Cometabolism of cis-1,2-dichloroethene by aerobic cultures grown on vinyl chloride as the primary substrate.

Article abstract of DOI:10.1021/es011220v

An aerobic enrichment culture was grown on vinyl chloride (VC) as the sole source of carbon and energy. In the absence of VC, the enrichment culture cometabolized cis-1,2-dichloroethene (cDCE) and, to a lesser extent, trans1,2-dichloroethene (tDCE), beginning with oxidation to the corresponding DCE-epoxides. When provided with VC (1.3 mM) and cDCE (0.2-0.3 mM), the enrichment culture cometabolized repeated additions of cDCE for over 85 days. Cometabolism of repeated additions of tDCE was also demonstrated but at a lower ratio of nongrowth substrate to VC. VC-grown Pseudomonas aeruginosa MF1 (previously isolated from the enrichment culture) also readily cometabolizes cDCE, with an observed transformation capacity (Tc,obs) of 0.82 micromol of cDCE/mg of total suspended solids (TSS). When provided with VC and cDCE, MF1 did not begin cometabolizing cDCE until nearly all of the VC was consumed. The presence of cDCE reduces the maximum specific rate of VC utilization. A kinetic model was developed that describes these phenomena via Monod parameters for substrate and nongrowth substrate, plus inactivation and inhibition coefficients. MF1 did not show any cometabolic activity on tDCE or trichloroethene and very limited activity on 1,1-DCE (Tc,obs = 2 x 10(-5) micromol/mg TSS). Above 40 microM, tDCE and TCE noticeably increased the maximum specific rate of VC utilization, even though neither compound was consumed during or after VC consumption. High concentrations of 1,1-DCE (950 microM) completely inhibited VC biodegradation. As there is currently no evidence for aerobic biodegradation of cDCE as a sole source of carbon and energy, the results of this study provide a potential explanation for in situ disappearance of cDCE when the only other significant substrate available is VC. It is fortuitous that the VC-grown cultures tested exhibit their highest cometabolic activity toward cDCE, because it is the predominant DCE isomer formed during anaerobic reductive dechlorination of trichloroethene and tetrachloroethene.

Full text of DOI:10.1021/es011220v

Environ. Sci. Technol. 2002, 36, 2171-2177
Introduction
Cometabolism of
Anaerobic reductive dechlorination of tetrachloroethene and
trichloroethene (TCE) often leads to accumulation of cis-
cis-1,2-Dichloroethene by Aerobic
Cultures Grown on Vinyl Chloride as
the Primary Substrate
1
,2-dichloroethene (cDCE) and vinyl chloride (VC). VC
presents the greatest concern because it is a known car-
cinogen. As contaminated groundwater containing cDCE and
VC migrates away from a source area, electron-acceptor
conditions may transition from anaerobic to aerobic, due to
either engineered or naturally occurring conditions. Disap-
pearance of cDCE and VC at the aerobic fringe of contaminant
plumes has been documented in field (1, 2) and microcosm
studies (3-5) of natural attenuation. Currently, several
explanations exist for this observation. First, methane gener-
ated in the anaerobic source area that migrates with (or
somewhat ahead of) the daughter products can be used as
a primary substrate for the cometabolism of VC and cDCE
under aerobic conditions downgradient. Both VC and cDCE
are readily consumed by methanotrophs (6-13). Second,
ethene and ethane formed from reductive dechlorination of
VC in the anaerobic zone can be used as primary substrates
for the cometabolism of cDCE (14) and VC (14-18) under
aerobic conditions. Third, VC can be degraded by aerobes
that use it as a growth substrate (19-21). No organisms have
been isolated thus far with the ability to use cDCE as a growth
substrate.
Another possible explanation for disappearance of VC
and cDCE at the aerobic fringe of a contaminant plume is
cometabolism of cDCE by organisms using VC as a primary
substrate. Mycobacterium aurum L1 was the first VC-grown
organism reported to cometabolize DCEs (20). Initial oxida-
tion rates of cDCE, trans-1,2-dichloroethene (tDCE), and 1,1-
dichloroethene (1,1-DCE) were 50-80% lower than the initial
VC oxidation rate. However, the apparent ability ofM. aurum
L1 to cometabolize DCEs is somewhat obscured by the
statement that degradation of polychlorinated ethenes in
the absence of VC was “not significant” (20). Furthermore,
cDCE cometabolism in the presence of VC was not char-
acterized.
