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Reductive Dechlorination of Tetrachloroethylene and Trichloroethylene Catalyzed by Vitamin B12 in Homogeneous and Heterogeneous Systems D A V I D R . B U R R I S , * ,† CARRIE A. DELCOMYN,‡ MARK H. SMITH,† AND A. LYNN ROBERTS§ Armstrong Laboratory, AL/EQ, 139 Barnes Drive, Tyndall Air Force Base, Florida 32403-5323, Applied Research Associates, Inc., 139 Barnes Drive, Tyndall Air Force Base, Florida 32403-5323, and Department of Geography and Environmental Engineering, The Johns Hopkins University, 313 Ames Hall, Baltimore, Maryland 21218-2686
The reduction of tetrachloroethylene (PCE) and trichloroethylene (TCE) catalyzed by vitamin B12 was examined in homogeneous and heterogeneous (B12 bound to agarose) batch systems using titanium(III) citrate as the bulk reductant. The solution and surface-mediated reaction rates at similar B12 loadings were comparable, indicating that binding vitamin B12 to a surface did not lower catalytic activity. No loss in PCE reducing activity was observed with repeated usage of surface-bound vitamin B12. Carbon mass recoveries were 81-84% for PCE reduction and 89% for TCE reduction, relative to controls. In addition to sequential hydrogenolysis, a second competing reaction mechanism for the reduction of PCE and TCE by B12, reductive β-elimination, is proposed to account for the observation of acetylene as a significant reaction intermediate. Reductive β-elimination should be considered as a potential pathway in other reactive systems involving the reduction of vicinal polyhaloethenes. Surface-bound catalysts such as vitamin B12 may have utility in the engineered degradation of aqueous phase chlorinated ethenes.
Introduction The discharge of chlorinated solvents (e.g., PCE and TCE) into subsurface environments has led to extensive groundwater contamination. Chlorinated solvent-contaminated wastewaters are also generated by industrial processes. The * Author to whom correspondence should be addressed; e-mail address:
[email protected]. † Armstrong Laboratory. ‡ Applied Research Associates. § The Johns Hopkins University.
S0013-936X(96)00116-2 CCC: $12.00
1996 American Chemical Society
treatment of aqueous solutions containing chlorinated solvents has gained considerable attention in recent years. Reductive dehalogenation of chlorinated solvents catalyzed by metallocoenzymes, as one approach, has been examined by a number of researchers (e.g., refs 1-8). The metallocoenzyme vitamin B12 is produced by anaerobic bacteria and contains a corrin ring that coordinates a cobalt atom. The common commercially available form of vitamin B12 is cyanocobalamin. The cyano group (β-ligand) can be replaced by water to form aquo-cob(III)alamin (vitamin B12a). Either of these compounds can be reduced by stepwise addition of electrons to form cob(II)alamin (vitamin B12r) and cob(I)alamin (vitamin B12s). Dithiothreitol (DTT) reduces vitamin B12a to vitamin B12r (4) and titanium(III) citrate to vitamin B12s (1). The Co(I) center of vitamin B12s is one of the most powerful nucleophiles known (9). Gantzer and Wackett (2) showed that the reduction of chlorinated ethenes was catalyzed by vitamin B12 in the presence of titanium(III) citrate. Sequential hydrogenolysis products (PCE f TCE f cis-1,2-dichloroethylene f vinyl chloride f ethene) were observed. Experimental data detailing product distributions and mass recoveries over time, which might assist in clarifying details of the pathways through which dehalogenation proceeds, were not reported. Schanke and Wackett (3) showed that reductive β-elimination reactions can occur via vitamin B12 in similar experiments with vicinal polychlorinated ethanes, thus producing ethenes. Roberts et al. (10) recently reported that reductive β-elimination of polyhalogenated organics is not restricted to polyhaloalkanes but can also occur for reactions of vicinal polychlorinated ethenes with zero-valent metals in aqueous solution to result in the formation of ethynes. A similar reductive β-elimination reaction of chloroalkenes involving vitamin B12 does