Kinetics and inhibition of reductive dechlorination of chlorinated ethylenes by two different mixed cultures

Article abstract of DOI:10.1021/es0496773

Kinetic studies with two different anaerobic mixed cultures (the PM and the EV cultures) were conducted to evaluate inhibition between chlorinated ethylenes. The more chlorinated ethylenes inhibited the reductive dechlorination of the less chlorinated ethylenes, while the less chlorinated ethylenes weakly inhibited the dechlorination of the more chlorinated ethylenes. Tetrachloroethylene (PCE) inhibited reductive trichloroethylene (TCE) dechlorination but not cis-dichloroethylene (c-DCE) dechlorination, while TCE strongly inhibited c-DCE and VC dechlorination. c-DCE also inhibited vinyl chloride (VC) transformation to ethylene (ETH). When a competitive inhibition model was applied, the inhibition constant (K_I) for the more chlorinated ethylene was comparable to its respective Michaelis-Menten half-velocity coefficient, K_S. Model simulations using independently derived kinetic parameters matched the experimental results well. k _max and K_S values required for model simulations of anaerobic dechlorination reactions were obtained using a multiple equilibration method conducted in a single reactor. The method provided precise kinetic values for each step of the dechlorination process. The greatest difference in kinetic parameters was for the VC transformation step. VC was transformed more slowly by the PM culture (k_max and K_S values of 2.4 ± 0.4 μmol/mg of protein/day and 602 ± 7 μM, respectively) compared to the EV culture (8.1 ± 0.9 μmol/mg of protein/day and 62.6 ± 2.4 μM). Experimental results and model simulations both illustrate how low K_S values corresponded to efficient reductive dechlorination for the more highly chlorinated ethylenes but caused strong inhibition of the transformation of the less chlorinated products. Thus, obtaining accurate K _S values is important for modeling both transformation rates of parent compounds and their inhibition on daughter product transformation.

Full text of DOI:10.1021/es0496773

Environ. Sci. Technol. 2005, 39, 195-205
persistence of intermediate products such as c-DCE and VC
is of concern, since VC is a known carcinogen.
Kinetics and Inhibition of Reductive
Dechlorination of Chlorinated
Ethylenes by Two Different Mixed
Cultures
The competition for H2 by dechlorinating microorganisms,
nitrate and sulfate reducers, methanogens, and acetogens
has previously been reported (5-7). However, few studies
have been performed on the inhibition among chlorinated
ethylenes during reductive dechlorination. Inhibition of VC
dechlorination by other chlorinated ethylenes was indi-
cated by modeling studies of Tandoi et al. (8). Haston (9)
reported competitive inhibition between c-DCE and VC and
showed the inhibition constants were comparable to their
respective half-velocity coefficients. Garant and Lynd (10)
showed that a competitive inhibition model fit experimental
data better than the noncompetitive model for a culture
capable of complete dechlorination of PCE to ETH. The
kinetic parameters used in their study were obtained from
statistical fits of the model to the experimental data, not
independently determined. Cupples et al. (11) recently
showed competitive inhibition between c-DCE and VC with
a dechlorinating culture that grows on c-DCE and VC.
However, inhibition studies on each step of the dechlori-
nation process are needed.
Reported here are the results of the kinetic characterization
of two different mixed cultures that completely dechlorinate
PCE to ETH. A simple multiequilibration method was de-
veloped to determine kmax and KS values using a single batch
reactor. Batch kinetic experiments were also conducted to
study chlorinated aliphatic hydrocarbon (CAH) inhibition
for each step of reductive dechlorination from PCE to ETH.
The inhibition of more chlorinated ethylenes on less chlo-
rinated products was evaluated and vice versa. A model
including terms for competitive inhibition was employed
and compared with the experimental results using inde-
pendently determined kinetic parameters.
S E U N G H O Y U , M A R K E . D O L A N , A N D
L E W I S S E M P R I N I *
Department of Civil, Construction, and Environmental
Engineering, Oregon State University, Corvallis, Oregon 97331
Kinetic studies with two different anaerobic mixed
cultures (the PM and the EV cultures) were conducted to
evaluate inhibition between chlorinated ethylenes. The
more chlorinated ethylenes inhibited the reductive
dechlorination of the less chlorinated ethylenes, while the
less chlorinated ethylenes weakly inhibited the dechlo-
rination of the more chlorinated ethylenes. Tetrachloroethylene
(PCE) inhibited reductive trichloroethylene (TCE) dechlo-
rination but not cis-dichloroethylene (c-DCE) dechlorination,
while TCE strongly inhibited c-DCE and VC dechlorination.
c-DCE also inhibited vinyl chloride (VC) transformation
to ethylene (ETH). When a competitive inhibition model was
applied, the inhibition constant (KI) for the more chlorinated
ethylene was comparable to its respective Michaelis-
Menten half-velocity coefficient, KS. Model simulations using
independently derived kinetic parameters matched the
experimental results well. kmax and KS values required for
model simulations of anaerobic dechlorination reactions
were obtained using a multiple equilibration method conducted
in a single reactor. The method provided precise kinetic
values for each step of the dechlorination process.