M A T T H E W F . V E R C E , †
C L A U D I A K . G U N S C H ,
A N T H O N Y S . D A N K O , A N D
D A V I D L . F R E E D M A N *
‡
Department of Environmental Engineering and Science,
Clemson University, Clemson, South Carolina 29634
An aerobic enrichment culture was grown on vinyl
chloride (VC) as the sole source of carbon and energy. In
the absence of VC, the enrichment culture cometabolized
cis-1,2-dichloroethene (cDCE) and, to a lesser extent, trans-
1
,2-dichloroethene (tDCE), beginning with oxidation to
the corresponding DCE-epoxides. When provided with VC
1.3 mM) and cDCE (0.2-0.3 mM), the enrichment culture
(
cometabolized repeated additions of cDCE for over 85 days.
Cometabolism of repeated additions of tDCE was also
demonstrated but at a lower ratio of nongrowth substrate
to VC. VC-grown Pseudomonas aeruginosa MF1 (previously
isolated from the enrichment culture) also readily
cometabolizes cDCE, with an observed transformation
capacity (Tc,obs) of 0.82 µmol of cDCE/mg of total suspended
solids (TSS). When provided with VC and cDCE, MF1 did
not begin cometabolizing cDCE until nearly all of the VC was
consumed. The presence of cDCE reduces the maximum
specific rate of VC utilization. A kinetic model was developed
that describes these phenomena via Monod parameters
for substrate and nongrowth substrate, plus inactivation and
inhibition coefficients. MF1 did not show any cometabolic
activity on tDCE or trichloroethene and very limited
activity on 1,1-DCE (Tc,obs ) 2 × 10 µmol/mg TSS).
Above 40 µM, tDCE and TCE noticeably increased the
maximum specific rate of VC utilization, even though neither
compound was consumed during or after VC consumption.
High concentrations of 1,1-DCE (950 µM) completely
inhibited VC biodegradation. As there is currently no
evidence for aerobic biodegradation of cDCE as a sole
source of carbon and energy, the results of this study provide
a potential explanation for in situ disappearance of
cDCE when the only other significant substrate available
is VC. It is fortuitous that the VC-grown cultures tested exhibit
their highest cometabolic activity toward cDCE, because
it is the predominant DCE isomer formed during anaerobic
reductive dechlorination of trichloroethene and tetrachlo-
roethene.
The objective of this study was to evaluate the ability of
VC-grown cultures to transform cDCE, tDCE, 1,1-DCE, and
TCE either as growth substrates or as nongrowth substrates
following growth on VC. Preliminary tests were conducted
with a VC-grown enrichment culture, followed by similar
tests with a pure culture of strain MF1 that was isolated from
the enrichment culture. The effect of polychlorinated ethenes
on the kinetics of VC utilization by MF1 were also evaluated.
-
5
Materials and Methods
Chem icals and Medium . VC gas (99.5%, containing <0.5%
phenol to inhibit polymerization), cDCE (97%), tDCE (98%),
and 1,1-DCE (99%) were obtained from Aldrich. TCE (99.5%)
was obtained from Fisher. All other chemicals used were of
reagent grade. cDCE, tDCE, 1,1-DCE, and TCE were added
as either neat compounds or saturated water solutions. The
minimal salts medium (MSM) described by Hartmans et al.
(
22) was used but with the amount of (NH
4 2 4
) SO reduced to
0
.67 g/ L. No vitamins or other complex growth factors were
added to the MSM. cDCE- and tDCE-epoxide were chemically
synthesized by reacting the parent compounds (1.1 g) with
3
-chloroperoxybenzoic acid (1.6 g) dissolved in chloroform-d
*
Corresponding author phone: (864) 656-5566; fax: (864) 656-
(6 mL) and heating (50-60 °C) for 8-12 h (23).
0
672; e-mail: dfreedm@clemson.edu.
Analytical Methods. Consumption of VC, DCEs, and TCE
was monitored by gas chromatographic analysis of headspace
samples (0.1-mL sample), as previously described (21, 24).
The gas chromatograph (GC) response was calibrated to give
†
Present address: Environmental Restoration Division, Lawrence
Livermore National Laboratory, Livermore, CA 94550.
‡
Present address: Department of Civil Engineering, University of
Texas, Austin, TX 78712.
