Cobalamin-mediated reduction of cis- and trans-dichloroethene, 1,1- dichloroethene, and vinyl chloride in homogeneous aqueous solution: Reaction kinetics and mechanistic considerations

Article abstract of DOI:10.1021/es9701220

Since cobalamin is involved in the enzymatic reduction of halogenated ethenes by a variety of anaerobic bacteria and since cobalamin has been suggested as electron transfer mediator for the treatment of halogenated solvents, its reactions with such compounds are presently of great interest. In this paper, it is shown that, in homogeneous aqueous solution containing titanium(III) citrate as the bulk electron donor, superreduced cobalamin reductively dechlorinated cis- and trans-dichloroethene (cis-DCE and trans-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC) in pH-dependent reactions to ethene and ethane. Evidence is given that the initial step was the addition of cob(I)alamin to the chlorinated ethenes (CEs) with simultaneous protonation. Only for 1,1-DCE at high pH, a dissociative electron transfer mechanism as suggested for tetrachloroethene (PCE) and trichloroethene (TCE) in earlier work was important. 1,1-DCE reacted about 30 times faster than VC, 600 times faster than trans-DCE, and 3000 times faster than cis-DCE. Acetylene and ethene were found to react at similar rates as 1,1-DCE and VC, respectively. However, at more positive redox potentials, the reductive cleavage of the addition products, particularly of the adducts of acetylene, ethene, and VC with cob(I)alamin, may become very slow, thus preventing the regeneration of cob(I)alamin. The results of this study demonstrate that, at more negative potentials and at low pH, cobalamin is a potent electron transfer mediator for the complete dehalogenation of PCE and TCE without significant accumulation of VC. Since cobalamin is involved in the enzymatic reduction of halogenated ethenes by a variety of anaerobic bacteria and since cobalamin has been suggested as electron transfer mediator for the treatment of halogenated solvents, its reactions with such compounds are presently of great interest. In this paper, it is shown that, in homogeneous aqueous solution containing titanium(III) citrate as the bulk electron donor, superreduced cobalamin reductively dechlorinated cis- and trans-dichloroethene (cis-DCE and trans- DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC) in pH-dependent reactions to ethene and ethane. Evidence is given that the initial step was the addition of cob(I)alamin to the chlorinated ethenes (CEs) with simultaneous protonation. Only for 1,1-DCE at high pH, a dissociative electron transfer mechanism as suggested for tetrachloroethene (PCE) and trichloroethene (TCE) in earlier work was important 1,1-DCE reacted about 30 times faster than VC, 600 times faster than trans-DCE, and 3000 times faster than cis-DCE. Acetylene and ethene were found to react at similar rates as 1,1-DCE and VC, respectively. However, at more positive redox potentials, the reductive cleavage of the addition products, particularly of the adducts of acetylene, ethene, and VC with cob(I)alamin, may become very slow, thus preventing the regeneration of cob(I)alamin. The results of this study demonstrate that, at more negative potentials and at low pH, cobalamin is a potent electron transfer mediator for the complete dehalogenation of PCE and TCE without significant accumulation of VC.

Full text of DOI:10.1021/es9701220

Environ. Sci. Technol. 1997, 31, 3154-3160
reduction of such halogenated compounds by a variety of
Cobalamin-Mediated Reduction of
phylogenetically diverse anaerobic bacteria (1-4). Secondly,
since superreduced corrinoids are potent reductants for
halogenated compounds in aqueous solution (5-14), they
have a great potential to be used as electron transfer mediators
in the treatment of halogenated solvent wastes or in reme-
diation approaches of contaminated soils and aquifers (15).
cis- and trans-Dichloroethene,
1
,1-Dichloroethene, and Vinyl
Chloride in Homogeneous Aqueous
Solution: Reaction Kinetics and
Mechanistic Considerations
To date, detailed kinetic studies of reactions of halogenated
solvents with superreduced cobalamin or other corrinoids in
aqueous solutions have focused mainly on highly chlorinated
substances including tetrachloroethene (PCE), trichloro-
ethene (TCE), and tetrachloromethane (6-9). In previous
work (8), we have shown that PCE was transformed quan-
titatively to TCE by superreduced corrinoids, e.g., cob(I)-
alamin, cob(I)inamid, and cob(I)amid, while the primary
products of TCE reduction were cis- and trans-1,2-dichlo-
roethene (cis-DCE and trans-DCE) and acetylene. In addition,
during TCE reduction, we have found that at more positive
redox potentials the corrinoid species may be blocked by the
formation of addition products, which could be very disad-
vantageous for the use of such species as electron transfer
mediators in treatment processes.