The greatest difference in kinetic parameters was for the
VC transformation step. VC was transformed more
Materals and Methods
Chemicals. Commercially available PCE, TCE, and VC (no
stabilizers) were used in the kinetic experiments. PCE (99.9%,
spectrophotometric grade), TCE (99.9%), and c-1,2-DCE
(97%) were obtained from Acros Organics (Pittsburgh, PA),
and VC and ETH (both 99.5%, Aldrich Chemical, Milwaukee,
WI) were used in the preparation of analytical standards and
for addition to the batch reactors. 1-Butanol (99.8%, HPLC
grade, Aldrich Chemical, Milwaukee, WI) and hydrogen (99%,
Airco, Inc., Albany, OR) were used as the electron donors.
Analytical Methods. PCE, TCE, c-DCE, VC, and ETH in
the reactors were measured with an HP-6890 gas chromato-
graph (GC) equipped with a photoionization detector (PID)
and flame ionization detector (FID) connected in series. The
headspace samples were obtained and transferred using 100
to 250 µL gastight syringes (Hamilton, Leno, NV). Chro-
matographic separation of 100-200 µL reactor headspace
samples was achieved using a 30 m × 0.53 mm GS-Q column
(J&W Scientific, Folsom, CA) with helium as the carrier gas
(15 mL/min). The GC oven was initially set at 80 °C for 1.5
min, heated at 65 °C/min to 170 °C and 40 °C/min to 220 °C,
and kept at 220 °C for 2.7 min. Hydrogen concentrations in
headspace gas samples (200 µL) were determined using an
HP-5890 GC with a thermal conductivity detector (TCD),
operated isothermally at 220 °C. The hydrogen samples were
chromatographically separated with a Carboxen 1000 column
(15 ft × 1/8 in, Supelco, Bellefonte, PA) using argon gas as
a carrier gas at 15 mL/min. The hydrogen detection limit
was 4 nM (aqueous concentration).
slowly by the PM culture (kmax and KS values of 2.4 ( 0.4
µmol/mg of protein/day and 602 ( 7 µM, respectively)
compared to the EV culture (8.1 ( 0.9 µmol/mg of
protein/day and 62.6 ( 2.4 µM). Experimental results and
model simulations both illustrate how low KS values
corresponded to efficient reductive dechlorination for the
more highly chlorinated ethylenes but caused strong
inhibition of the transformation of the less chlorinated
products. Thus, obtaining accurate KS values is important
for modeling both transformation rates of parent compounds
and their inhibition on daughter product transformation.
Introduction
Tetrachloroethylene (PCE) and trichloroethylene (TCE) are
among the most commonly observed groundwater con-
taminants (1, 2). Many laboratory studies have shown that
PCE acting as an electron acceptor can be reductively
dechlorinated to TCE, cis-dichloroethylene (c-DCE), vinyl
chloride (VC), and finally to ethylene (ETH) using hydrogen
as an electron donor (3, 4). However, the accumulation and
Culture Enrichment and Growth. Two different mixed
cultures were enriched and used in this study. Microbial
* Corresponding author phone: (541)737-6895; fax: (541)737-3099;
e-mail: Lewis.Semprini@orst.edu.
9
10.1021/es0496773 CCC: $30.25
Published on Web 12/03/2004
2005 American Chemical Society
VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 195

mixed cultures obtained from Point Mugu Naval Weapon
Facility, CA (PM) and the Evanite site in Corvallis, OR (EV)
were enriched in two separate batch reactors (total volume
1.2 L with liquid volume of 1 L) fitted with gray chlorobutyl
rubber septa (Wheaton Industries, Millville, NJ). The PM
culture was originally enriched under anaerobic conditions
with aquifer solids and groundwater from the site (12). After
1.5 years, supernatant (300 mL) was transferred into sterile
basal medium described by Yang and McCarty (6). The
medium was modified to double its buffer capacity (1 g/L of
K2HPO4 and 3 g/L of Na2CO3), and sodium chloride and
ammonium chloride were replaced with sodium bromide
and ammonium bromide, respectively, to make a low-
chloride content medium. Initially, 10 mg/L of TCE was added
with 5-10 mg/L of butanol. Over a 1 year period, TCE
concentrations were increased in steps up to 100 mg/L while
maintaining complete reductive dechlorination to ETH. The
batch enrichment reactor was operated on a fill-and-draw
mode with a hydraulic residence time of about 45 days. After
50 to 100% of the TCE was reductively dechlorinated to ETH,
the batch reactor was purged with a furnace-treated gas
consisting of N2 (90%) and CO2 (10%). Cells were harvested
by replacing 300 mL of the liquid culture with fresh anaerobic
medium. At the final operating conditions, neat TCE and
butanol were added to achieve aqueous concentrations of
100 mg/L and 40 mg/L, respectively. The batch enrichment
reactor was maintained at 20 °C with continuous shaking at
200 rpm. The PM culture was fed TCE as the electron acceptor
because the culture was originally unable to transform PCE.
After about 3 years of enrichment on TCE, the PM mixed
culture started to show PCE dechlorination ability.
FIGURE 1. Measurements of c-DCE transformation (a) and VC
production (b) by the EV mixed culture using the multiple
equilibration method.