1
0.1021/es011220v CCC: $22.00

2002 Am erican Chem ical Society
VOL. 36, NO. 10, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2 1 7 1
Published on Web 04/02/2002

the total mass of the compound in that bottle (25). Assuming
the headspace and aqueous phases were in equilibrium, the
total mass present was converted to an aqueous-phase
concentration
and absence of growth substrate (VC). For a completely mixed
batch system, the coupled differential equations for substrate
and nongrowth substrate use, respectively, are
dS
dt
C0
S
C
KC
-
) k - k
X
(2)
M
S
S,C(C0 + KI))
(
C )
(1)
l
K 1 +
+ S
V + H V
(
S
)
l
c
g
(
)
where C
total mass present (µmol/ bottle); V
in the bottle (L); V ) volume of the headspace in the bottle
l
) concentration in the aqueous phase (µM); M )
dC
dt
-
)
l
) volume of the liquid
g
KS
C
-3
(
(
L); and H
c
) Henry’s constant ((mol‚m gas concentration)/
mol‚m aqueous concentration)), calculated for 23 °C (25).
Aqueous-phase detection limits ranged from 10 nM for 1,1-
DCE to 50 nM for cDCE. The validity of assuming equilibrium
between headspace and aqueous phases was verified during
kinetic experiments (see the following discussion).
(k ′ - k
(C - C))
X (3)
C
inact
0
(
)
K + S
S
-
3
S
KC 1 +
+ C
(
)
(
KS)
where S is the growth substrate concentration (µM); k
maximum specific growth substrate utilization rate (µmol of
S
is the
cDCE- and tDCE-epoxide were identified by analysis of
headspace samples with a GC/ mass spectrometer (MS)
system, consisting of a Hewlett-Packard 5890 GC with a 60-m
substrate/ mg of TSS/ d); K
coefficient for growth substrate (µM); C is the nongrowth
substrate concentration (µM); C is the initial nongrowth
substrate concentration (µM); k ′ is the theoretical maximum
specific nongrowth substrate utilization rate (µmol of non-
growth substrate/ mg of TSS/ d); K is the Monod half-
saturation coefficient for nongrowth substrate (µM); kS,C is
the specific rate of substrate inactivation (µmol of nongrowth
I
substrate/ mg of TSS/ d); K is a nongrowth substrate inhibition
coefficient (µM); kinact is the specific rate of nongrowth
substrate inactivation (L/ mg TSS/ d); and X is the biomass
concentration (mg of TSS/ L). The increase in biomass
concentration during kinetic experiments was estimated to
be less than 2%, permitting the use of initial biomass
S
is the Monod half-saturation
0
1
-µm DB-5 column, a Ruska Instruments thermal desorption
C
trap for kryofocusing of samples, and a 5988 Hewlett-Packard
MS. Confirmation of epoxide formation by the VC-grown
enrichment culture exposed to cDCE and tDCE involved
purging the headspace of a serum bottle with nitrogen
through a solution containing 0.094M 4-(4-nitrobenzyl)-
pyridine in ethylene glycol. Aliquots of the solution were
then combined with 0.1 M Tris-HCl buffer and acetone,
heated at 37 °C, and analyzed in a scanning UV/ visible
spectrophotometer. The 4-(4-nitrobenzyl)pyridine and ep-
oxide reaction product absorbs at 550-570 nm (26). Chemi-
cally synthesized cDCE- and tDCE-epoxide were used to
validate the GC/ MS and 4-(4-nitrobenzyl)pyridine proce-
dures.
C
0
concentrations (X ) in place of X in eqs 2 and 3.
Parameter estimates were obtained with a stepwise
approach described by Chang and Criddle (28). Parameters
Standard methods (27) were used to determine total
suspended solids (TSS).
k
S
and K
only VC (21). Parameters k
simultaneously by fitting eq 3 to depletion data from cultures
fed only cDCE. Parameters kS,C and K were obtained
S
were determined previously in experiments using
C
′, K , and kinact were obtained
C
Culture Maintenance and Kinetic Experim ents. A VC-
grown enrichment culture (18) was maintained in a 2.5-L
glass bottle (Bellco Biotechnology) capped with a gray butyl
rubber septum (30-mm), held in place with a screw cap.