G U Y G L O D , † U R S B R O D M A N N , †
‡
W E R N E R A N G S T ,
C H R I S T O F H O L L I G E R , † A N D
R E N EÄ P . S C H W A R Z E N B A C H * ,
‡
Swiss Federal Institute for Environmental Science and
Technology (EAWAG) and Swiss Federal Institute of
Technology (ETH), CH-8600 D u¨ bendorf, Switzerland, and
Limnological Research Center, EAWAG, CH-6047
Kastanienbaum, Switzerland
In the work presented in this paper, we have investigated
the effect of pH and redox potential on the cobalamin-
mediated reduction of cis-DCE, trans-DCE, 1,1-dichloro-
ethene (1,1-DCE), and vinyl chloride (VC) in homogeneous
aqueous solution using titanium(III) citrate or titanium(III)-
NTA as bulk electron donors. Except for 1,1-DCE, these
compounds have generally been found to be intermediates
in the biological as well as abiotic reduction of PCE and TCE
(16-22). 1,1-DCE is used as raw material for plastics (23),
and it has also been reported to be a transformation product
of another important chlorinated solvent, 1,1,1-trichloro-
ethane (24, 25). Among these mono- and dichloroethenes,
vinyl chloride is of particular environmental concern because
of its high carcinogenicity. The major goal of this study was
to assess the kinetics of the reduction of the four CEs by
superreduced cobalamin when present as electron transfer
mediator in aqueous solution and to get hints on the
mechanism(s) of these reactions. Such knowledge is not only
important from a basic scientific point of view (e.g., for better
understanding of enzymatic transformations of such com-
pounds), it is also essential for selecting optimal conditions
for the treatment of PCE and TCE wastes and contaminations
with corrinoids as electron transfer mediators.
Since cobalamin is involved in the enzymatic reduction
of halogenated ethenes by a variety of anaerobic bacteria
and since cobalamin has been suggested as electron
transfer mediator for the treatment of halogenated solvents,
its reactions with such compounds are presently of great
interest. In this paper, it is shown that, in homogeneous
aqueous solution containing titanium(III) citrate as the
bulk electron donor, superreduced cobalamin reductively
dechlorinated cis- and trans-dichloroethene (cis-DCE and
trans-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride
(VC) in pH-dependent reactions to ethene and ethane.
Evidence is given that the initial step was the addition of
cob(I)alamin to the chlorinated ethenes (CEs) with
simultaneous protonation. Only for 1,1-DCE at high pH, a
dissociative electron transfer mechanism as suggested
for tetrachloroethene (PCE) and trichloroethene (TCE) in
earlier work was important. 1,1-DCE reacted about 30
times faster than VC, 600 times faster than trans-DCE, and
3000 times faster than cis-DCE. Acetylene and ethene
were found to react at similar rates as 1,1-DCE and VC,
respectively. However, at more positive redox potentials, the
reductive cleavage of the addition products, particularly
of the adducts of acetylene, ethene, and VC with cob(I)alamin,
may become very slow, thus preventing the regeneration
of cob(I)alamin. The results of this study demonstrate that,
at more negative potentials and at low pH, cobalamin is
a potent electron transfer mediator for the complete deha-
logenation of PCE and TCE without significant accumulation
of VC.
Experimental Section
Chem icals and Stock Solutions. cis-Dichloroethene (purum),
vinyl chloride (VC) (puriss.), ethanol (puriss.), trisodium citrate
dihydrate (MicroSelect), nitrilotriacetic acid (NTA) (puriss.),
pentane (Burdick & Jackson), vitamin B12 (cyanocobalamin)
(
>98%), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)
for pH 7 solutions and glycine for pH 9 solutions were
purchased from Fluka AG (Buchs, Switzerland). 1,1-Dichloro-
ethene (99%) and trans-dichloroethene were purchased from
Aldrich Chemie, Switzerland. 2-Amino-2-(hydroxymethyl)-
1
,3-propanediol (Tris) for pH 8 solution, TiCl
3
(15% in 10%
HCl), and Na CO were from Merck AG (Dietikon, Switzer-
land). A gas mixture containing methane, acetylene, ethene,
ethane, carbon monoxide, and carbon dioxide (1% each in
2
3
Introduction
The reduction of halogenated solvents by superreduced
cobalamin (cob(I)alamin) and other corrinoids has recently
drawn considerable attention, mainly for two reasons. First,
it has been shown that cobalamin is involved in the enzymatic
N
2
) (Scott Specialty Gases) was purchased from Supelco.
Isopropyl alcohol-d (99.5%) and D O were from Armag AG
D o¨ ttingen, Switzerland). All chemicals were used as received.
