Environ. Sci. Technol. 1991, 25, 715-722
(10) Grosjean, D. Enuiron. Sci. Technol. 1982, 16, 254. (11) Salas, L. J.; Singh, H. B. Atmos. Enuiron. 1986,20, 1301. (12) Grosjean, ID.,Fung, K. J . Air Pollut. Control Assoc. 1984, 34, 537. (13) Rogozen, M. B.; Maldonado, G.; Grosjean, D.; Shochet, A.; Rapaport, R. Formaldehyde: a survey of airborne concentrations and sources. Contract A2-052-32, California Air Resources Board, Sacramento, CA, 1984. (14) Grosjean, 13. Atmos. Enuiron. 1988, 22, 1637. (15) Lawson, D. R.; Biermann, H. W.; Tuazon, E. C.; Winer, A. M.; MacKay, G. I.; Schiff, H. I.; Kok, G. L.; Dasgupta, P. K.; Fung, I tetrachloroethylene > hexachlorobenzene. For hematin, the order of reductive dechlorination rates was carbon tetrachloride > hexachlorobenzene > tetrachloroethylene. Within each class of compounds, rates of dechlorination decreased with decreasing chlorine content. Regio- and stereospecificity were observed in these reactions. In the reductive dechlorination of trichloroethylene, cis-1,2-dichloroethylene was the predominant product formed with vitamin B,,, coenzyme F430, and hematin. Pentachlorobenzene and pentachlorophenol were each dechlorinated by vitamin BI2to yield two out of three possible isomeric tetrachlorobenzenes. Similar relative kinetics and dechlorination products have been observed in anaerobic cultures, suggesting a possible role of transition-metal coenzymes in the reductive dechlorination of polychlorinated compounds in natural and engineered environments.
Some aerobic microorganisms can biodegrade a limited number of chlorinated organic molecules, but often fail to metabolize the most heavily chlorinated compounds. For example, several bacteria capable of oxidizing toluene, methane, or ammonia can cooxidize trichloroethylene, dichloroethylenes, and vinyl chloride by using nonspecific catabolic oxygenases (4-7). Tetrachloroethylene is not oxidized by any of these enzyme systems. Similarly, aerobic bacteria that rapidly biodegrade monochlorinated benzenes are usually unable to oxidize heavily chlorinated aromatic compounds (8). It has been suggested that anaerobic and aerobic biodetoxification of chlorinated wastes could be used in a sequential treatment process. For example, methanogens and other anaerobes in consortia can catalyze a slow reductive dechlorination of tetrachloroethylene to trichloroethylene, making the pollutant available for rapid oxidative degradation (9-13). A similar trend is observed with chlorinated benzenes. Highly chlorinated benzenes such as the fungicide hexachlorobenzene and polychlorinated biphenyls (PCBs) undergo reductive dechlorination in anaerobic environments (14,15). The products are congeners bearing fewer chlorine substituents, which are more susceptible to biodegradation by aerobic bacteria
Introduction Chlorinated organic molecules constitute the largest single group of compounds on the list of priority pollutants compiled by the 1J.S.Environmental Protection Agency (EPA) (1). As is true for most organic compounds, the turnover of chlorinated molecules in the environment is largely determined by their susceptibility to biotransformation by microorgpnisms (2). Many of the chloroorganics that are not biodegraded by bacteria and fungi have the potential to persist in the environment and to express their toxicity over extended periods of time ( 3 ) .
The environmental and public health significance of microbial-catalyzed reductive dechlorination reactions has fostered efforts to understand the molecular basis of these biotransformations. A unique anaerobic bacterium, denoted DCB-1, has been studied in pure culture and demonstrated to reduce tetrachloroethylene to trichloroethylene (13). DCB-1 can biosynthesize ATP by coupling hydrogen oxidation to reduction of the carbon-chlorine bond of 3-chlorobenzoate (18, 19), suggesting the involvement of specific enzymes in that reaction. In contrast, Krone et al. (20,211 have presented evidence that suggests the reduction of carbon tetrachloride to methane by
0013-936X/91/0925-0715$02.50/0
(16, 17).
