as controls without
HD during the first 7 days and somewhat more VC than
cultures amended with the “high” PCE/ HD solution. Sig-
nificantly, cultures receiving the “low” PCE/ HD solution, in
which the PCE concentration was maintained below 0.3
mmol/ L, produced about 4-fold more ETH than the cultures
amended with the “high” PCE/ HD solution. ETH began
accumulating by Day 6 in the “low” PCE cultures, long before
the concentration of the PCE decreased significantly. It is
unlikely that the actual aqueous concentrations were much
lower than those measured in the culture headspaces at this
time since the cultures were vigorously agitated and the rate
of PCE utilization was still slow at this time. By Day 20, product
formation stopped in all cultures, presumably due to nutrient
depletion or possibly buildup of toxic products.
Thus, the interaction between VC utilization and that of
other chloroethenes used for dehalorespiration by strain 195
is complex. Since strain 195 cannot grow using VC, it needs
an electron acceptor it can use for dehalorespiration and
growth such as PCE to reach high enough cell densities to
cause appreciable VC utilization. However, at the same time,
high concentrations of these chloroethenes inhibit VC
conversion to ETH although the mechanism is unclear. At
chloroethene-contaminated sites at which reductive dechlo-
rination is occurring, VC is produced biogenically from more
highly chlorinated chloroethenes, so that there should be
sufficient growth of D. ethenogenes or similar organisms. It
is also unlikely that the concentrations of these chloroethenes
would exceed 600 µM or higher needed to fully inhibit VC
dechlorination (Figure 6), except perhaps immediately at
the source zone. Moreover, there may be organisms present
more proficient at using VC than D. ethenogenes (14, 21) so
that the prospects for complete dechlorination of chloro-
ethenes at most contaminated sites with appropriate electron
donors and organisms would be good.
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Supported by the U. S. Air Force Armstrong Laboratory,
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Florida and the “La Caixa” Foundation, Catalonia, Spain (X.
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Received for review May 22, 2000. Revised manuscript re-
ceived October 17, 2000. Accepted November 8, 2000.
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