not appear to have been previously reported. PCE is resistant to metabolism by aerobic microorganisms, although the lesser chlorinated ethenes can be degraded aerobically by nonspecific oxygenases (11). One possible treatment approach for waters contaminated with PCE may therefore be to alternate anaerobic with aerobic conditions, using B12 to reduce PCE to lesser chlorinated ethenes, which can subsequently be degraded under aerobic conditions to harmless products. The success of such a scheme would rely, in part, on developing an ability to rapidly and reliably reduce PCE to products amenable to aerobic degradation. Treatment approaches based upon the use of solution phase (homogeneous) catalytic macrocycles may be impractical if the catalyst cannot be separated from the waste stream for reusage. Immobilization of the catalytic macrocycles to solid supports can allow retention of the catalyst within the reactor system. Catalytic activity of the immobilized catalysts would have to be stable over time for this treatment approach to be practical. Several researchers (6-8, 12) have recently examined the use of immobilized catalytic macrocycles (e.g., corrins and porphyrins) to reduce chlorinated organics. Marks and Maule (6) examined the use of immobilized cobalamins and porphyrins to dehalogenate a number of organochlorine pollutants in column systems using dithiothreitol as the bulk reductant. TCE was found to be
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unreactive. Habeck and Sublette (8) investigated the reduction of PCE in batch and column systems using B12 immobilized by adsorption onto Duolite S-761 resin and titanium(III) citrate as the bulk reductant. Significant sorption of PCE onto the resin occurred, creating an additional process to account for. Reaction kinetics and carbon mass recoveries were not reported. The reduction of chlorinated methanes by immobilized cobalamins (immobilized by adsorption onto talc) using titanium(III) citrate as the reductant in batch systems was investigated by Matheson (7). Reuse of the talc-immobilized B12 resulted in successive decreases in the observed reaction rate. Loss of the cobalamin by desorption or deactivation of the cobalamin may have contributed to the reduction in rate. In this study, the reduction of PCE and TCE catalyzed by vitamin B12 in batch homogeneous and heterogeneous systems is examined. Vitamin B12 was immobilized by covalent bond formation to agarose, a solid support that does not adsorb either chlorinated substrate. Particular attention is paid to volatile organic reaction products and intermediates and to carbon mass recoveries. The product/ intermediate formation and reduction kinetics of the two batch systems were compared to assess the effect of immobilization of vitamin B12 on catalytic activity. The reusage potential of the immobilized vitamin B12 was also examined. Evidence will be presented in support of a reductive β-elimination reaction mechanism, a previously unreported reaction pathway in the reduction of PCE and TCE by vitamin B12.
Materials and Methods Chemicals. PCE was obtained from J. T. Baker. TCE was supplied by Fisher Scientific. cis-1,2-Dichloroethylene (cisDCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and sodium citrate (citric acid, trisodium salt) were obtained from Aldrich. Vitamin B12 (cyanocobalamin), sodium hydroxide, and Trizma base were obtained from Sigma. Vitamin B12 immobilized on 4% beaded agarose (1.2 mg/mL packed gel) was also supplied by Sigma. In this product, vitamin B12 was attached at the corrin ring to the agarose by binding monocarboxyl derivatives of B12 to cyanogen bromideactivated agarose via hexanediamine linkages (i.e., a C8 spacer) (13). Pentane was supplied by Burdick and Jackson. Vinyl chloride (VC) in nitrogen and a gas mixture containing acetylene, ethene, ethane, methane, carbon dioxide, and carbon monoxide were obtained from Alltech Associates. Titanium trichloride (13% in 20% HCl) was obtained from Fluka. Stock solutions of 250 mM titanium(III) citrate in 660 mM Tris buffer (pH 8.2) were prepared in serum