0 1991 American Chemical SOC:iety
Environ. Sci. Technol., Vol. 25, No. 4, 1991
715
(A) METAL COORDINATED TO NON-PROTEIN LIGANDS
HCH I HCH I
C%H
Coenzyme F430
HCH I
HCH I C%H
Hematin CH H'
c-c
1
H>H
HO-CH2 I \ 0 /
Vitamin BI2 (B) METAL COORDINATED DIRECTLY TO PROTEIN LIGANDS
,s-cys S-Fe'
NS
NS
q-7:
N
NS
A
?,Met
-
/s-cys Fe&
Cluster
Fe4S4 Cluster
is
&CH3
Cu Center of Azurin
Figure 1. Biological metal centers examined for reductive dechlorination activity.
methanogens is mediated nonspecifically by cobalamins, by the nickel-containing coenzyme F430,or by both coenzymes. The slow biological reductive dechlorination of polychlorinated alkenes and benzenes may also be mediated by nonspecific processes involving metallocofactors. Figure 1 illustrates two different classes of transition-metal cofactors found in bacteria that grow under anaerobic conditions (22). In the first type, the metal is coordinated by a stable macrocyclic ligand system, which in turn can be bound by proteins. In the second type, metal(s) is(are) directly coordinated to protein ligands. Both types of redox-active metal centers display great versatility in their biological functions. The cobalt-containing cobalamins (23) and the iron coenzyme hematin ( 2 4 ) show catalytic reactivities in addition to their biological role as electron carriers. Iron-sulfur clusters, which also function in electron transfer, are now implicated as key participants in several enzyme-catalyzed hydrolytic reactions (25,26). The structure of coenzyme F430, found uniquely in methanogens ( 2 7 ) ,has only recently been elucidated (28). In nature, an axially reactive nickel center of coenzyme F,,, may particpate in the major biogenic reaction producing methane ( 2 9 ) . Determining the ability of coenzyme F430 to catalyze other reactions is an active field of research (30). Cobalamins, coenzyme F,,,, and hematin have recently been shown to dehalogenate chlorinated methanes in the presence of a reductant (20,21,31). Thus, transition-metal cofactors may mediate nonspecific reactions with hydrophobic chlorinated pollutants that gain entry into bacterial 716
Environ. Sci. Technol., Vol. 25, No. 4, 1991
cells by partitioning through membranes. In this study, we investigated whether the transitionmetal cofactors shown in Figure 1 would catalyze the reductive dehalogenation of chlorinated ethylenes and benzenes. For those cofactors that were reactive, the rates of dechlorination were determined and compared as a function of compound class (alkane, alkene, and benzene) and the number of chlorine substituents. In those reactions where regiospecific dechlorination could occur, the isomeric distribution of the products was determined. The product distribution and modeled rate data were used to compare these coenzymatic dechlorination reactions against previously observed biological dechlorination reactions conducted with the same chlorinated substrates. Materials and Methods Chemicals and Coenzymes. Hematin, vitamin BI2 (cyanocobalamin),chloroplast ferredoxin (Fe2S, cluster), clostridial ferredoxin (Fe& cluster), and azurin were obtained from Sigma Chemical Co. (St. Louis, MO). Cyanoaquacobinamide was kindly provided by Dr. Harry Hogenkamp, Department of Biochemistry, University of Minnesota. Coenzyme F430 was a gift from Drs. Scott Raybuck and Christopher Walsh of the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School. Analytical standards were obtained commercially and as gifts from other laboratories. Tetrachloroethylene, trichloroethylene, cis-dichloroethylene, trans-dichloroethylene, 1,l-dichloroethylene, hexachlorobenzene, pen-