Maintenance consisted of pH adjustment (7.0 ( 0.1) with 8
M NaOH, purging the headspace with oxygen, and adding
VC (100 mL) every 3-5 days. After the consumption of the
VC, aliquots were periodically removed and replaced with
MSM. Samples (100 mL) were placed in serum bottles (160
mL) to evaluate cDCE and tDCE biodegradation in the
presence and absence of VC. The bottles were capped with
Teflon-faced rubber septa. Autoclaved controls (121 °C for
I
simultaneously by fitting eqs 2-3 to depletion curves from
additional cultures fed VC plus cDCE. The effect of mass
transfer on parameter estimation was evaluated with a
previously described method (29), by comparing solutions
of eqs 2-3 to solutions of similar equations that include
mass transfer. For example, the impact of mass transfer on
k ′, K , and kinact was evaluated by comparing the solution
C C
to eq 3 without growth substrate present to the following
equation:
1
5 min) and water controls (WC) were used to evaluate abiotic
losses.
Strain MF1 (isolated from the aforementioned enrichment
dC
dt
C
-
) (k ′ - k
(C - C))
X - K a(C - Cact)
L
C
inact
0
(
)
K + C
C
(4)
culture) was grown in a VC-fed reactor operated in a
semicontinuous draw-and-fill batch mode, as previously
described (21). The VC provided contains a trace level of
phenol (see previous discussion), but MF1 is unable to use
phenol as a sole carbon and energy source (21). The slow
growth rate of MF1 necessitated that the reactor be run at
a relatively long retention time (140 days). The TSS con-
centration in the reactor averaged 162 mg/ L. After the
consumption of VC, reactor effluent samples (25 mL) were
distributed to serum bottles (70 mL) for the kinetic experi-
ments with cDCE, tDCE, 1,1-DCE, and TCE in the presence
and absence of VC. Bottles were agitated on a gyratory shaker
table (150 rpm) between headspace sampling.
The endogenous decay coefficient for cometabolic activity
c
(b ) was determined for cDCE in the same manner described
by Chang and Criddle (28) (details provided in Supporting
Information).
Kinetic Modeling. A model was developed to describe
the ability of MF1 to cometabolize cDCE, both in the presence
where K a is the mass transfer coefficient for cDCE (17.6 (
L
-
1
2.47 h ) and Cact is the actual liquid-phase concentration of
cDCE experienced by the culture (µM). K a for VC was
L
measured previously (21). A similar analysis was performed
to assess the impact of mass transfer on estimation of
parameters kS,C and K . In all cases, solutions including mass
I
transfer became indistinguishable from solutions assuming
equilibrium before the first data points for VC and cDCE
were collected, indicating that mass transfer did not affect
the estimation of parameters.
Fitting of model equations to substrate depletion data
was performed numerically using the software Aquasim 2.0
(30). Fitting was done in a simultaneous manner, in which
all available depletion curves were fitted together to obtain
one set of parameters, rather than fitting each curve
individually and reporting an arithmetic average. A reiterative
weighting procedure (31) was used to obtain an adequate fit
of the cDCE-only data. Standard deviations of parameter
2
1 7 2
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 10, 2002

FIGURE 2. Sustained cometabolism of cDCE (a) and tDCE (b) by the
VC-grown enrichment culture when VC is simultaneously provided
as the growth substrate. Results for one bottle are shown in each
panel; duplicates behaved similarly.
FIGURE 1. Cometabolism of cDCE (a) and tDCE (b) by the VC-grown
enrichment culture. A standard was not available for quantification
of the cDCE-epoxide. Error bars represent one standard deviation
of replicate bottles.
estimates and correlation matrix elements were also com-
puted by Aquasim.
of the cDCE was consumed, headspace samples were
analyzed by GC/ MS. The resulting spectra matched what
has been reported for chemically synthesized and biologically
produced cDCE-epoxide (32). Further confirmation of cDCE-
epoxide in the headspace of the serum bottle was obtained
by trapping the contents in 4(4-nitrobenzyl)pyridine. The
reaction product absorbed at 550-570 nm, consistent with
chemically synthesized cDCE-epoxide.