vials as described in Smith and Woods (14). Working stock vials were stored in the anaerobic chamber (10% H2 in N2) prior to use. Milli-Q (Millipore) distilled deionized water, argonsparged (using an in-line O2 trap) for at least 1 h, was used to prepare all solutions in this study. Reaction Systems. The batch systems (foil-wrapped 160-mL serum vials, crimp-sealed with Teflon-lined septa, with final volumes of 100 mL of aqueous solution and 60 mL of headspace) were prepared, in duplicate, in the anaerobic chamber. Vapor/liquid exchange in these systems is rapid (90% of equilibrium for chloroethenes is reached within 5 min). Air-water partitioning may safely be assumed to be at pseudo-equilibrium for compounds whose reactions require many hours or more, as in the
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present study. A 5 mM vitamin B12 solution served as a stock reagent for the homogeneous system. For the heterogeneous system, the required volume of gel was added to a graduated pipet and washed with 66 mM Tris. The final vitamin B12 concentrations were 10 and 9.7 µM for the homogeneous and heterogeneous systems, respectively (heterogeneous system B12 concentrations are reported in terms of µmol of B12/L suspension; 0.97 ( 0.05 µmol of vitamin B12 immobilized on agarose was added to each serum vial). The stated masses of TCE or PCE and 400 µg of pentane were spiked into each vial and allowed to equilibrate between the aqueous and vapor phases for several days prior to commencing the reactions by placing vials on a roller drum and rotating (g8 rpm) vertically as the vial axis remained horizontal at 20 °C. Methanol was used as a co-solvent in some cases and did not exceed 2 µL per vial. Care was taken to ensure that the septa were not in contact with the vial headspace in order to minimize losses. Reduction reactions were initiated by spiking with the titanium(III) citrate stock solution to yield a final concentration of 15 mM Ti[III], and the reactions were allowed to continue on the roller drum. Positive controls omitted vitamin B12. Negative controls consisted of the respective chloroethylene in water (no titanium(III) citrate or B12). In controls using only the vitamin B12 agarose (results not shown), it was determined that sorption of PCE and TCE to agarose was negligible. At selected intervals, 200-µL headspace samples were taken from the reaction vials for analysis. In heterogeneous system studies in which the immobilized B12 was reused, after an 8-h time course, the reaction vials were opened and allowed to sit overnight in a fume hood. The vitamin B12 agarose was then washed seven times with 75 mL of water, allowing the agarose to settle by gravity for 45 min between washes to ensure minimal loss of agarose. After the final wash, the system was prepared for reaction as described previously. The vitamin B12 immobilized on agarose was used to reduce PCE four times in this fashion. Sampling was conducted as described above. A loss of less than 5% of the initial B12 on agarose was confirmed volumetrically at the end of this immobilized B12 reusage experiment. Analytical Methods. Calibration standards containing each organohalide and hydrocarbon gas component were prepared using the 160-mL crimp-top serum vials with 100 mL of water and 60 mL of headspace volume. Quantification using headspace samples was by the internal standard method (using pentane as the internal standard). The concentrations could be determined as mass per vial, since the solution/headspace ratio was the same as that used in the reaction systems thus accounting for vapor/water partitioning. Headspace samples (200 µL) were analyzed by a dual-column, column sequence reversal gas chromatographic method (15) using a Hewlett-Packard 5890 GC. The chlorinated ethenes and C2 hydrocarbon gases were separated on 1% SP-1000 (60/80 mesh Carbopack B, 8 ft × 1/8 in. stainless steel (ss), Supleco) and Carboxen 1000 (60/80 mesh, 4 ft × 1/8 in. ss, Supelco) packed columns, respectively. Initial column sequence was the SP-1000 followed by the Carboxen 1000. Samples were injected splitless at 200 °C. Carrier gas was He at 30 mL/min. Oven temperature program was 60 °C for 1 min, ramp 18 °C/min to 210 °C, and hold for 3 min. The column switching valve was rotated at 1 min runtime. Flame ionization detector temperature was 240 °C. Due to co-elution of cis- and trans-