tachlorobenzene, 1,2,4,5-tetrachlorobenzene, 1,2,3,4-tetrachlorobenzene, pentachlorophenol, (2,4,5-trichlorophenoxy)acetic acid, (2,4-dichlorophenoxy)aceticacid, and (3,4-dichlorophenoxy)aceticacid were purchased from Aldrich Chemical Co. (Milwaukee, WI). 1,2,3,5-Tetrachlorobenzene, 2,3,4,6-tetrachlorophenol, and 2,3,5,6tetrachlorophenol were obtained from American Tokyo Kasei, Inc. (Portland, OR). Vinyl chloride was from Fluka Chemical Corp. (Ronkonkoma, NY). Ethylene and ethane were obtained from Matheson Gas Products (Secaucus, NJ). 2,3,4,5-Tetrachlorophenolwas provided by Dr. Ronald Crawford, Department of Microbiology and Biochemistry, University of Idaho. (2,5-Dichlorophenoxy)acetic acid was a gift from Dr. Joseph Suflita, Department of Botany and Microbiology, University of Oklahoma. Dechlorination Reaction Conditions. The products of, and the pseudo-first-order rate constants for, the reductive dehalogenation of carbon tetrachloride and chlorinated ethylenes were determined by analysis of reactions conducted in foil-wrapped 10-mL serum vials crimp-sealed with Teflon-faced rubber septa. The vials contained a 9-mL argon headspace and a 1-mL degassed reaction mixture consisting of 2.2 pmol of the tested chlorinated aliphatic compound, 27 pmol of titanium(II1) citrate or dithiothreitol as the reductant, and 46 nmol of the tested cofactor in a 0.66 M Tris buffer a t pH 8.2 (20, 21). The vials were incubated a t 22 "C on a shaker platform operating a t 225 rpm. Batch experiments examining the reductive dechlorination of pentachlorophenol and (2,4,5-trichlorophenoxy)acetic acid were conducted with the same reaction mixture as described above except with 0.375 pmol of the initial substrate. For product identification, 0.5-mL samples of the reaction mixtures were acidified, extracted into n-pentane, and analyzed by HPLC or GC as described below. The kinetics for the reductive dechlorination of hexachlorobenzene were determined in 10-mL serum vials with a reaction mixture consisting of 2 mL of Tris buffer a t pH 8.2,l mL of tetrahydrofuran, 100 nmol hexachlorobenzene, 15.2 pmol of titanium(II1) citrate, and 150 nmol of the tested cofactor. The tetrahydrofuran was added to increase the solubility of the hexachlorobenzene. Because hexachlorobenzene was considered to be nonvolatile (1.09 x Torr a t 20 "C), pseudo-first-order rate constants (K,) were equal in magnitude to the slope of the line produced when the natural logarithm of hexachlorobenzene concentration in the reaction mixture was plotted versus time. Reaction progress was monitored by withdrawing 900-pL samples from the reaction mixture and extracting with 100 pL of toluene. Because cobalamins may react with tetrahydrofuran under some conditions (321, the above method may underestimate the hexachlorobenzene dechlorination rate values in the presence of vitamin Biz. Analytical Methods. In all experiments using HPLC and GC, authentic standard compounds were used for product identification and quantification. For the chlorinated aliphatics, reaction progress was followed by monitoring the disappearance of the chlorinated compound in the reaction vial headspace with a Hach-Carl AGC-100 gas chromatograph with a flame-ionization detector. It was fitted with various 6-m columns designated below a t nitrogen carrier gas flows of 30 mL min-l. Standard curves were developed by relating the total mass of the tested aliphatic in the reaction vial to the gas chromatograph peak area obtained from a 0.1-mL headspace injection. Headspace samples containing carbon tetrachloride, tet-
