Environ. Sci. Technol. 1997, 31, 3154-3160
Cobalamin-Mediated Reduction of cis- and trans-Dichloroethene, 1,1-Dichloroethene, and Vinyl Chloride in Homogeneous Aqueous Solution: Reaction Kinetics and Mechanistic Considerations GUY GLOD,† URS BRODMANN,† WERNER ANGST,‡ CHRISTOF HOLLIGER,† AND 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 Du ¨ bendorf, Switzerland, and Limnological Research Center, EAWAG, CH-6047 Kastanienbaum, Switzerland
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.
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 * Corresponding author e-mail address:
[email protected]; telephone: 41 1 8235109; fax: 41 1 8235471. † EAWAG, Kastanienbaum. ‡ EAWAG and ETH, Du ¨ bendorf.
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reduction of such halogenated compounds by a variety 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 remediation approaches of contaminated soils and aquifers (15). 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), trichloroethene (TCE), and tetrachloromethane (6-9). In previous work (8), we have shown that PCE was transformed quantitatively 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-dichloroethene (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 disadvantageous for the use of such species as electron transfer mediators in treatment processes. In the work presented in this paper, we have investigated the effect of pH and redox potential on the cobalaminmediated reduction of cis-DCE, trans-DCE, 1,1-dichloroethene (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-trichloroethane (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 compounds), it is also essential for selecting optimal conditions for the treatment of PCE and TCE wastes and contaminations with corrinoids as electron transfer mediators.
Experimental Section Chemicals 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-Dichloroethene (99%) and trans-dichloroethene were purchased from Aldrich Chemie, Switzerland. 2-Amino-2-(hydroxymethyl)1,3-propanediol (Tris) for pH 8 solution, TiCl3 (15% in 10% HCl), and Na2CO3 were from Merck AG (Dietikon, Switzerland). A gas mixture containing methane, acetylene, ethene, ethane, carbon monoxide, and carbon dioxide (1% each in N2) (Scott Specialty Gases) was purchased from Supelco. Isopropyl alcohol-d7 (99.5%) and D2O were from Armag AG (Do¨ttingen, Switzerland). All chemicals were used as received. Stock solutions of the halogenated ethenes (0.1 M) were 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
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1997 American Chemical Society
dark. The exact concentrations of the corrinoids were determined spectrophotometrically as dicyano complexes using the 367 value of 30500 M-1 cm-1 (26). All solutions were N2 saturated and anaerobic. 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% N2 and 4% H2 according to the procedure described in ref 27. The final concentration of citrate was 300 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 Na2CO3. These solutions were then kept in the glovebox at room temperature. Experimental Procedures. The kinetic experiments were conducted in triplicates using 59-mL serum flasks sealed with a Viton stopper (Maagtechnik, Du ¨ bendorf, Switzerland) and an aluminum crimp cap. The reaction mixtures were prepared in the glovebox. The pH buffer concentration was 90 mM, and the titanium(III) citrate or titanium(III)-NTA concentrations were 10 mM. The concentrations of cobalamin 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 of the sample, 100 µL of clean N2 was injected. Any reaction due to the bulk reductant was evaluated using control vials that did not contain cobalamin. 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 gastight 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. 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 alcohol-d7 (0.1%, 0.5 or 2% of the total volume of the solution) 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 D2O. The 100 mM stock solutions of titanium(III) citrate (pH 7) and the 100 mM PIPES buffer solution were lyophylized and then resuspended in D2O. Cobalamin stock solutions were prepared in D2O. The experimental setup was the same as for the experiments with isopropyl alcohol-d7. An anaerobic ethanolic stock solution of 1,1-DCE (4 µL) was added to start the reaction. After 3 half-lives, the products were analyzed by GC-MS.
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. Analytical Procedures. The pentane extracts were analyzed 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 (FID40, 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. The relative amounts of the deuterated versus nondeuterated 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. Spectrophotometric Studies. Spectrophotometric measurements 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 2050 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.
Results and Discussion 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. At pH 7, a fast initial decrease in CE concentration followed by a much slower process was observed. As shown for cisDCE 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 9 by the addition of Na2CO3 to the reaction solution (see 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
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FIGURE 2. Reduction of 400 nmol of cis-DCE (b) in 100 µM 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. 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 alkylcobalamins (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 2b), 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 titanium(III)-NTA, which can be explained with the more
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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 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.
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
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′add (M-1 s-1) PCEa TCEa 1,1 DCE VC trans-DCE cis-DCE
pH 7.05
pH 8.05
pH 9.0
kaddd (M-2 s-1)
(1.2 ( 0.07) × 102 (2.4 ( 0.2) × 100 (8 ( 1) × 101 (2 ( 1) × 100 (1.3 ( 0.1) × 10-1 (2.8 ( 0.5) × 10-2
(1.4 ( 0.4) × 102 (2.1 ( 1) × 100 (7 ( 1) × 100 (4.4 ( 1) × 10-1 b (1.1 ( 0.3) × 10-2 b (5 ( 0.3) × 10-3
(1.4 ( 0.2) × 102 (2.1 ( 0.5) × 100 (7.5 ( 2) × 10-1 b (6 ( 1) × 10-2 (2.4 ( 0.5) × 10-3 c (6 ( 1) × 10-4 c
(7.5 ( 2) × 108 (4.1 ( 2) × 107 (1.6 ( 0.7) × 106 (4.3 ( 1.6) × 105
a k values determined at 35 °C in titanium(III) citrate (7); see also eqs 1 and 2. 2 titanium(III)-NTA. c Determined in titanium(III)-NTA. d kadd ) 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 firstorder 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 pHdependent addition of the cob(I)alamin to the CE forming an alkylcobalamin followed by a reductive cleavage of the Cocarbon bond: k′add
kred
CE + cob(I)alamin 9 8 alkylcobalamin 9 8 + H
e
cob(II)alamin + products (1) 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
-
d[CE] ) k′add[CE][cob(I)alamin] dt
(2)
and
d[alkylcobalamin] ) k′add[CE][cob(I)alamin] dt kred[alkylcobalamin] (3) Table 1 summarizes the k′add values derived for all compounds using only data from the initial phase (0.02 >0.02 >0.02 0.02 0.01 0.001
>0.02 >0.02 >0.02 0.02 0.02 0.003
>0.005 >0.01 0.01a 0.001a
a Determined by modeling the data of cis- and trans-DCE at pH 8 in titanium(III)-NTA.
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 transDCE (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. Table 2 indicates that kred of the DCEs (>0.02 h-1) was slightly larger than kred of vinyl chloride and acetylene (0.02 h-1) and that they were almost unaffected by changes in redox 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 32). Mechanistic Considerations. cis-DCE, trans-DCE, and 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
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FIGURE 5. Proposed pathway for the reduction of cis-DCE, trans-DCE, and VC. reaction of chlorinated methanes with cob(I)alamins (12, 33, 34), or (ii) via an electron transfer mechanism leading to the formation of a CE radical and cob(II)alamin, which subsequently 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, 13). 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-d7, which is an excellent D• donor, in contrast to PCE, TCE, TCEF (8), and 1,1-DCE (see below), no significant amounts (