TABLE 1
Apparent Pseudo-First-Order Reduction Rate Coefficients for TCE and PCE in 10 and 9.7 µM Vitamin B12 (in Solution or Suspension) for Homogeneous and Heterogeneous Systems (100 mL of aqueous phase, 60 mL of vapor phase), Respectively, Using Excess Titanium(III) Citrate as Bulk Reductant initial mass in system compd (µmol) TCE
15
TCEc
15
TCE TCE PCE
5 0.5 13
PCE PCE
4 0.4
homogeneous system apparent pseudo-first-order rate coefficient (h-1)
heterogeneous system apparent pseudo-first-order rate coefficient (h-1)
0.0181 ( 0.0015a (n ) 18)b 0.0301 ( 0.0035 (n ) 14) 0.0227 ( 0.0026 (n ) 6) 0.0365 ( 0.0058 (n ) 8) 0.0318 ( 0.0034 (n ) 11) ndd 0.0316 ( 0.0033 (n ) 11) nd 0.802 ( 0.095 0.940 ( 0.113 (n ) 12) (n ) 12) 0.758 ( 0.035 (n ) 25) nd nd 0.929 ( 0.055 (n ) 13)
a 95% confidence limits. b n ) number of observations. c Based upon TCE concentrations from experiment with PCE as initial substrate. d nd, not determined.
DCE on the dual-column system, their relative proportions were determined on a Hewlett-Packard 5890 GC with flame ionization detection using a 30-m megabore GSQ-PLOT column (J&W). The oven temperature program was 40 °C for 3 min, ramp 25 °C/min to 200 °C, hold for 12 min, and ramp again at 30 °C/min to 230 °C. Injector and detector temperatures were 210 and 240 °C, respectively. Peak identifications from the dual-column GC system were confirmed on the megabore capillary GC system. Chloroacetylene was determined by GC/MS (HP 5890 GC equipped with an HP 5971 quadrupole mass selective detector) using the GSQ megabore column. Reaction vials were prepared as discussed above using 45 µmol of initial substrate (TCE or PCE). The initial search was made using the selective ion monitoring mode (m/z 60 and 62). Identification was based upon comparison of the full-scan electron impact spectra with library spectra. A search for dichloroacetylene was made in a similar fashion. Kinetic Analysis. Apparent pseudo-first-order reaction rate coefficients (kobs) values were obtained by fitting MT (total moles of component per vial) to a linearized form of the exponential decay expression (ln MT ) ln MoT - kobst), where MoT is the initial mass present and t is the elapsed time. The relative constancy of the rate coefficients over a wide concentration range confirmed first-order kinetics with respect to the chloroethenes (see Table 1). Assuming that reaction takes place only in the solution phase, the apparent pseudo-first-order reaction rate coefficient, kobs, that pertains to the reaction of a volatile constituent capable of rapid partitioning between the solution and the headspace phases is equivalent to the rate coefficient that would be obtained in a headspace-free system, k′obs, modified by the factor fw (i.e., kobs ) k′obsfw), where fw is the fraction of the total mass of the reactive component present in the the aqueous phase. The term fw is related to the Henry’s law constant for the system and the ratio of headspace to solution volumes via the expression fw ) 1/(1 + (Vg/Vw)Hc), where Vg, Vw, and Hc are the headspace volume, water volume, and “dimensionless” Henry’s law constant (in mol L-1air/mol L-1water), respectively. Using the Henry’s law
FIGURE 1. Reduction of TCE by vitamin B12 (10 µM) in solution with titanium(III) citrate (15 mM) as the bulk reductant in batch system (100 mL of aqueous phase; 60 mL of vapor phase) at 20 °C.
FIGURE 2. Reduction of TCE by vitamin B12 (9.7 µM in suspension) on agarose with titanium(III) citrate (15 mM) as the bulk reductant in batch system (100 mL of aqueous phase; 60 mL of vapor phase) at 20 °C.
constants from Gossett (16), the fw values for the compounds and conditions used in this study are 0.754 for PCE, 0.849 for TCE, 0.660 for 1,1-DCE, 0.932 for cis-DCE, 0.847 for transDCE, and 0.649 for vinyl chloride.