rachloroethylene, and trichloroethylene were determined a t 150 "C with a Graphpac AT-1000 column (Alltech Associates, Deerfield, IL). Trichloroethylene, cis-dichloroethylene, trans-dichloroethylene, and 1,l-dichloroethylene were separated and determined with a GasChrom 254 column (Alltech Associates) a t 200 OC. Vinyl chloride and ethylene were determined with a VZ-10 column (Alltech Associates) a t 40 "C. Determination of the pseudo-first-order rate constants for the reductive dehalogenation of the volatile chlorinated aliphatics, except for vinyl chloride, was based on disappearance of the starting material. Because of the very slow rates, vinyl chloride K1 values could be obtained more accurately by monitoring the appearance of the product ethylene. The amount of material in the aqueous phase was derived from the determined quantity in the headspace by using published Henry's law constants (33) and by comparison with standard calibration vials. In rate and product determination experiments, aromatic compounds were extracted into organic solvents and analyzed by HPLC and/or GC. Pentachlorophenol, (2,4,5-trichlorophenoxy)aceticacid, and their dechlorination products were extracted with pentane following acidification of the reaction mixture to pH < 3 with HC1. Toluene was used to extract hexachlorobenzene and pentachlorobenzene. For (2,4,5-trichlorophenoxy)acetic acid, the extracts were injected into a Hewlett-Packard 1084B liquid chromatograph containing a 5-pm Adsorbosphere CI8column (Alltech Associates) with a UV detector a t 285 nm. The isocratic eluent was 30% acetonitrile in water containing 5% acetic acid (v/v). A t a flow rate of 1 mL min-l, the elution times for (2,4,5-trichlorophenoxy)acetic acid, (2,4-dichlorophenoxy)aceticacid, (3,4-dichlorophenoxy)acetic acid, and (2,5-dichlorophenoxy)aceticacid standards were 13.0,8.7,8.5, and 8.0 min, respectively. For pentachlorophenol, the isocratic eluent was 70 70 acetonitrile in water containing 5% acetic acid (v/v) and the UV detector was operated a t 300 nm. At a flow rate of 1 mL min-', the elution times for pentachlorophenol and 2,3,4,5-,2,3,4,6-, and 2,3,5,6-tetrachlorophenol standards were 13.5, 9.4, 9.4, and 9.7 min, respectively. Because 2,3,4,5- and 2,3,5,6-tetrachlorophenol comigrated on the HPLC column, pentachlorophenol and tetrachlorophenol isomer concentrations in the pentane extracts were also determined by using a Hewlett-Packard 5790A Series gas chromatograph with an electron capture detector and a HP-5 (cross-linked 5 % phenyl methyl silicone) capillary column operated at 100 "C for 5 min and increased to 220 "C a t a rate of 5 "C min-'. Total carrier gas flow was 80 mL m i d with 0.25 mL min-l flow down the column. The retention times for the pentachlorophenol and 2,3,5,6-, 2,3,4,6-,and 2,3,4,5-tetrachlorophenolstandards were 22.6, 17.7,17.6, and 17.5 min, respectively. Hexachlorobenzene and pentachlorobenzene concentrations in the toluene solution were determined by using a Spectra Physics HPLC with a 5-pm Adsorbosphere C18column with a UV detector operated at 300 nm. The isocratic eluent was 90% acetonitrile and 10% water. At a flow rate of 1 mL m i d , the elution times for the hexachlorobenzene and pentachlorobenzene standards were 8.4 and 6.9, respectively. The formation of pentachlorobenzene from hexachlorobenzene was also confirmed by GC/MS with a Kratos MS-25 instrument operated by Thomas Krick a t the University of Minnesota Biochemistry Analytical Facility. When pentachlorobenzene was the initial substrate, its dechlorination products in the toluene extracts were determined by using a Hewlett-Packard 5790A Series gas chromatograph with a HP-5 capillary column and an Environ. Sci. Technol., Vol. 25, No. 4, 1991
717
I
2 3 Elapsed Time (hr)
0
1