The EV culture was enriched from groundwater obtained
from the Evanite site in Corvallis, OR (12, 13), using similar
enrichment procedures as the PM culture; however, enrich-
ment was on PCE instead of TCE. The batch enrichment
reactor was maintained for 3 years in the same manner as
the PM culture, but PCE was added as the electron acceptor
at 100 mg/L with 40 mg/L of butanol, since the EV culture
had the ability to dechlorinate PCE.
when amplified with the Dehalococcoides specific primers
sets and produced no bands when amplified with the
Desulfuromonas specific primers indicating that Dehalo-
coccoides-like microorganisms were present in both cultures
and were likely responsible for the complete reductive
dechlorination to ETH. This result was also in agreement
with a previous T-RFLP analysis that showed 65% of the
universally amplified DNA from the EV culture had a TFL
consistent with that predicted from Dehalococcoides se-
quence information available in GenBank.
Determination of Cell Concentration. The total cell
concentration, X, in the study was determined by protein
analysis using the Pierce Micro BCA Protein Assay Reagent
Kit (Pierce, Rockford, IL). All samples were pretreated with
the Compat-Able Protein Assay Preparation Reagent Set
(Pierce, Rockford, IL) to eliminate potentially interfering
substances prior to total protein quantitation. The protein
concentration detection limit was 0.5 mg/L.
Kinetic Study for Determination of kmax and KS. An
experimental procedure was developed to determine kmax
and KS values for PCE, TCE, c-DCE, and VC dechlorination
using sequential equilibrations in batch reactors. The method
involved stepwise increases of the CAH concentration over
a short time interval and measuring parent compound
disappearance and daughter product production at each
concentration step (Figure 1). The batch kinetic studies were
conducted in 150 mL serum bottles containing 125 mL of
liquid media and culture. The culture added to the reactor
was harvested from the enrichment batch reactor 10 to 14
days after a batch feeding of PCE or TCE (50 to 100% converted
to ETH). The amount of culture used was varied, depending
on the test, to yield protein concentrations ranging from 2
to 50 mg/L. The batch reactors were constructed in an
anaerobic glovebox and then purged with a furnace-treated
mixed gas of N2 (90%) and CO2 (10%), followed by the addition
of 10 µL of neat butanol (99.8%) and 0.5-2.0 mL of H2 gas
Both of the cultures initially showed very active metha-
nogenesis with butanol added as an electron donor (12).
However, no methanogenesis was observed after 2-3 years
of enrichment on the high PCE or TCE concentrations.
A PCR assay targeting genes encoding for 16S ribosomal
RNA was performed to determine if Dehalococcoides-like
microorganisms were present in the mixed cultures. DNA
was extracted from the PM and EV cultures using FastDNA
spin kits for soil (Qbiogene, Inc., Carlsbad, CA). For each
culture, amplification reactions were performed using 5 µL
of template DNA and three different primer pairs. One primer
pair, developed by Lo¨ffler et al. (14), was specific to the
hypervariable regions of the 16S rRNA genes of the PCE-
dechlorinating Desulfuromonas sp., including D. chloro-
ethenica and D. sp. strain BB1, and consisted of forward
primer Dsm-205F, 5′-AACCTTCGGGTCCTACTGTC-3′, and
the reverse primer Dsm-1051R, 5′-GCCGAACTGACCCCTAT-
GTT-3′. Another primer pair, also developed by Lo¨ffler et al.
(14), was specific to the hypervariable regions of the 16S
rRNA genes of two members of the Dehalococcoides genus,
D. ethenogenes and D. sp. strain FL2, and consisted of forward
primer Dhg-728F, 5′-AAGGCGGTTTTCTAGGTTGTCAC-3′,
and the reverse primer Dhg-1155R, 5′-CGTTTCGCGGGGC-
AGTCT-3′. The final primer pair, developed by Hendrickson
et al. (15), was based on consensus variable and hypervariable
regions in the 16S rRNA gene sequences of D. ethenogenes
and Dehalococcoides sequences from the Victoria and Pinellas
cultures and consisted of forward primer DHC-1F, 5′-
GATGAACGCTAGCGGCG-3′, and the reverse primer DHC-
1377R, 5′-GGTTGGCACATCGACTTCAA-3′. Both the PM and
EV cultures produced bright bands of the expected length
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196 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 1, 2005

(99%) as electron donors. The reactors were initially amended
with the lowest concentration of a chlorinated ethylene to
be tested and shaken at 300 rpm at 20 °C. The rates of parent
compound disappearance and daughter product production
were generally measured over a period of less than 1 h by
repeated headspace sampling. Total CAH mass in the reactor
was computed using published Henry’s Law constants (16,
17). Most of the kinetic parameters in the experiments were
obtained based on the measurement of dechlorination
products due to the greater sensitivity in measuring product
production rates. The rates were determined at early time
before the product was significantly transformed. After
measuring the dechlorination rate at the lowest CAH
concentration, the reactor was purged with the mixed gas
(90% N2 and 10% CO2) for 5-10 min to remove all the
dechlorination products from the reactor. H2 was added,
along with a higher concentration of the chlorinated ethylene,
and the transformation rates were again measured using the
procedures previously described.