Results and Discussion The reduction of TCE in the homogeneous and heterogeneous vitamin B12 systems are shown in Figures 1 and 2, respectively. The TCE and product/intermediate concentrations as a function of time for both systems were similar. The products (or intermediates) observed were cis-DCE, acetylene, ethene, and smaller amounts of trans-DCE, 1,1DCE, and vinyl chloride. A trace amount of chloroacetylene was also observed. Carbon mass recovery at the end of the experiment was approximately 89% relative to the positive control for both systems. The less than total carbon mass recovery suggests that an unidentified product(s) may exist. The production of ethene was significantly greater for the
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FIGURE 3. Reduction of PCE by vitamin B12 (10 µM) in solution with titanium(III) citrate (15 mM) as the bulk reductant in batch system (100 mL of aqueous phase; 60 mL of vapor phase) at 20 °C.
FIGURE 4. Reduction of PCE by vitamin B12 (9.7 µM in suspension) on agarose with titanium(III) citrate (15 mM) as the bulk reductant in batch system (100 mL of aqueous phase; 60 mL of vapor phase) at 20 °C.
heterogeneous system over the reaction time period examined. In the heterogeneous system, ethene concentrations increase even during the latter part of the time course (after all of the TCE is reduced), a period in which cis- and trans-DCE concentrations remain stable and acetylene concentrations decrease. This suggests that ethene production results, at least in part, from the reduction of acetylene. Implications of the production of acetylene are discussed below. The apparent pseudo-firstorder reduction rate coefficients for TCE are shown in Table 1. TCE appears to degrade slightly more rapidly in the heterogeneous system than in the homogeneous system. Since TCE is very rapidly produced from the rapid degradation of PCE (as shown in Figures 3 and 4 for the homogeneous and heterogeneous systems, respectively), the TCE pseudo-first-order rate coefficients were also determined using the TCE concentrations after all the PCE was reduced. In that case, the TCE reduction rate coefficients were essential identical in the two systems. These
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FIGURE 5. Apparent pseudo-first-order rate plots for the sequential consecutive reduction of 15 µmol of PCE by vitamin B12 (9.7 µM in suspension) on agarose with titanium(III) citrate (15 mM) as the bulk reductant in batch system (100 mL of aqueous phase; 60 mL of vapor phase) at 20 °C. The agarose with immobilized vitamin B12 was washed extensively between each usage.
resultsindicate that immobilizing vitamin B12 by covalent linkage of the corrin ring onto a solid support such as agarose (using a C8 spacer) does not lower catalytic activity. The results for PCE reduction in the homogeneous and heterogeneous vitamin B12 systems are shown in Figures 3 and 4, respectively. PCE undergoes reduction much more rapidly than TCE. As with TCE, the initial substrate and product/intermediate concentrations over time are quite similar for the two systems. Dichloroacetylene was not found; however, a trace of chloroacetylene was observed. Carbon mass recovery at the end of the experiment was approximately 81-84% relative to the positive control for both systems. The less than total carbon mass recovery suggests that an unidentified product(s) may exist, as with the TCE experiments. In order to obtain more accurate apparent pseudo-first-order rate coefficient estimates, the reaction was repeated to obtain more observations during the interval when PCE was being depleted. The apparent pseudo-first-order rate coefficients for the two systems are essentially identical (see Table 1), again consistent with the notion that immobilizing vitamin B12 on a solid support does not decrease its reactivity. The PCE reduction products or intermediates observed were TCE, cis-DCE, acetylene, ethene, and minor amounts of trans- DCE, vinyl chloride, and 1,1-DCE. For both PCE systems, as with the reactions conducted with TCE, a comparison of the behavior of acetylene and ethene during the latter part of the time course (after all of the PCE and TCE has been degraded and the cis- and trans-DCE concentrations remain stable) suggests that ethene originates, at least in part, from the reduction of acetylene. The apparent pseudo-first-order rate plots for four consecutive reductions of PCE using vitamin B12 immobilized on agarose is shown in Figure 5. The agarose with immobilized vitamin B12 was washed extensively between each usage. The results show no loss of PCEreducing activity. These results agree qualitatively with those obtained by Habeck and Sublette (8) concerning the reduction of PCE using immobilized B12 in column experiments. These researchers observed no loss of catalytic activity after several days of continuous operation. The apparent stability of immobilized vitamin B12 is encouraging in terms of its potential utility in a reactor system.