Stock solutions of the halogenated ethenes (0.1 M) were
7
2
(
*
Corresponding author e-mail address: schwaba@eawag.ch;
prepared in ethanol and were found to be stable for at least
5 days. Stock solutions of cobalamin were prepared in water
at a concentration of about 5 mM and stored at 4 °C in the
telephone: 41 1 8235109; fax: 41 1 8235471.
†
‡
EAWAG, Kastanienbaum.
EAWAG and ETH, D u¨ bendorf.
3
1 5 4
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 11, 1997
S0013-936X(97)00122-3 CCC: $14.00
 1997 Am erican Chem ical Society

dark. The exact concentrations of the corrinoids were
determined spectrophotometrically as dicyano complexes
-
1
-1
using the ꢀ367 value of 30500 M cm (26). All solutions
were N saturated and anaerobic.
2
Solutions of 100 mM buffer were titrated to the desired
pH at 35 °C using HCl or NaOH. Stock solutions of 100 mM
titanium(III) citrate were prepared in an anaerobic glovebox
containing 96% N
2 2
and 4% H according to the procedure
described in ref 27. The final concentration of citrate was
3
00 mM. Titanium(III)-NTA stock solutions of 100 mM were
prepared as described in ref 28. The pH of the stock solutions
of titanium(III) citrate and titanium(III)-NTA was adjusted
with Na CO . These solutions were then kept in the glovebox
2 3
at room temperature.
Experim ental Procedures. The kinetic experiments were
conducted in triplicates using 59-mL serum flasks sealed with
a Viton stopper (Maagtechnik, D u¨ bendorf, Switzerland) and
an aluminum crimp cap. The reaction mixtures were
prepared in the glovebox. The pH buffer concentration was
FIGURE 1. Reduction of 310 µM cis-DCE in 150 µM cobalamin and
10 mM titanium(III) citrate at 35 °C. The lines are best fits using the
model described by eq 1 and including the competition of the products
(for details see text). (2) At pH 7; (b) at pH 8; (9) at pH 8 in 10 mM
titanium(III)-NTA; (1) at pH 9.
9
0 mM, and the titanium(III) citrate or titanium(III)-NTA
concentrations were 10 mM. The concentrations of cobal-
amin were adjusted by adding the appropriate aliquot from
the stock solution. The concentration of the cobalamin was
in the range of 100-250 µM for all kinetic experiments and
was always smaller than the initial concentration of the
halogenated compound. The headspace of the reaction
mixture was less than 2 mL.
The experiment was started by adding 57 µL of an
anaerobic ethanolic stock solution of the dichloroethene with
a gas-tight Hamilton syringe through the Viton stopper or by
adding pure VC. The flasks were incubated at 35 °C in the
dark. At appropriate time-intervals, 100-µL samples were
withdrawn with a gas-tight syringe and extracted with 0.8 mL
of pentane containing a standard. Prior to the withdrawal
Analytical Procedures. The pentane extracts were ana-
lyzed on a Carlo Erba HRGC 5160 equipped with an electron
capture detector (Carlo Erba ECD 400) with a Ni-63 source
or on a Carlo Erba MFC Mega Series GC equipped with a
QMD 1000 mass spectrometer. The column used was a 30-m
fused-silica DB-624 from J&W Scientific. Concentrations were
calculated from a calibration curve using cis- or trans-DCE
as a standard.
Headspace samples were analyzed by GC on a Carlo Erba
HRGC 5160 equipped with a flame ionization detector (FID-
40, Carlo Erba) or on a Fisons Instruments GC 8165 equipped
with a Fisons MD 800 mass spectrometer. The column was
a 30-m megabore GS-Q from J&W Scientific. Quantification
was done with pentane as the standard and by comparing
with a calibration curve prepared at 35 °C.
of the sample, 100 µL of clean N
2
was injected. Any reaction
due to the bulk reductant was evaluated using control vials
that did not contain cobalamin.
The relative amounts of the deuterated versus non-
deuterated products were calculated from the intensity ratios
of the respective molecular ions, i.e., m/z 62 and 63 for VC,
m/z 30 and 31 for ethane, m/z 28 and 29 for ethene, and m/z
26 and 27 for acetylene.
Spectrophotom etric Studies. Spectrophotometric meas-
urements were conducted to study the kinetics of the oxidation
of the cobalamin during the reaction. In the glovebox, 7 µL
of an oxygen-free ethanol solution of the DCE or 15 µL of
pure VC was added to a 1-cm quartz cuvette with a gas-tight
syringe. The cuvette containing 1.4 mL of a solution of 20-
50 µM cobalamin, 10 mM titanium(III) citrate, and 90 mM
buffer was then sealed with a Teflon stopper. Spectra were
recorded on a Kontron Uvikon 810 spectrophotometer at 35
°C.