The set of kinetic experiments with a particular chlorinated
ethylene were usually completed within 10 h to minimize
microbial growth. Doubling times for dechlorinating cultures
have been reported to be approximately 2 days (18, 19). Thus,
the 10 h time period to complete the test was relatively short
compared to the doubling time. The kinetic batch experi-
ments were conducted in duplicate or triplicate to ensure
that kinetic parameters were statistically reliable. Biogenic
c-DCE was used for c-DCE dechlorination rate measurements,
since chloroform present in commercialc-DCE has inhibitory
effects on CAH utilization (9, 20). Biogenic c-DCE was
produced from TCE dechlorination by a mixed anaerobic
culture obtained from Site 300 at Lawrence Livermore
National Laboratory, CA (LLNL), which dechlorinates TCE
to c-DCE (12). An enrichment reactor of the LLNL culture
was fed TCE to produce c-DCE. c-DCE dissolved in liquid
was obtained from centrifuging the reactor at 8000 rpm and
filtering the liquid suspension through a 0.2 µm syringe filter.
The filtrate was used as the biogenic source of c-DCE in the
kinetic experiments.
Inhibition Study. Inhibition of chlorinated ethylenes on
transformation of each other was also studied. For example,
PCE and TCE were tested for inhibitory effects on each other.
The same aqueous concentration of TCE was added to 3 to
4 batch reactors with different concentrations of PCE for the
evaluation of PCE inhibition of TCE transformation. Similarly,
inhibition of TCE on the reductive PCE dechlorination was
evaluated at the same aqueous PCE concentration with
different concentrations of TCE. For the experiments of PCE
inhibition on TCE dechlorination, the production rates of
c-DCE formation were used to determine rates of TCE
transformation, since c-DCE transformation to VC was not
significantly observed during the test. However, decreases
in PCE concentrations were used to quantify TCE inhibition
on PCE dechlorination because high concentrations of TCE
as an inhibitor were already present. Mass balances showed
that at the highest TCE concentration there was less than a
20% difference in rates determined by parent compound
disappearance and daughter product production. At lower
TCE concentrations the differences were 3 to 7%. For these
inhibition studies, several sets of chlorinated ethylenes were
chosen including PCE T TCE, PCE T c-DCE, TCE T c-DCE,
TCE T VC, and c-DCE T VC. Both inhibitions of the more
chlorinated ethylene on the less and the less on the more
were studied. PCE inhibition of VC transformation was not
studied after it was established that PCE did not inhibit c-DCE
transformation and thus would not likely inhibit a further
step in the transformation process. The concentrations of
CAHs as reactants (not as inhibitors) ranged from 30 to 251
µM. These concentrations were chosen in order to complete
the kinetic experiments within 3-4 h. The VC concentration
of 251 µM was the highest used because of the high KS values
for both cultures.
Model Development
Kinetic Model for Reductive Dechlorination. The dechlo-
rination rate of a given CAH can be described using
Michaelis-Menten kinetics (21, 22). Reductive dechlorination
of CAHs can be affected by several factors, including
inhibition among chlorinated ethylenes, electron donor
concentrations, and CAH toxicity (9). However, for these
kinetic experiments the chlorinated ethylene concentration
was considered to influence only the kinetic parameters, kmax
and KS. This was regarded as a valid assumption because the
initial dechlorination rates were measured before concen-
trations of the product CAHs could accumulate to inhibitory
or toxic levels (23). Electron donor limitations were also
excluded in the experiments by adding H2 and butanol in
excess. Thus, a Michaelis-Menten model based on electron
acceptor concentrations was used including terms for the
transformation and production of the CAH of interest
dC
kmax,YXCL,Y
1
L,Y ) -
+
dt
KS,Y + CL,Y
VG
1 + HCC,Y
(
)
VL
k
max,XXCL,X
1
(1)
KS,X + CL,X
VG
1 + HCC,X
(
)
VL
where CL is the CAH aqueous concentration (µmol/L), kmax
is the maximum specific CAH dechlorination rate (µmol/mg
of protein/day), X is the biomass concentration (mg of
protein/L), and KS is the half-velocity coefficient (µmol/L).
Since the kinetic experiments were conducted with batch
bottles consisting of gas and aqueous phases, the Michaelis-
Menten equation was modified (24), where VG and VL are the
volumes of gas and aqueous phases, respectively, and the
dimensionless Henry’s constant (HCC) was used for each
chlorinated ethylene (16, 17). For measurement of the
dechlorination rates, the total CAH mass in the bottle was
computed from headspace measurement using published
Henry’s constants (16, 17). The aqueous CAH concentration
in eq 1 is related to the total mass of each CAH via a mass
balance equation (CL ) M/(VL + VGHCC)). For multiple
equilibration tests with eq 1, the values of kmax and KS were
estimated without the production term, using a nonlinear
least-squares regression fitting program [Solver in Excel
(Microsoft, Redmond, WA) and S-PLUS (Insightful, Seattle,
WA)].
Competitive Inhibition among Chlorinated Ethylenes.
Competitive inhibition among chlorinated ethylenes was
included in the modeling analysis (9, 10). The reductive
dechlorination rate of a CAH was modeled with competitive
inhibition kinetics using eq 2
dC
dt
kmax XCL
L ) -
(2)
CI,1 CI,2
KS 1 +
+
+ CL
(
)
KI,1 KI,2
where inhibition constants of each chlorinated ethylene are
expressed as KI,1 and KI,2 (µmol/L). As will be described in the
Results and Discussion, the respective half-velocity coef-
ficients were used for the inhibition constants. The model
included TCE, c-DCE, and VC as the major intermediates in
PCE dechlorination to ETH, not 1,1-DCE or t-DCE.