Results
Enrichm ent Culture. The enrichment culture was main-
tained on VC as a sole source of carbon and energy for over
3
years prior to the present experiments. Samples were
distributed to duplicate serum bottles and fed either cDCE
or tDCE. Subcultures fed cDCE rapidly consumed one
addition but did not completely consume a second (Figure
tDCE-epoxide was not detectable when low amounts of
tDCE were transformed by the enrichment culture (Figure
1
b). However, when a high biomass concentration was used
1
a). By comparison, the enrichment culture transformed a
along with a high initial concentration of tDCE (as described
previously in Results for cDCE), formation of tDCE-epoxide
was confirmed by GC/ MS and the headspace trapping
procedure with 4(4-nitrobenzyl)pyridine.
much smaller amount of tDCE (Figure 1b). When higher initial
amounts of cDCE and tDCE were added than shown in Figure
1
, similar amounts of both compounds were consumed,
leaving a residual amount of cDCE and tDCE in the bottles
data not shown). The autoclave controls demonstrated that
(
Simultaneous addition of cDCE and VC was also exam-
ined. The enrichment culture consumed repeated additions
of both compounds (Figure 2a). Over an 85 day period, 143
µmol of cDCE and 1420 µmol of VC were biodegraded.
Autoclaved controls indicated minimal loss of VC with the
Teflon-faced septa (8.0% in 38 days). Experiments with
simultaneous additions of VC and tDCE yielded similar
results, although a much lower amount of tDCE was
consumed relative to VC (Figure 2b). Over a 24 day period,
2.17 µmol of tDCE and 913 µmol of VC were biodegraded.
These results demonstrated the potential to sustain cDCE
and tDCE cometabolism when the enrichment culture was
maintained on VC as a growth substrate.
the disappearance of cDCE and tDCE was a biotic process
and that the Teflon-faced rubber septa adequately retained
cDCE and tDCE. The enrichment culture did not transform
1
,1-DCE (130 µM) or TCE (170 µM) after 13 days, as compared
to abiotic losses (data not shown).
Concurrent with cDCE biodegradation was accumulation
of a volatile compound, subsequently identified as cDCE-
epoxide. Identification required a much higher headspace
concentration than that shown in Figure 1a. This was achieved
by growing the enrichment culture on VC in a 2.5-L bottle
to a biomass concentration of approximately 740 mg of TSS/
L, centrifuging the biomass (20 min at 16 000g), resuspending
the biomass in MSM (100 mL) in a serum bottle (160 mL),
and adding a high dose of cDCE (6.1-12.2 mM). Once most
Com etabolism of cDCE by MF1. The kinetics of cDCE
cometabolism were examined further with VC-grown strain
VOL. 36, NO. 10, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2 1 7 3

FIGURE 3. cDCE cometabolism by VC-grown MF1. These data were
used simultaneously to determine k ′, K , and kinact. Solid lines
C
C
represent the fit of eq 3. The observed transformation capacity was
determined from bottles with incomplete cDCE utilization. The inset
provides an expanded view of the data and model fit for the three
lowest cDCE concentrations.
TABLE 1. Summary of Kinetic Parameter Estimates for
Metabolism of VC and Cometabolism of cDCE by MF1
parameter
value
kS (µm ol of VC/m g of TSS/d)
KS (µM VC)
kC′ (µm ol of cDCE/m g of TSS/d)
KC (µM cDCE)
kS,C (µm ol of VC/m g of TSS/d)
KI (µM cDCE)
kinact (L/m g of TSS/d)
0.41 ( 0.003a
0.26 ( 0.037a
0.44 ( 0.011
22.0 ( 0.8
0.31 ( 0.002
1.0 ( 0.15
0.0032 ( (9 × 10-5)
a
From Verce et al. (21).
MF1, isolated from the VC enrichment culture (21). Con-
sumption of cDCE in the absence of VC by strain MF1 is
shown in Figure 3. Fitting of eq 3 to these data permitted
estimation of k
off-diagonal elements of the resulting correlation matrix were
C
greater than 0.9, indicating that estimates of k ′, K , and kinact
were not highly correlated (33). The finite amount of cDCE
that can be cometabolized by strain MF1, expressed as an
observed transformation capacity (Tc,obs), is 0.82 ( 0.06 µmol
of cDCE/ mg of TSS.
C C
′, K , and kinact (Table 1). Some, but not all,
FIGURE 4. Metabolism of VC and cometabolism of cDCE by MF1
when (a) [VC] . [cDCE] ; (b) [VC] ; and (c) [VC]
≈ [cDCE]
[cDCE] . Data from these plus three other bottles were used
simultaneously to determine kS,C and K . Solid lines represent the
I
fit of eqs 2-3. The dashed lines represent the fit of the Monod
model when VC is present as the sole substrate.