The product studies were carried out in 8-mL sealed serum
flasks containing 2 mL of reaction mixture. The preparation
of the reaction mixture was identical to that for the kinetic
experiments. The concentration of cobalamin was 20-100
µM. The reaction was started by adding 10 µL of the ethanolic
stock solution of the compound to the mixture. The stock
solution contained pentane as the internal standard. The
vials were incubated at 35 °C in the dark. The 200-µL samples
of the headspace were taken at appropriate times with a gas-
tight syringe. Control vials contained 2 mL of buffer without
titanium(III) and cobalamin to estimate the loss through the
stopper and the loss due to sorption. To account for the
products formed from the reaction with the bulk reductant,
control experiments with vials containing only 10 mM
titanium(III) and no cobalamin were also performed.
•
Results and Discussion
D abstraction experiments were conducted in duplicates
in 8-mL sealed serum flasks containing 4 mL of the reaction
mixture. Experiments with 1,1-DCE and VC were carried out
at pH 9.0 and pH 7.0; experiments with cis- and trans-DCE
were carried out at pH 7.0. Different volumes of isopropyl
Reaction Kinetics and Product Distribution. As illustrated
in Figure 1 for cis-DCE and found for all chlorinated ethenes
(CEs) studied, distinctly different disappearance patterns were
observed at different pH values.
alcohol-d
7
(0.1%, 0.5 or 2% of the total volume of the solution)
At pH 7, a fast initial decrease in CE concentration followed
by a much slower process was observed. As shown for cis-
DCE and 1,1-DCE in Figures 2a and 3a, respectively, in the
initial phase, almost no volatile reaction products could be
detected. Some products appeared with increasing time, but
an almost complete recovery of the reacted CE in volatile
products was only achieved after the pH was increased above
•
were added as D donor to the reaction mixture. To start the
reaction, 4 µL of an ethanolic stock solution of the chlorinated
ethene was given to the solution. Headspace samples (200
µL) were taken during 2-3 half-lives of the halogenated
compounds and analyzed by GC-MS.
Experiments in D
titanium(III) citrate (pH 7) and the 100 mM PIPES buffer
solution were lyophylized and then resuspended in D O.
Cobalamin stock solutions were prepared in D O. The
experimental setup was the same as for the experiments with
isopropyl alcohol-d . An anaerobic ethanolic stock solution
2
O. The 100 mM stock solutions of
9 by the addition of Na
2 3
CO to the reaction solution (see
2
arrows in Figures 2a and 3a). Obviously the nonvolatile
reaction intermediate(s) that was(were) formed at the more
positive redox potential at pH 7 reacted only very slowly to
yield the volatile products. We suspect that these intermediate
products were alkylcobalamin species formed by addition of
cob(I)alamin to the CEs. In fact, as is illustrated in Figure 4
2
7
of 1,1-DCE (4 µL) was added to start the reaction. After 3
half-lives, the products were analyzed by GC-MS.
VOL. 31, NO. 11, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3 1 5 5

FIGURE 3. (a) Reduction of 200 nmol of 1,1-DCE (b) in 70 µM
cobalamin and 10 mM titanium(III) citrate at pH 7 and 35 °C. Time
courses of the masses of 1,1-DCE and its transformation products;
VC (O), acetylene (4), ethene (9), and ethane (2). The arrow marks
the time at which the pH of the solution was increased to above
FIGURE 2. Reduction of 400 nmol of cis-DCE (b)in 100µM cobalamin
and 10 mM titanium(III) citrate at 35 °C. Time courses of the masses
of cis-DCE and its transformation products; VC (O), ethene (9), and
ethane (2). (a) At pH 7; (b) at pH 8. Note that in the mass balances
only volatile compounds are included. The arrows indicate the
increase of pH above 9.
9. (b) Reduction of 400 nmol of 1,1-dichloroethene in 25 µM cobalamin
and 10 mM titanium(III) citrate at pH 9 and 35 °C. Time courses of
the masses of 1,1-DCE and its transformation products. Note that in
the mass balances only volatile compounds are included.
for cis-DCE, when a given CE was added to a solution of
cob(I)alamin in titanium(III) citrate at pH 7, the intensive
absorption band at 390 nm that is characteristic for cob(I)-
alamin disappeared, and a new band appeared with a
maximum around 520 nm, which is typical for alkylcobal-
amins (26). These findings suggest that a cobalt-carbon bond
was formed in the initial reaction step. When the pH was
increased above 9, the band at 520 nm disappeared, and the
band at 390 nm increased again, indicating cleavage of the
Co-carbon bond and regeneration of cob(I)alamin.