Based on the results of the inhibition experiments, a
conceptual inhibition model was developed (Figure 2). The
equations for reductive dechlorination by the anaerobic
cultures including appropriate inhibition terms are presented
9
VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 197

(day-1). Values for growth yield, Y () 0.006 mg of protein/
µmol of Cl- dechlorinated), and decay constant, kd () 0.024
day-1), were obtained from the literature (25, 21). For our
tests, we do not know the actual biomass, X, of the
dechlorinating microorganisms, and whether different dechlo-
rinators are active at different steps of the process. Since kmax
values were determined on a total protein basis, the values
were normalized to the total mixed culture biomass. The
simultaneous solution of the model equations was obtained
using STELLA Research 5.0 (High Performance Systems,
Lebanon, NH).
FIGURE 2. Proposed inhibition model between chlorinated ethylenes
for reductive dechlorination based on the inhibition studies of Figures
4-8. Arrows represent inhibition directions. Dashed arrows indicate
very weak inhibition.
Results
in eqs 3-6. The proposed inhibition patterns (Figure 2) and
resulting equations assume PCE inhibits TCE transformation,
TCE inhibits both c-DCE and VC transformation, and c-DCE
inhibits VC transformation. As the inhibition results will show,
the less chlorinated ethylene inhibition on more chlorinated
ethylenes was very weak and therefore was not included in
the model equations.
Determinations of Kinetic Parameters. The Michaelis-
Menten curves for PCE, TCE, c-DCE, and VC transforma-
tion by the PM culture determined using the multiequili-
bration kinetic method are shown in Figure 3. The triplicate
determinations show statistically reliable results were ob-
tained for all the chlorinated ethylenes tested. The Michaelis-
Menten curves in Figure 3 show good fits for all chlorinated
ethylenes. Table 1 presents kmax and KS values for the PM and
EV cultures for each step of the dechlorination process. The
standard deviation of kmax and KS based on triplicate
determinations were approximately 11 and 16% of the
parameter value, respectively. For comparison, Haston and
McCarty (22) reported standard deviations in kmax and KS of
17% and 60% of the parameter values, respectively, using
single equilibrations in multiple reactors.
Table 1 also presents the kmax and KS values obtained by
Fennell and Gossett (21) for a mixed culture containing
Dehalococcoides ethenogenes strain 195 and those obtained
by Haston and McCarty (22), for comparison purposes. The
PM and EV cultures have comparable kmax and KS values for
PCE and TCE (Table 1). For both cultures, the TCE kmax values
are a factor of 10 greater than those for PCE. The KS value
for PCE of the PM culture is a factor of 2 greater than the EV
culture, while the kmax values are essentially equal, indicating
that the EV culture transforms PCE more rapidly at con-
centrations below the KS values (1.6 µM). The kmax and KS
values for c-DCE and VC show significant differences between
the two cultures. The kmax for c-DCE of the EV culture was
40% lower than that of the PM culture, while the KS values
were almost the same, indicating slower c-DCE biotrans-
formation by the EV culture.
The kmax value for VC by the EV culture was a factor of
3 greater, while the KS was a factor of 10 lower than the PM
culture, indicating much more rapid VC transformation with
the EV culture. This result was consistent with the assumption
of potential growth of the EV culture using VC as an electron
acceptor as discussed by Pon and et al. (13). Recently, VC-
grown dechlorinating cultures (Dehalococcoides sp. strain
BAV1 and Dehalococcoides sp. strain VS) were isolated [He
et al. (18, 26) and Cupples et al. (19)]. The KS value (62.6 µM)
of the EV culture is closer to those of the VC-grown cultures
(5.8 µM and 2.6 µM, respectively) than that of Dehalo-
coccoides ethenogenes strain 195 (290 µM) (21), which co-
metabolically transforms VC. The slower maximum trans-
formation rate and higher KS value (602 µM) for VC with the
PM culture are consistent with cometabolic VC transforma-
tion (21, 25). The transformation characteristics of the PM
culture are similar to that of Dehalococcoides ethenogenes
strain 195 that has a high KS value (21).
dCPCE -kmax,PCEXCPCE
)
(3)
(4)
dt
KS,PCE + CPCE
dCTCE
dt
-kmax,TCEXCTCE
kmax,PCEXCPCE
)
)
+
CPCE
KS,PCE + CPCE
KS,TCE 1 +
+ CTCE
(
)
KI,PCE
dCc-DCE
dt
-kmax,c-DCEXCc-DCE
+
CTCE
KS,c-DCE 1 +
+ Cc-DCE
(
)
KI,TCE
kmax,TCEXCTCE
(5)
(6)
CPCE
KS,TCE 1 +
+ CTCE
(
)
KI,PCE
dCVC
-kmax,VCXCVC
CTCE Cc-DCE
)
+
dt
KS,VC 1 +
+
+ CVC
(
)
KI,TCE KI,c-DCE
k
max,c-DCEXCc-DCE
CTCE
KS,c-DCE 1 +
+ Cc-DCE
(
)
KI,TCE
Equations 3-6 were combined to generate the series of
rate equations that include transformation, production, and
partitioning, as shown in eq 1.