(1) hydrogenolysis (yielding the lesser chlorinated analogs as mentioned above) and (2) hydrolysis (either to yield the same acetylene products as with hydrogenolysis; or acetate or possibly chloroacetate, which are nonvolatile and water soluble). In relation to the potential for hydrolysis to yield the lesser chlorinated analogs, chlorinated acetylenes are reported (18) to hydrolyze in hydroxylic solvents under alkaline conditions via nucleophilic attack at Cl (instead of C). The reaction under aqueous alkaline conditions for chloroacetylene would be: H2O
HsCtCsCl + OH- 98 HsCtCsH + HOCl
FIGURE 6. Proposed pathways for the reduction of chloroethenes by vitamin B12 using titanium(III) citrate as the bulk reductant. Pathway a corresponds to reductive β-elimination: R(X)dR(X) + 2e- f RtR + 2X-. Pathway b corresponds to hydrogenolysis: RX + 2e- + H+ f RH + X-. Pathway c corresponds to reduction of triple bond to olefin: RtR + 2e- + 2H+ f R(H)dR(H). Not shown: hydrolysis reactions of chlorinated acetylenes to carboxylic acids.
Acetylene has not been previously reported in the reduction of PCE or TCE by vitamin B12 using titanium(III) citrate as a bulk reductant, even though the concentrations observed (as shown in Figures 1-4) are quite substantial. In most cases, the concentration profiles for acetylene, which display an initial increase followed by a decline, are typical of behavior anticipated by reaction intermediates. Roberts et al. (10) observed the production of acetylene during the reduction of trans-DCE and (to a lesser extent) cis-DCE with Fe(0). A reductive β-elimination pathway was proposed to account for the production of acetylene from the vicinal DCEs. Acetylene could also arise from reactions of chlorinated acetylenes, themselves the products of reductive β-elimination of more highly chlorinated ethenes. Roberts et al. (10) also observed production of acetylene during the reduction of PCE by Zn(0) even though cis- and trans-DCE were relatively stable under reaction conditions. In this case, it was proposed that the PCE had undergone reductive β-elimination to dichloroacetylene followed by sequential hydrogenolysis to chloroacetylene and then finally acetylene. In the case of TCE, reductive β-elimination would initially produce chloroacetylene, which would subsequently undergo hydrogenolysis to acetylene. The pathways shown in Figure 6 could account for the products observed during the reduction of PCE (and TCE) by vitamin B12 using titanium(III) citrate as the bulk reductant. The formation of chloroacetylene and possibly dichloroacetylene (not detected in this study) during reductive β-elimination of PCE or TCE may be of environmental significance since they have been reported to be toxic (17, 18). The potential fate of the chloroacetylenes in aqueous solution has been postulated by Roberts et al. (10) to include
Due to the potential environmental significance of the chloroacetylenes, efforts should be made to detect their presence and evaluate their fate. Belay and Daniels (19) observed the production of acetylene by several pure cultures of methanogenic bacteria incubated with 1,2-dibromoethylene (isomer unspecified). These researchers did not speculate as to the pathway or reactive agent(s) involved in the production of acetylene. In light of the evidence presented in this study and that of Roberts et al. (10) on zero-valent metals, we consider reductive β-elimination to be the likely reaction pathway. We encourage researchers to consider the possibility of reductive β-elimination reactions of vicinal polyhaloalkenes in reduction reaction systems. This consideration requires, at a minimum, the use of analytical methodology that includes the detection of acetylene. In this study, the reduction of PCE and TCE catalyzed by vitamin B12 was examined in homogeneous and heterogeneous batch systems using titanium(III) citrate as the bulk reductant. Immobilization of vitamin B12 onto the solid support did not result in any reduction of catalytic activity since the reaction rates for the two systems were comparable. Consecutive usage of the immobilized vitamin B12 also did not adversely influence catalytic activity. Carbon mass recoveries ranging from 81-89% were obtained, indicating that the majority of organic reaction products have been identified. The distribution of reaction products indicated that the reactions took place, in part, via sequential hydrogenolysis. A second competing reaction pathway involving reductive β-elimination is proposed to account for the observation of acetylene as a significant reaction intermediate. The observation of a trace amount of chloroacetylene further supports the reductive β-elimination route. The results of this study and that of Roberts et al. (10) suggest that reductive β-elimination should be considered as a potential pathway in other systems involving the reduction of vicinal polyhaloethenes. Engineered treatment systems utilizing surface-bound catalysts such as vitamin B12 may prove useful in the remediation of waters contaminated with chlorinated ethenes.