The behavior observed at pH 8 was qualitatively similar
to that observed at pH 7 (Figures 1 and 2b). However, the
rate of the initial reaction, i.e., the addition of the cob(I)-
alamin to the CEs, was significantly lower than at pH 7.
Nevertheless, volatile products appeared much earlier (Figure
2
b), indicating that the difference between the relative rates
of formation and cleavage of the Co-carbon bond was smaller
as compared with the rates at pH 7.
In contrast to the cobalamin (29), the reduction potential
of the Ti(IV)/ Ti(III) couple is strongly pH-dependent between
pH 7 and pH 9 [i.e., ∆E/ ∆pH ) -60 mV at 25 °C (30)].
Consequently, as already discussed in a previous paper (8),
the rate of regeneration of superreduced cobalamin increases
with increasing pH. The earlier assumption that the rate of
regeneration of the cob(I)alamin increases with decreasing
redox potential is also illustrated by the differences in the
disappearance pattern of cis-DCE in titanium(III) citrate and
titanium(III)-NTA, respectively, at pH 8 (Figure 1). In the
initial phase that was dominated by the addition reaction, no
significant differences were observed. Later on, however,
when the regeneration of the cob(I)alamin became more
important, the decrease in cis-DCE was slower in tita-
nium(III)-NTA, which can be explained with the more
FIGURE 4. UV/VIS spectra of 40 µM cobalamin in 10 mM titanium(III)
citrate solution at pH 7 at 35 °C. Spectra before and after the addition
of 1200 µM cis-DCE.
positive redox potential of titanium(III)-NTA as compared
to titanium(III) citrate (8).
At pH 9, finally, the reduction of CEs was even slower than
at pH 7 and pH 8, and it followed pseudo-first-order kinetics.
Most likely, at the low redox potential at pH 9, the regeneration
of the cob(I)alamin was fast as compared to the much slower
formation of the addition product and was never rate-limiting.
Thus, a constant steady-state concentration of superreduced
cobalamin was established. An additional possibility is that,
at this pH, some of the CEs, particularly 1,1-DCE (see below),
were reduced by a dissociative one-electron transfer as we
3
1 5 6
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 11, 1997

TABLE 1. Pseudo-Second-Order Rate Constants, k′add, and Third-Order Rate Constants, kadd, for Reaction of 1,1-DCE, VC, trans-DCE,
and cis-DCE with Cob(I)alamin
k′ (M- s-1
1
)
add
kadd (M s-1
)
d
-2
pH 7.05
pH 8.05
pH 9.0
a
a
2
2
2
0
PCE
TCE
1
(1.2 ( 0.07) × 10
(1.4 ( 0.4) × 10
(1.4 ( 0.2) × 10
(2.1 ( 0.5) × 10
0
0
(2.4 ( 0.2) × 10
(2.1 ( 1) × 10
1
0
-1 b
8
,1 DCE
(8 ( 1) × 10
(7 ( 1) × 10
(7.5 ( 2) × 10
(7.5 ( 2) × 10
(4.1 ( 2) × 10
0
-1 b
-2
7
VC
(2 ( 1) × 10
(4.4 ( 1) × 10
(6 ( 1) × 10
-
1
2
-2 b
-3 c
6
5
trans-DCE
cis-DCE
(1.3 ( 0.1) × 10
(1.1 ( 0.3) × 10
(2.4 ( 0.5) × 10
(1.6 ( 0.7) × 10
-
-3
-4 c
(2.8 ( 0.5) × 10
(5 ( 0.3) × 10
(6 ( 1) × 10
(4.3 ( 1.6) × 10
a
b
k
2
values determ ined at 35 °C in titanium (III) citrate (7); see also eqs 1 and 2. Average value from experim ents with titanium (III) citrate and
titanium (III)-NTA. c Determ ined in titanium (III)-NTA. d
k
add ) k′add/[H + ].
have suggested for the reduction of PCE and TCE (8).