(111) citrate as the reductant. As shown in Figure 2, concomitant with the disappearance of tetrachloroethylene, a 1:l stoichiometric quantity of trichloroethylene was formed, with small amounts of cis-dichloroethylene appearing later in the batch experiments. Control experiments omitting vitamin Blz or titanium(II1) citrate showed no appreciable tetrachloroethylene disappearance or trichloroethylene production. Experiments performed with dithiothreitol as the reductant instead of titanium(II1) citrate also indicated the stoichiometric dechlorination of tetrachloroethylene to trichloroethylene, although a t greatly reduced rates. These controls were performed because titanium salts are known to reduce a-halo ketones to ketones (34). Additional evidence suggesting the direct participation of the titanium-reduced cobalt center in the reductive dechlorination of tetrachloroethylene was obtained spectrophotometrically. UV/vis absorption spectra were recorded for reduced cyanoaquacobinamide before and after the addition of tetrachloroethylene. Upon addition of titanium(II1) citrate, cyanoaquacobinamide(II1) was reduced to its Co(1) form, indicated by the absorbance band a t 386 nm in Figure 3. Addition of tetrachloroethylene led to a decrease in the 386-nm peak with a concomitant increase in absorbance a t 470 nm and clear isosbestic points. These data are similar to previous electronic spectral changes observed with reduced cobinamide and chloroalkanes, which in those cases were correlated with the formation of a direct carbon-cobalt bond (20, 35). Previously, the reaction of Co(1) cobalamins with vinyl bromide and tetrafluoroethylene had been reported to yield vinylcobalamin (36) and (tetrafluoroethy1)cobalamin (37),respectively. Further experiments will need to be conducted to determine if the product detected in Figure 3 arises from a substitution or an addition reaction. Reductive Dehalogenation Kinetics for Chlorinated Ethylenes. As a benchmark for the dehalogenation of chlorinated alkenes, we first determined the rate at which vitamin BIZ,coenzyme and hematin mediated the reductive dechlorination of carbon tetrachloride to chloroform. The ability of the three cofactors to dechlorinate carbon tetrachloride was previously reported by Krone et al. (20,21) and Klecka and Gonsior (31). The reaction in each case was first order with respect to the metallocofactor and the chlorocarbon. While all three were capable of dechlorinating carbon tetrachloride in the presence of an excess amount of titanium(II1) citrate, the pseudo-firstorder rate constants (K,) describing the dechlorination kinetics in the water phase varied by 40-fold (Table I). Of the three cofactors, F,,, was the fastest and hematin was the slowest. The K , value for vitamin Blz was 75% of that for coenzyme Fd3,,,an observation consistent with the reaction progress data presented by Krone et al. (20, 21). By performing a series of experiments with different initial chlorinated ethylene species, it was demonstrated that vitamin B12 and coenzyme F,,, had the capacity to mediate the eight-electron reduction of tetrachloroethylene to ethylene (Figure 4). Hematin catalyzed the reductive
4
Figure 2. Reaction progress curves for the vitamin B,,-catalyzed dechlorination of tetrachloroethylene (PCE) to trichloroethylene (TCE) and cis-l,2dichloroethylene (CIS). The reactions were conducted as described in Materials and Methods.
0 z 1.8 W
a K
0 v)
2
~
1 2 t
1
i
0.6 I
~
300
400
600
500
700
W A V E L E N G T H (nm) Figure 3. Visible absorption spectra of reduced (A) and alkylated (B and C) cyanoaquacobinamide. Spectrum A was cyanoaquacobinamide in buffer containing excess titanium(II1) citrate, and spectra B and C were recorded 5 and 10 min, respectively, after the addition of tetrachloroethylene.