Acknowledgments Thanks are extended to Tim Campbell and Baolin Deng for their assistance and helpful suggestions. This work was funded, in part, by the Air Force Office of Scientific Research and the Strategic Environmental Research and Development Program (SERDP) of the DoD, DoE, and EPA.
Literature Cited (1) Krone, U. E.; Thauer, R. K.; Hogenkamp, H. P. C. Biochemistry 1989, 28, 4908-4914.
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(2) Gantzer, C. J.; Wackett, L. P. Environ. Sci. Technol. 1991, 25, 715-722. (3) Schanke, C. A.; Wackett, L. P. Environ. Sci. Technol. 1992, 26, 830-833. (4) Assaf-Anid, N.; Hayes, K. F.; Vogel, T. M. Environ. Sci. Technol. 1994, 28, 246-252. (5) Chiu, P.; Reinhard, M. Environ. Sci. Technol. 1995, 29, 595-603. (6) Marks, T. S.; Maule, A. Appl. Microbiol. Biotechnol. 1992, 38, 413-416. (7) Matheson, L. J. Ph.D. Dissertation, Oregon Graduate Institute of Science and Technology, 1994. (8) Habeck, B. K.; Sublette, K. L. Appl. Biochem. Biotechnol. 1995, 51/52, 747-759. (9) Schrauzer, G. N.; Deutsch, E. J. Am. Chem. Soc. 1969, 91, 33413350. (10) Roberts, A. L.; Totten, L. A.; Arnold, W. A.; Burris, D. R.; Campbell, T. J. Environ. Sci. Technol. 1996, 30, 2654-2659. (11) Fetzner, S.; Lingens, F. Microbiol. Rev. 1994, 58, 641-685. (12) Ukrainczyk, L.; Chibwe, M.; Pinnavaia, T. J.; Boyd, S. A. Environ. Sci. Technol. 1995, 29, 439-445.
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(13) Frances, G. L.; Smith, G. W.; Toskes, P. P.; Sanders, E. G. Gastroenterology 1977, 1304-1307. (14) Smith, M. H.; Woods, Appl. Environ. Microbiol. 1994, 60, 41074110. (15) Campbell, T. J.; Burris, D. R. Int. J. Environ. Anal. Chem. 1996, 63, 119-126. (16) Gossett, J. M. Environ. Sci. Technol. 1987, 21, 202-208. (17) Piganiol, P. Acetylene Homologs and Derivatives; Mapleton House: New York, 1950. (18) Rutledge, T. F. Acetylenic Compounds: Preparation and Substitution Reactions; Reinhold: New York, 1968. (19) Belay, N.; Daniels, L. Appl. Environ. Microbiol. 1987, 53, 16041610.
Received for review February 8, 1996. Revised manuscript received May 6, 1996. Accepted May 30, 1996.X ES960116O X
Abstract published in Advance ACS Abstracts, August 1, 1996.