Kinetic Model for Biomass Growth. Since VC inhibition
experiments lasted several days, the potential growth of
culture was included in the model using eq 7. Microbial
growth was calculated with respect to the transformation
rate of each chlorinated ethylene. The PM culture was
assumed to grow on PCE, TCE, and c-DCE. VC transformation
to ETH was assumed to be cometabolic, since the measured
KS value for VC transformation of 602 µmol/L was in the
range of that reported for Dehalococcoides ethenogenes strain
195, which transforms VC cometabolically. Thus, no biomass
growth was coupled with kinetic modeling (25). Unlike the
PM culture, microbial growth on each dechlorination step
was considered for the EV culture (13).
Competitive Inhibition among Chlorinated Ethylenes.
Results of inhibition studies are shown in Figures 4-8. The
lines shown in each figure represent model simulations using
the series of equations previously described. The model used
the independently determined kinetic parameters listed in
Table 1 with the initial biomass determined by protein
analysis. The measured KS values were used as putative
inhibition constants. Figure 4(a),(b) presents PCE inhibition
dX
dt
dC
dt
) Y
- kdX
(7)
In eq 7, Y is the growth yield of dechlorinating micro-
organisms, dC/dt is the rate of concentration change of each
chlorinated ethylene over time (µmol/L/day), and kd is the
decay constant of the dechlorinating microorganisms
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198 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 1, 2005

FIGURE 3. Initial reductive dechlorination rates of CAHs by the PM culture. The simple multi equilibration method was used to determine
kmax and KS values. The results of replicate experiments are represented by the different symbols.
Inhibition of TCE on c-DCE dechlorination is presented
TABLE 1. Kinetic Parameters Obtained from the Experiments
and Used in Model Simulations and Reported Values from
Fennell and Gossett (21) and Haston and McCarty (22)a
in Figure 4(c),(d). TCE inhibition of c-DCE transformation
was observed with both cultures, with the EV culture being
more strongly inhibited. The KS value for TCE of the EV culture
was a factor of 1.5 lower than the PM culture, which was
consistent with the greater TCE inhibition with the EV culture.
Simulations based on the inhibition model given in eq 5
showed good agreement with the inhibition experimental
results. TCE inhibition on VC transformation was also
examined, as shown in Figure 4(e),(f) for the PM and EV
cultures, respectively. TCE strongly inhibited VC transforma-
tion to ETH with both cultures. The EV culture more rapidly
transformed VC (by a factor of 10) than the PM culture (note
the different y axis scales), even though similar protein
concentrations were used. This result is consistent with the
EV’s kmax value being a factor of 3 higher and the KS value
being a factor of approximately 10 lower compared to the
PM culture (Table 1). The strong inhibition of TCE on VC
transformation is consistent with the much lower KS value
for TCE compared to the KS values of VC for both the PM and
EV cultures. The modeling results using eqs 4-6 provide
excellent matches to the experimental data using the
independently derived kinetic values given in Table 1, and
with the KS values representing the inhibition constants.
Neither culture showed clear VC inhibition on TCE dechlo-
rination (data not shown), which was consistent with the
findings of Nielsen and Keasling (27).
PM
kmax (µmol/mg
of protein/day) KS (µmol/L) of protein/day) KS (µmol/L)
EV
kmax (µmol/mg
PCE
TCE
c-DCE
VC
13.3 ( 1.8
124 ( 17
22.0 ( 2.0
2.4 ( 0.4
3.9 ( 1.4
2.8 ( 0.3
1.9 ( 0.5
602 ( 7
12.4 ( 0.7
125 ( 14
13.8 ( 1.1
8.1 ( 0.9
1.6 ( 0.2
1.8 ( 0.4
1.8 ( 0.3
62.6 ( 2.4
Fennell and Gossett (21)
Haston and McCarty (22)
kmax (µmol/mg
of VSS/day)
kmax (µmol/mg
of VSS/day)
KS (µmol/L)
KS (µmol/L)
PCE
TCE
c-DCE
VC
43.2
72.0
72.0
72.0
0.54
0.54
2.0 ( 0.1
1.6 ( 0.3
0.37 ( 0.08
0.34 ( 0.08
0.11 ( 0.04
1.4 ( 0.9
3.3 ( 2.2
2.6 ( 1.9
0.54
290
a All kmax and KS values for the PM and EV cultures were determined
using the multiple equilibration method in a single bottle on triplicate
samples. The range of initial CAH concentrations tested with the PM
and EV cultures, respectively, were PCE (µM): 0.2-10 and 0.5-20, TCE
(µM): 0.4-30 and 0.6-10, c-DCE (µM): 1.0-25 and 0.2-30, and VC
(µM): 50-2200 and 30-420.
on TCE transformation. As PCE concentrations increased,
rates of c-DCE production decreased for both cultures,
indicating that TCE dechlorination was inhibited by PCE.
The EV culture was more strongly inhibited by PCE than the
PM culture. A PCE concentration approximately 2.5 times
higher than that used for the EV culture was required to
obtain similar inhibition of the PM culture. The effect of TCE
on PCE dechlorination was also evaluated (data not shown).