On the basis of these observations and on the findings of
Glod et al. (8) that for PCE, TCE, and 1,1-DCE (data not shown),
over a reasonably large mediator concentration (0.1-10 µM
cobalamin), the initial part of the reaction was always first-
order in both the CEs and the cobalamin, we postulate that
under the conditions investigated the reduction of the DCEs
and VC occurred primarily by a two-step process, i.e., a pH-
dependent addition of the cob(I)alamin to the CE forming an
alkylcobalamin followed by a reductive cleavage of the Co-
carbon bond:
TABLE 2. Estimates of Pseudo-First-Order Rate Constants, kred
-
1
(
h ), for Cleaving of Co-Carbon Bond of Addition Products
of cis-DCE, trans-DCE, 1,1-DCE, VC, Acetylene, and Ethene
with Cob(I)alamin As Determined with Software Package
AQUASIM (31)
titanium(III) citrate
titanium(III)-NTA
pH 7.05
pH 8.05
pH 8.05
cis-DCE
trans-DCE
>0.02
>0.02
>0.02
0.02
>0.02
>0.02
>0.02
0.02
>0.005
>0.01
1
,1-DCE
VC
0.01a
k′
kred
add
acetylene
ethene
0.01
0.001
0.02
0.003
CE + cob(I)alamin H+ 8 alkylcobalamin
8
e-
0.001a
cob(II)alamin + products (1)
a
Determ ined by m odeling the data of cis- and trans-DCE at pH 8 in
titanium (III)-NTA.
where, for given pH and redox-conditions, k′add is the
pseudo-second-order and kred is the pseudo-first-order rate
constant, respectively, for the two reaction steps. The
corresponding rate laws can be written as
Figure 2) does not imply that VC was not a major initial
transformation product.
In order to be able to model the disappearance kinetics
of cis- or trans-DCE or VC particularly over longer time periods
(see Figure 1), competition for the cob(I)alamin between the
DCE and the reduction products including one major
transformation product, ethene (see Figure 2), had to be taken
into account. With this assumption, the experimental data
could be fitted very well for both cis-DCE (Figure 1) and trans-
DCE (data not shown). The kadd and kred values used for VC
and ethene were obtained from independent experiments
with vinyl chloride. The kred values determined with the
software package AQUASIM (31) are summarized in Table 2.
Note that these values should be taken as rough estimates
determined using the data for pH 7 and pH 8 only, because
at pH 9 the addition reaction was primarily rate-limiting.
d[CE]
dt
-
) ka′dd[CE][cob(I)alamin]
(2)
and
d[alkylcobalamin]
)
ka′dd[CE][cob(I)alamin] -
dt
kred[alkylcobalamin] (3)
Table 1 summarizes the k
a
dd values derived for all
compounds using only data from the initial phase (<50-100
h) of the reaction, where for all pH values it can be assumed
that the disappearance rate of a given CE can be described
solely by eq 2. Also included in Table 1 for comparison are
the more or less pH-independent second-order rate constants,
-
1
Table 2 indicates that kred of the DCEs (>0.02 h ) was
slightly larger than k of vinyl chloride and acetylene (0.02
red
-1
k
2
, for the reaction of PCE and TCE with cob(I)alamin that
h ) and that they were almost unaffected by changes in redox
were determined for the same reaction conditions in an earlier
work (8).
From the data in Table 1, an important conclusion can be
drawn. For all three DCEs and for VC, an increase in pH by
potential. The calculated kred of ethene was about 10 times
smaller than the kred of VC and was more influenced by
changes in redox potential. Thus, at lower pH values, where
the addition of cob(I)alamin is faster, the decomposition of
these addition products may become rate-limiting. This
conclusion is also supported by results obtained from UV/ vis
and mass spectroscopic analyses of the adducts that were
isolated from the reaction mixtures. These measurements
indicated that vinylcobalamin and mainly ethylcobalamin
were major reaction intermediates (data not shown, see ref
1
1
pH unit resulted in a decrease in kadd by about a factor of
0. This suggests that a proton was involved in the rate-
determining step. Thus, eq 2 can be rewritten as
d[CE]
dt
+
-
) kadd[H ][CE][cob(I)alamin]
(4)
3
2).
Mechanistic Considerations. cis-DCE, trans-DCE, and
where kadd ) k
a
dd/ [H + ] is the third-order rate constant for the
-
2
VC. As mentioned above and illustrated in Figure 2, the major
volatile products that accumulated from cis- and trans-DCE
and VC were ethene and ethane.