electron capture detector. The column was operated a t 120 "C for 2 min and increased to 200 "C a t a rate of 8 "C min-'. Pentachlorobenzene and 1,2,3,4-, 1,2,4,5-, and 1,2,3,5-tetrachlorobenzenestandards had retention times of 9.4, 7.2, 6.4, and 6.4 min, respectively. To separate 1,2,4,5- and 1,2,3,5-tetrachlorobenzene, the column was operated isothermally a t 90 "C and the respective standards had retention times of 27.8 and 27.4 min. Spectrophotometric determinations were made with a Beckman DU-70 spectrophotometer. A 2-mL anaerobic cuvette contained 1 mL of 0.66M Tris buffer a t pH 8.2, 100 nmol of cyanoaquacobinamide, and 3 pmol of titanium(II1) citrate at 22 "C under an argon atmosphere. After the absorption spectrum between 250 and 750 nm was recorded, 5 mmol tetrachloroethylene was added and spectra were recorded a t 5-min intervals. Results and Discussion Initial experiments examined the ability of vitamin BIZ (cyanocobalamin) to catalyze the reductive dechlorination of tetrachloroethylene (perchloroethylene) with titaniumCI
\
CI I
I
\ CI
c=c
CI
H \
---+
Tetrachloroethylene (PCE)
CI
csc
I
CI I
\
d \
CI
Trlchloroethylene (TCE)
I
c=c
I CI
\ CI
\
I
I
\
c=c
d
cis -Dlchloroethylene (CIS)
H
H ..
H ..
H
H
CI
Vinyl Chloride (VCL)
-
H
H
\
H
I
c=c
I
\
H
Ethylene
(ETH)
Figure 4. Sequential reductive dechlorination of tetrachloroethylene to ethylene as mediated by vitamin B,, and coenzyme .F ,, 718
Environ. Sci. Technol., Vol. 25, No. 4, 1991
Table I. Pseudo-First-Order Rate Constants for Dehalogenation of Carbon Tetrachloride and Chlorinated Ethylenesa
compound carbon tetrachloride
74 f 4 ( n = 2)
tetrachloroethylene
7.3 f 1.5 ( n = 5) 0.28 f 0.10 ( n = 9) 0.0017 f 0.0006 (n= 5) 0.0049 f 0.0007 ( n = 4) 0.0039 f 0.0016 ( n = 5) 0.00085 f 0.00010 (n = 2)
trichloroethylene cis-dichloroethylene trans-dichloroethylene 1,l-dichloroethylene vinyl chloride Ii
K1, h-I coenzyme F430
Vitamin BI2
hematin 2.4 f 0.0 (n = 2)
100 f 4 ( n = 2) 3.7 f 0.5 ( n = 4) 0.20 f 0.01 (n = 2) 0.011 f 0.002 ( n = 4) 0.017 f 0.007 ( n = 2)
0.16 f 0.05
(n = 2) 0.024 f 0.003 (n = 5 ) 0.0016 f 0.0008 ( n = 2)
ND ND
0.018 ( n = 1)
0.00033 f 0.00022 ( n = 2)
ND
ND, not determined; n number of determinations.