PCE dechlorination was not clearly inhibited by TCE,
suggesting there is very weak inhibition of TCE on PCE
dechlorination.
VC dechlorination rates to ETH in the presence of c-DCE
were measured for a longer time compared to other inhibition
experiments, due to the slower rates of VC transformation
(Figure 5). The initial protein concentration of the EV culture
for this test was a factor of 24 greater than the PM culture.
This resulted in a much shorter time for c-DCE and VC
transformation, as predicted by the model simulations. ETH
production rates for the PM culture increased (in bottles
amended with c-DCE), after all c-DCE was essentially
transformed to VC (Figure 5(a)-(c)). After complete trans-
formation of c-DCE to VC by the PM culture, ETH production
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VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 199

FIGURE 4. Competitive inhibition studies between CAHs. Lines represent the model simulations. Average initial cell concentrations
measured and used in model simulations are 1.5-2.0 (a), 1.3-1.4 (b), 2.0-2.5 (c), 3.9-4.2 (d), 2.2-2.5 (e), and 1.4-1.6 mg/L (f).
rates were greater in c-DCE amended reactors. The addition
and transformation of c-DCE resulted in higher VC concen-
trations which led to greater rates of ETH production, as was
confirmed in model simulations. VC transformations were
very well predicted based on the proposed inhibition model.
The VC concentrations used here were well below the KS
value for the PM culture. Thus, an increase in VC concentra-
tion would be expected to result in faster VC transformation,
as was observed in the experimental results and model
simulations. The simulated increase in biomass concentra-
tions over the several day-period of the test are presented in
Figure 5(d). A greater relative increase in biomass was
predicted for the PM culture, which would result in increased
rates of ETH production.
The inhibition of c-DCE on VC dechlorination by the EV
culture is shown in Figure 5(e)-(h). Over the time scale of
these tests, the simulated increase in cell mass was less than
3% (Figure 5(h)). Although c-DCE was added as an inhibitor,
c-DCE was much more rapidly transformed than VC resulting
in transient increases of VC concentrations. Figure 5(e)-(h)
shows that VC transformation to ETH was increasingly
inhibited as the initial aqueous c-DCE concentration was
increased. The lags in ETH production are clearly associated
with higher concentrations of c-DCE, with ETH production
initiated when c-DCE concentrations were reduced to low
values. Unlike the PM culture, after complete transformation
of c-DCE to VC, the rate of VC dechlorination to ETH was
similar in all cases. Due to the lower KS value for VC with the
EV culture, the increase in VC concentrations had little effect
on transformation rates, since the initial VC concentration
(290 µM) was significantly higher than the KS value (62.6
µM). Also, the higher initial cell mass and short duration of
the tests resulted in minimal biomass growth, as predicted
by the model simulations. The model simulations did a very
good job capturing the temporal changes in c-DCE, VC, and
ETH concentrations.
Experiments were performed with both cultures to study
PCE inhibition on c-DCE dechlorination (Figure 6). Although
the PCE concentration was increased up to 395 µM and 491
µM for the PM and EV cultures, respectively, essentially no
difference was observed in the rate of c-DCE transformation
to VC. Simulations were performed with and without PCE
inhibition on c-DCE transformation and compared with the
experimental results. Simulations without PCE inhibition
more closely matched experimental data than those with
PCE inhibition, where inhibition was represented by the
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200 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 1, 2005

FIGURE 5. Competitive inhibition studies of c-DCE on VC dechlorination by the PM culture (a, b, c, d) and the EV culture (e, f, g, h). Lines
represent model simulations. Cell concentrations from model simulations are shown in (d) and (h).
measured KS value. The experimental results and model
simulations indicate that reductive c-DCE dechlorination is
unaffected or very weakly inhibited by PCE. c-DCE dechlo-
rination rates were a factor of about 7 higher with the PM
culture, due to the higher initial protein content used, and
the higher kmax value of the culture.
Potential VC inhibition on c-DCE dechlorination was also
evaluated. Model simulations were performed with and
without VC inhibition and were compared to the experimental
results (Figure 7). For both cultures, the experimental ob-
servations showed no apparent VC inhibition on c-DCE
dechlorination, and there was also no clear difference be-
tween the different model simulations. The results show that
VC had a very weak inhibitory effect on c-DCE transformation
for both cultures, even at VC concentrations as high as 290
µM, approximately 5 times the KS value for the EV culture.
This result is consistent with the high VC KS values of the PM
and EV cultures of 602 and 62.6 µM, respectively, and the low
KS values for c-DCE of 1.9 and 1.8 µM. Thus, VC inhibition
on c-DCE transformation was shown to be very weak in our
proposed model (Figure 2).
Batch kinetic experiments were performed to studyc-DCE
inhibition on TCE dechlorination (Figure 8). Interestingly,
although the KS values for c-DCE were similar to those of
TCE, rates of TCE dechlorination showed no clear inhibition
by c-DCE. The model without c-DCE inhibition simulated
the experimental data much more closely than that with
inhibition. The differences in the simulations for 0 µM initial
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VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 201

FIGURE 6. Comparison of simulations with and without PCE inhibition and the experimental data for c-DCE dechlorination by the PM culture
(a and b) and the EV culture (c and d). Initial c-DCE concentrations were 124 and 107 µM with cell concentrations of 5.0 and 1.3 mg/L
for the PM and EV cultures, respectively.
c-DCE with and without inhibition results from c-DCE formed
upon TCE transformation. Some inhibition was observed at
the highest c-DCE concentration tested, but it was very weak.