The formation of the CE-cob(III)alamin intermediate may,
in principle, proceed via two alternative pathways: (i) via a
nucleophilic addition mechanism, which is analogous to the
nucleophilic substitution mechanism postulated for the
-
1
s
. The kadd values reveal large differences in reactivities
between the four CEs (see Table 1). Of particular importance
is the finding that VC, a possible transformation product of
cis- and trans-DCE, reacted much faster with the cob(I)alamin
than its precursors. Thus, the fact that almost no VC was
detected in the experiments with cis- and trans-DCE (see
VOL. 31, NO. 11, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3 1 5 7

FIGURE 5. Proposed pathway for the reduction of cis-DCE, trans-DCE, and VC.
reaction of chlorinated methanes with cob(I)alamins (12, 33, these two compounds. Furthermore, the addition of cob(I)-
3
4), or (ii) via an electron transfer mechanism leading to the
alamin to cis- and trans-DCE may be slowed down because
of the presence of a bulky chlorine atom on each carbon. If
it is assumed that the addition of Co(I) follows a trans-mode,
the following conformational analysis can be made:
formation of a CE radical and cob(II)alamin, which subse-
quently recombine again, as has been suggested by Toscano
and Marzilli (35) for Co(I) substitution of certain alkylhalides.
Such a recombination was also postulated to occur during
the reaction of chlorinated methanes with cob(II)alamin (9,
1
3). The latter reaction mechanism seems, however, rather
unlikely for the reaction of cis- and trans-DCE and VC. First,
in experiments carried out in the presence of various amounts
•
of isopropyl alcohol-d
7
, which is an excellent D donor, in
contrast to PCE, TCE, TCEF (8), and 1,1-DCE (see below), no
significant amounts (<2%) of deuterated volatile products
were detected at pH 7 and pH 9 (data not shown), indicating
that no significant amounts of free radicals were formed in
solution during the reaction. Second, it is difficult to
rationalize the observed pH dependence with a radical
mechanism. Thus, we postulate that the reaction proceeded
by an addition of the cob(I)alamin to the CE with simultaneous
protonation to form the corresponding R,â-dichloroethyl-
cobalamin in the case of cis- and trans-DCE and â-chloro-
ethylcobalamin in the case of VC (see Figure 5). These
alkylcobalamins were then probably cleaved via â-elimination
into a chloride and an intermediary cobalt(III)-π-complex
that decomposed into vinyl chloride or ethene, respectively.
Such cobalt(III)-π-complexes have been postulated as
intermediates in the reaction of olefins carrying a nucleofugal
group at the â-carbon (33-35). In addition, the increase in
rate with increasing proton concentration has also been
observed before for the reaction of cob(I)alamin with isolated
olefins (36) and with R,â-unsaturated nitriles (37).
The adduct of the trans-DCE is slightly more stable than
the corresponding adduct of cis-DCE due to a gauche
interaction of the two chlorine atoms. Thus, according to
Hammond’s postulate, the transition state of the trans-DCE
adduct is energetically more favorable and therefore more
rapidly formed. This is in accordance with the results
observed by Johnson and Meeks (41) for the reaction of the
bis(dimethylglyoximinato)pyridinecobalt(I) anion with cis-
and trans-â-bromostyrenes. Consequently, the kadd of 1,1-
DCE was largest because the anion formed was best stabilized
by the two chlorines on the R-carbon atom.
1,1-DCE. In contrast to cis- and trans-DCE, in the case of
1,1-DCE quite different product patterns were found at
different pH values. At pH 7 (Figure 3a), acetylene was
detected as an important intermediate, and only a minor
amount of VC accumulated. Since VC reacts more slowly
with cob(I)alamin than 1,1-DCE (Table 1), one can conclude
that the reduction of 1,1-DCE to VC was not a major pathway
Table 1 shows that cis-DCE exhibits the smallest kadd of all
compounds studied. Due to the π-donor effect of â-Cl, the
carbanions formed from cis-and trans-DCE were probably
the least stable, which could explain the lower reactivity of
3
1 5 8
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 11, 1997

TABLE 3. Percent of Deuterated Ethene and VC formed from 1,1-DCE at pH 7 and pH 9 as a Function of Amount of Isopropyl
Alcohol-d Present in Solution
7
%
of deuterated
amount of isopropyl
alcohol-d7 (in % of total vol)
% of deuterated
ethene at pH 7a
% of deuterated
VC at pH 7b
% of deuterated
ethene at pH 9a
VC the product of
1,1-DCE at pH 9
b
0
0
2
.1
.5
2.3 ( 0.2
2.4 ( 0.1
18 ( 0.5
6.7 ( 0.8
8.1 ( 0.8
31.3 ( 0.4
2.1 ( 0.1
4.1 ( 0.3
4.5 ( 0.1
10 ( 2
15 ( 4
52 ( 0.5
a
Total ethene ) 100%. b Total VC ) 100%.
as intermediate (see Figure 6), is likely to involve also an
alternative mechanism with a radical intermediate. In analogy
to the reaction of PCE and TCE with cob(I)alamin (8), a likely
mechanism is therefore a dissociative electron transfer
yielding a chlorovinyl radical as intermediate, which could
also account for the formation of the minor amounts of VC
detected at pH 7 (Figure 3a, Table 3).