C
,
o
o
o
o
o
o
When fed VC and cDCE, strain MF1 did not begin
consuming cDCE until virtually all of the VC was utilized
Initial zeroth-order rates of cDCE cometabolism by MF1
were measured just after the culture finished consuming VC
(
Figure 4). This is consistent with MF1 having a much smaller
half-saturation coefficient for VC than for cDCE (i.e., K
, Table 1), indicating that VC should effectively out-compete
(
r
C(0)) and after increasing amounts of time in the absence
C
>
of VC (rC(t)), to determine the endogenous decay coefficient
K
S
for nongrowth substrate activity (b
value of value of b
C
) (Figure 5). The resulting
is 0.041 ( 0.038 h . Similar experiments
cDCE for consumption. The presence of cDCE reduces the
rate of VC utilization, as indicated by cultures with and
without cDCE present (Figure 4). The complete kinetics model
-1
C
were not performed with tDCE, 1,1-DCE, and TCE because
MF1 exhibits comparatively little or no activity on them (see
the following section).
Evaluation of tDCE, 1,1-DCE, and TCE Com etabolism
by MF1. Cometabolism of tDCE (1-90 µM), 1,1-DCE (5-20
µM), and TCE (5-12 µM) by VC-grown MF1 was examined
in the absence of VC. In contrast to cDCE, MF1 exhibited no
(
eqs 2-3) was fit to depletion curves from cultures fed VC
plus cDCE to obtain estimates of kS,C and K (Table 1), using
the values for k ′, K , and kinact determined with cDCE-only
data. Parameters kS,C and K were not correlated, as indicated
by correlation matrix elements less than 0.9 (33).
I
C
C
I
2
1 7 4
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 10, 2002

tDCE by the VC enrichment culture to their respective
epoxides is consistent with this pathway in MF1. Given the
substrate range of other alkene monooxygenases, such as
that used by propylene-grown Xanthobacter strain Py2 (34,
3
5), cometabolism of polychlorinated ethenes may be
expected. However, the alkene monooxygenase possessed
by VC-grown MF1 shows a distinct preference for cDCE, while
L1 and Py2 show high levels of cometabolic activity with
both cDCE and tDCE. Py2 also readily cometabolizes TCE,
while MF1 and L1 show no activity on it. Several butane
grown organisms also cometabolize cDCE but not tDCE (36,
3
7).
It is not yet known why the VC-grown enrichment culture
from which MF1 was isolated) exhibits a low level of
(
cometabolic activity on tDCE, while MF1 shows none. One
possible explanation examined is a rapid loss of cometabolic
activity in the absence of primary substrate, a process
quantified by the endogenous decay coefficient for cometa-
C
bolic activity (b ). MF1 loses its capacity to cometabolize
cDCE nearly twice as fast as a methanotrophic enrichment
culture that cometabolizes TCE (28). However, a rapid loss
of tDCE cometabolic capacity does not account for MF1’s
lack of activity on this compound. In experiments where VC
and tDCE were added, VC was readily metabolized (which
should have increased the capacity for cometabolism), but
there was still no activity on tDCE, either during or after VC
consumption. In addition to different levels of activity on
tDCE, the enrichment culture and MF1 also differ in their
growth rates on VC; the enrichment grows approximately 2
times faster.
Although MF1 showed little or no cometabolic activity on
tDCE, 1,1-DCE, and TCE, these compounds did have a
noticeable impact on the maximum rate of VC utilization at
concentrations above approximately 40 µM (see Supporting
Information). The inhibitory effect of 1,1-DCE is consistent
with other studies that have demonstrated the toxicity of
this compound or its metabolites (34, 38), and MF1 does
exhibit a low level of cometabolic activity toward this
compound. The mechanism by which tDCE and TCE increase
the maximum rate of VC metabolism, without themselves
being transformed, is not yet known.
FIGURE 5. Determining the endogenous decay coefficient for cDCE
activity (b ) for VC-starved MF1.
c
cometabolic activity with tDCE or TCE at any of the
concentrations tested. Some activity was observed with 1,1-
-5
DCE, but the transformation capacity was only 2 × 10 µmol
of 1,1-DCE/ mg of TSS (see Supporting Information for
representative results).