These results also indicate that substances inhibitory to TCE
transformation were not present in the biologically produced
c-DCE. These results show that KS values, when used as
inhibition constants, are not representative of less chlorinated
ethylene inhibition on more chlorinated ethylene transfor-
mation.
however, was equal to the KS value for c-DCE. Haston (9)
used KS values for c-DCE inhibition on VC transformation
when modeling the results of laboratory studies. In a recent
study, Cupples et al. (11) determined a competitive inhibition
constant (KI) for c-DCE inhibition on VC transformation. The
KI value generated for c-DCE was not statistically different
than the KS value of c-DCE (KS ) 3.3 ( 2.2, KI ) 3.6 ( 1.1).
Our results were consistent with these studies.
PCE did not inhibit c-DCE transformation. This likely
resulted from different microorganisms in the culture or
different enzyme systems being responsible for PCE and
c-DCE and VC transformation. Reductive dechlorination of
c-DCE and VC has been reported to be promoted by different
dechlorinators than those capable of reductive dechlorination
of PCE and TCE (28). The growth of Dehalococcoides sp. strain
VS and Dehalococcoides sp. strain BAV1 are coupled to the
dechlorination of c-DCE and VC but not PCE and TCE (18,
19, 26). Dehalococcoides sp. strain FL2 that cometabolically
dechlorinates PCE and VC uses only TCE and c-DCE as
metabolic electron acceptors (29). Dehalospirillum multi-
vorans, Dehalobacter restrictus strains PER-K23A, and De-
sulfuromonas sp. can reduce only PCE and TCE in terminal
electron accepting processes (14, 30). Another incompletely
dechlorinating bacterium (Desulfitobacterium sp. strain
PCE1) reductively dechlorinates only PCE to TCE (31). The
lack of PCE inhibition on c-DCE transformation is consistent
with different microorganisms being responsible for PCE and
c-DCE transformation. Different enzyme systems responsible
for different dechlorination reactions in the same microor-
ganism, as reported by Magnuson et al. (32) for Dehalococ-
coides ethenogenes, could also explain the lack of PCE
inhibition on c-DCE transformation.
Discussion
Several pairs of chlorinated ethylenes (PCE T TCE, PCE T
c-DCE, TCE T c-DCE, TCE T VC, and c-DCE T VC) were
investigated for transformation inhibition. The experimental
and modeling results indicate that more chlorinated ethylenes
inhibited reductive dechlorination of the less chlorinated
products, except that PCE did not inhibit c-DCE dechlori-
nation. Less chlorinated ethylenes, however, very weakly or
did not inhibit the transformation of more chlorinated
ethylenes. The KS values for VC for the PM and EV cultures
were much higher (602 µM and 62.6 µM, respectively) than
the KS values for the parent compounds. Thus, if inhibition
were related to the KS values, weak inhibition would be
expected, as shown in Figure 7 for VC inhibition on c-DCE
transformation. However, very weak inhibition of c-DCE on
TCE transformation was also observed, contrary to model
simulations using KS values as inhibition constants (Figure
8). Model simulations with inhibition constants much higher
than their KS values would be required to simulate the less
chlorinated ethylene inhibition on the more chlorinated
ethylene. Cupples et al. (11) reported that VC competitively
inhibited c-DCE dechlorination and that the VC inhibition
constant was three times higher than the KS value for VC.
The c-DCE inhibition constant on VC transformation,
Inhibition of the more chlorinated ethylenes on the less
chlorinated ethylenes was represented well using indepen-
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202 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 1, 2005

FIGURE 7. Comparison of simulations with and without VC inhibition and the experimental data for c-DCE dechlorination by the PM culture
(a and b) and the EV culture (c and d). Initial c-DCE concentrations were 96 and 121 µM with cell concentrations of 1.6 and 29 mg/L for
the PM and EV cultures, respectively.
FIGURE 8. Comparison of simulations with and without c-DCE inhibition and the experimental data for TCE dechlorination by the PM culture
(a and b) and the EV culture (c and d). Initial TCE concentrations were 21.8 and 21.9 µM with cell concentrations of 12 and 10.3 mg/L for
the PM and EV cultures, respectively.
dently determined KS values as the competitive inhibition
model simulations both illustrate how low KS values leading
constants in model simulations. Experimental results and
to efficient reductive dechlorination for each chlorinated
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VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 203

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product dechlorination. Thus, obtaining KS values is im-
portant for modeling both the rates of transformation of
parent compounds and their inhibition on the transformation
of daughter products.
A simple multiple equilibration method using a single
batch reactor was developed to obtain kmax and KS values for
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measured here are comparable to the values reported by
Fennell and Gossett (21) and Haston and McCarty (22). The
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Acknowledgments
The research was supported by a research grant from the
U.S. Environmental Protection Agency-sponsored Western
Region Hazardous Substance Research Center under agree-
ment R-828772. This article has not been reviewed by the
agency, and no official endorsement should be inferred.
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Received for review March 1, 2004. Revised manuscript re-
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ES0496773
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