dechlorination of tetrachloroethylene to vinyl chloride. Under the conditions examined, it was not possible to maintain Ti(II1) in the hematin vials long enough to demonstrate ethylene production rates above those observed in the control vials. For all three cofactors, cisdichloroethylene was the major dichloroethylene isomer formed from trichloroethylene dechlorination. Other biomolecules that contain transition metals and carry out important redox reactions in anaerobic bacteria were also examined for their ability to dechlorinate tetrachloroethylene with titanium(II1) citrate as the electron donor. Despite being reduced by the titanium, as shown spectrophotometrically, azurin (copper center), chloroplast ferrodoxin (Fe& cluster), and clostridial ferredoxin (Fe4S4 cluster) were unable to dechlorinate tetrachloroethylene under the conditions examined. As shown in Table 1,the Kl values for the dechlorination of carbon tetrachloride by vitamin BIZ,coenzyme F430, and hematin were a t least 1 order of magnitude greater than the K , values for the dechlorination of tetrachloroethylene. The increased resistance of chlorinated alkenes to reductive dechlorination has also been observed biologically in anaerobic reactors (9) and anaerobic freshwater-sediment microcosms (11). These results are consistent with the fact that chloride displacement via substitution mechanisms occurs more readily with chloroalkanes than with chlorinated alkenes and aromatics (38). In the reductive dehalogenation of chloroethylenes, vitamin BIZand coenzyme F430 had similar rate constants, while hematin was significantly slower (Table I). For all three of the cofactors, the displacement of each chlorine atom decreased the K1 values for the removal of the subsequent chlorine atom by approximately 1 order of magnitude (Table I). A similar trend was observed for the reductive dehalogenation of chlorinated methanes by cobalamins (20) and coenzyme F430 (21). Vogel et al. (39) have suggested that carbon-chlorine bond reductive cleavage rates may be proportional to the heat of formation of a resultant organic radical following one-electron input or may correlate with the standard reduction potential of the chlorinated aliphatic compound. As shown in Figure 5 , the kinetic data obtained with vitamin BIZ,coenzyme F430, and hematin are consistent with the latter relationship. Semilog plots of log10 K1 versus standard reduction potential can be described by straight lines ( r > 0.98). The rate diminution with decreasing chlorination is reminiscent of the kinetic course of tetrachloroethylene dehalogenation by consortia of anaerobic bacteria (11, 40). Modeling the Sequential Dechlorination of Tetrachloroethylene to Ethylene. By use of the K1 values
VCL 4ETH CIS 4 VCL TCE + CIS PCE -+ TCE 100 L
4
10
1
k z
-
M
.1
0
.01
,001 ,000 1 0.0
1.o
2.0
Standard Reduction Potential (volts) Flgure 5 . Relationship between the observed rate constants for dechlorination as a function of the half-reaction reduction potentials for the chlorinated ethylene series. Half-reaction reduction potentials for the reactions shown at the top of the graph were calculated as described in Vogel et al. (39). The catalysts were vitamin E,, ,).( coenzyme F430(O), and hematin (A).
listed on Table I, the time course for the sequential dechlorination of tetrachloroethylene to ethylene can be described by the following mass balance equations: dSpce/dt = -Kl,pceSpce
(1)
dStce/dt = K1,pceSpce - K1,tceStce
(2)
dScis/dt = K1,tceStce - K1,cisScis
(3)
dsvcddt = Kl,cisScis - Kl,vclSvcl
(4)
dSeth/dt = Kl,vclSvcl (5) in which S is the concentration of the indicated chloroethylene species. Use of the above equations assumes a 1:lstoichiometry in each dechlorination reaction, an excess of the reductant titanium(II1) citrate, and an environment consisting of a water phase exclusively. The five equations were solved simultaneously via a Runge-Kutta algorithm (41). As illustrated in Figure 6, trichloroethylene and cis-dichloroethyleneaccumulate to significant concentrations in 1day, vinyl chloride peaks after several weeks, and ethylene accumulates over several months. These time scales are similar to the observed time course of tetrachloroethylene dechlorination by anaerobic consortia (40). It is not currently clear what fraction of intracellular vitamin Blz and coenzyme F430 is available for reaction with halogenated compounds in vivo. Thus, a prediction of absolute rates as a function of total coenzyme content in
Environ. Sci. Technol., Vol. 25, No. 4, 1991 719
biocatalyst
K,, h-'
biocatalyst
vitamin B,,
1.53 f 0.13 ( n = 3) 0.34 f 0.00
coenzyme F,,, FezSz-ferredoxin Fe4S4-ferredoxin azurin
hematin
( n = 2)
Coenzyme F430
Cyanocobalamin
Table If. Pseudo-First-Order Rate Constants for Dehalogenation of Hexachlorobenzene"
101
I
K1, h-'
CIS-DCE