Transformation of tDCE, 1,1-DCE, and TCE was also tested
in the presence of VC to determine if VC consumption would
facilitate cometabolic activity and if the presence of these
compounds impacted the kinetics of VC utilization. During
and shortly after VC consumption, little or no cometabolism
of tDCE (15-800 µM), 1,1-DCE (8-950 µM), and TCE (8-800
µM) was observed (see Supporting Information for repre-
sentative results). However, at tDCE and TCE concentrations
above 40 µM, their presence increased the maximum specific
rate of VC utilization by 13-42% (statistically significant, R
)
0.05), even though tDCE and TCE were not consumed
during or after VC consumption (see Supporting Information
for representative results). On the other hand, high con-
centrations of 1,1-DCE (950 µM) completely inhibited VC
biodegradation. For most of the experiments, the presence
of tDCE, 1,1-DCE, or TCE did not have a statistically significant
The batch depletion kinetics of cDCE by MF1 are
characterized by several features. First, in the absence of VC,
the highest observed rate of cDCE utilization (0.26 µmol of
cDCE/ mg of TSS/ d based on the highest amount shown in
Figure 3b) was lower than the fitted value for k ′. Thus, k ′
S
impact on the K for MF1’s utilization of VC.
C
C
represents a “theoretical” maximum specific nongrowth
substrate utilization rate rather than an observed rate typified
by Monod kinetics. This suggests that MF1 suffers inactivation
that inhibits cDCE use before it can reach the maximum
Discussion
The findings reported here demonstrate that microorganisms
grown aerobically on VC as a primary substrate are capable
of cometabolizing cDCE and, to a lesser extent, tDCE and
C
theoretical rate (k ′). Although the nature of this inactivation
1
,1-DCE. VC-supported cometabolism of polychlorinated
is not known, it is more likely to be due to a depletion of
reducing power than to toxicity of reactive intermediates.
cDCE epoxide, observed in the headspace of the enrichment
culture, has the longest half-life of all chloroethene epoxides
(23) and is presumably the least toxic. Inactivation due to
ethenes was first suggested in studies with M. aurum L1 based
on initial oxidation rates of 43, 36, 14, and <1 µmol/ d/ mg
of cells for cDCE, tDCE, 1,1-DCE, and TCE, respectively (20).
However, nongrowth substrate depletion curves were not
provided for L1, and no experiments were conducted in which
VC and the nongrowth substrates were added at the same
time. Our results conclusively demonstrate the phenomenon
of VC serving as a primary substrate in support of come-
tabolism, especially with respect to cDCE. The observed
transformation capacity for cDCE by VC-grown MF1 is 14-
consumption of cDCE is described by the subterm “kinact(C
- C)” in eq 3, which was adapted from a previously developed
model (39). The k ′ value obtained for MF1 is 2 orders of
0
C
magnitude lower than the initial oxidation rate reported for
VC-grown L1 (20). Even higher maximum specific rates have
been reported for methanotrophic cometabolism of cDCE
(e.g., refs 8 and 13), although a much lower rate has been
observed with an ammonia oxidizer (40).
Second, when VC and cDCE are present together, cDCE
decreases the rate of VC use. The ability of the model to
adequately describe this phenomenon is due to subterm
4
3% of values reported for cDCE by methanotrophic cultures
(
8, 10). The transformation yield for MF1 (T
y
) TC,obs × Yobs
)
0.0026 µmol of cDCE/ µmol of VC) is considerably lower
than the comparable value for an ethene-grown enrichment
culture (14), in part because of the lower yield for VC as a
growth substrate.
“C
the maximum substrate utilization rate by an amount kS,C
when cDCE is present. The low value of K (Table 1) indicates
0
/ (C
0
I
+ K )” in eq 2, which acts as a “switch” to decrease
Both MF1 and L1 utilize an alkene monooxygenase to
initiate catabolism of VC (20, 21). Conversion of cDCE and
I
VOL. 36, NO. 10, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2 1 7 5

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(
Third, cDCE use is completely inhibited by VC. Preferential
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and nongrowth substrate. Another pattern, in which the
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2
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degree of correlation between k
′ and kinact, indicates that unique estimates may not have
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value for K is comparable to those for methanotrophic
C C
′ and K , as well as between
(
(
k
C
2
C
2
cometabolism of cDCE, based on studies that used a similar
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C C
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and L1 exhibit their highest cometabolic activity toward cDCE,
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assistance of Kim Ivey in the collection of MS data is greatly
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Supporting Information Available
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