−
The deuterated ethene found under these conditions origi-
nated probably from the transformation of deuterated VC
reacting further as shown in Figure 5.
Finally, the mass balances given in Figures 2 and 3 show
that even when the redox potential was lowered by increasing
the pH to over 9, only 80-90% of the reacted CE could be
recovered as volatile products. GC-MS measurements
indicated that also a more polar product, acetaldehyde, was
present in the reaction mixture (data not shown). Thus, it
is likely that the postulated cobalt(III)-π-complex (see above)
-
reacted with OH at elevated pH values to give hydroxy-
ethylcobalamin, which then decomposed to yield acetalde-
hyde or ethanol. Similar reactions have been postulated
before for cobalt(III)-π-complexes by Silvermann and Dol-
phin (36). In this study, the formation of these products has
not been investigated further.
Sum m arizing Rem arks and Environm ental Significance.
Together with our earlier work (8), this study provides
important information on the cobalamin-mediated reduction
of chlorinated ethenes in aqueous solution. The results
suggest that the highly chlorinated ethenes (i.e., PCE, TCE)
reacted primarily via a pH-independent dissociative one-
electron transfer, while the initial step for the less chlorinated
ethenes (i.e., cis- and trans-DCE, 1,1-DCE, VC) was a proton-
dependent addition of the cob(I)alamin to the CE. Figure 7
summarizes the reaction pathways and relative second-order
rate constants for the various CEs at pH 7, 8, and 9. Also
included are rough estimates of the rate constants for the
addition of cob(I)alamin to acetylene and ethylene. From
these data some important conclusions can be drawn. First,
in the pH range between 7 and 9, trans- and particularly
cis-DCE are by far the most unreactive compounds and will
therefore accumulate when reducing PCE or TCE by cob(I)-
alamin. These findings are consistent with the results of
laboratory and field studies on the microbial reduction of
PCE and TCE, where cis-DCE is commonly found as a major
metabolite (19, 21, 22, 42). Furthermore, if, as suggested by
the results of our earlier work (8), the reduction of PCE and
TCE does not yield any 1,1-DCE, it can be expected that no
significant amounts of VC, the most toxic CE, should be
produced. Thus, in cases in which the reduction of VC was
found to be the rate-limiting step in the complete microbial
dehalogenation of PCE to ethene (20, 43), either the addition
of the cobalamin to the CE was not rate-determining in the
enzymatic reaction or other cofactors such as factor F430,
which may react via an electron transfer mechansim (44, 45),
may have been involved instead of superreduced cobalamin.
FIGURE 6. Proposed pathway for the reduction of 1,1-DCE.
at pH 7. A possible mechanism is postulated in Figure 6.
After the addition of cob(I)alamin to 1,1-DCE, the deproton-
ated form of the â-dichloroethylcobalamin (Ib) loses a chloride
to yield a carbene intermediate (II), which is transformed
into â-chlorovinylcobalamin (III) by transfer of an R-H. In
analogy to the reactions of cis- and trans-DCE and VC, the
â-chlorovinyl cobalamin is then converted to acetylene by
â-elimination through a cobalt(III)-π-complex (IV). Some
preliminary evidence for this postulated H-shift was obtained
from experiments conducted in D
2
O. In these experiments,
the acetylene formed contained only H and no D, indicating
that no acetylene anion was formed during the reaction and
that the hydrogens probably originated from 1,1-DCE.
Further work using deuterated 1,1-DCE is presently in progress
to consolidate this hypothesis. The acetylene formed reacted
then further to ethene that was finally reduced to ethane.
At pH 9 (Figure 3b) where the overall reaction was much
slower than at pH 7, about equal amounts of VC and ethene
were formed simultaneously. Under these conditions, a
significant portion of the VC, but very little of the ethene that
was formed, was deuterated in the presence of isopropyl
alcohol-d
7
(Table 3). This indicates that the ethene originated
most likely from a fast reaction of acetylene. However, the
-
formation of VC, in addition to â-elimination of a Cl from
â-dichloroethylcobalamin (Ia) with a cobalt(III)-π-complex
VOL. 31, NO. 11, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3 1 5 9

(
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Acknowledgments
We thank Thomas Bucheli for help with GC/ MS measure-
ments, J o¨ rg Klausen for help with AQUASIM, Francisco
Vazquez for technical assistance, and Bernhard Kr a¨ utler for
valuable discussions. Johanna Buschmann, Sarra Gaspard,
J o¨ rg Klausen, Hans-Peter Kohler, and Wolfram Schumacher
are kindly acknowledged for reviewing the manuscript.
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