Effect of Hydrogen on Reductive Dechlorination of Chlorinated Ethenes

to study the effect of electron donor and PCE loading on chloroethene ...... (5) Tandoi, V.; DiStefano, T. D.; Browser, P. A.; Gossett, J. M.; Zinder,...
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Environ. Sci. Technol. 1997, 31, 1728-1734

Effect of Hydrogen on Reductive Dechlorination of Chlorinated Ethenes BHASKAR S. BALLAPRAGADA, H. DAVID STENSEL, J. A. PUHAKKA,† AND JOHN F. FERGUSON* Department of Civil Engineering, University of Washington, Seattle, Washington 98195-2700

A methanogenic fluidized bed reactor (FBR) fed with lactate and tetrachloroethene (PCE) was operated for 14 months to study the effect of electron donor and PCE loading on chloroethene dechlorination rates. Lactate was fed continuously at 200 mg/L (2.2 mmol/L), and the influent PCE feed concentration was increased stepwise from 3.5 to 160 µmol/L. Vinyl chloride (VC) and ethene accounted for 80% and 20%, respectively, of the PCE dechlorination. Batch tests with various electron donors showed that H2, propionate, and lactate supported dechlorination of PCE, trichloroethene (TCE), cis-dichloroethene (c-DCE), and VC, whereas no dechlorination was observed with acetate or in the absence of an electron donor. Different short-term steady H2 concentrations were obtained by adjusting the FBR influent lactate feed concentration, and the effect of H2 concentration on the rate of chloroethene dechlorination was determined. Dechlorination rates for PCE, TCE, c-DCE, and VC showed a Michaelis-Menten relationship with H2 partial pressure. The half-velocity coefficients for H2 utilization by dechlorinators ranged from 12 to 28 ppm for the chloroethenes and are at least an order of magnitude lower than values reported for methanogens. This implies that dechlorinating bacteria can out-compete methanogens for H2 utilization at low H2 concentration.

Introduction Chloroethenes have been widely used as solvents and chemical feedstocks, and their improper use and disposal has led to contamination of the subsurface, including soil and the groundwater (1). All chloroethenes are either known or suspected carcinogens (2), and therefore their presence in the environment is of concern. During the past decade, significant efforts have been directed to study microbial degradation of chlorinated ethenes and bioremediation of contaminated sites. All chloroethenes have been shown to be transformed under anaerobic conditions (3-5). Anaerobic transformation of chloroethenes occurs via reductive dechlorination, which involves sequential replacement of chlorines with hydrogen. Thus, transformation of tetrachloroethene (PCE) occurs via trichloroethene (TCE), cis-dichloroethene (c-DCE), and vinyl chloride (VC) to ethene. VC is slowest to degrade and often accumulates. The chlorinated ethenes serve as the electron acceptors in reductive dechlorination. Each dechlorination † Present address: Tampere University of Technology, Tampere, Finland. * Corresponding author telephone: (206)543-5176; fax: (206)5431543; e-mail: [email protected].

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step requires two electrons, and therefore an electron donor is necessary to complete the reaction. A number of organic compounds have been successfully used as electron donors for anaerobic dechlorination of chlorinated ethenes. Ethene formation from PCE was stimulated by acetate, glucose, formate, methanol (3), or lactate (4). Ballapragada et al. (6) showed that the fermentation of municipal wastewater sludge provided electron donors for PCE dechlorination to VC. Gibson and Sewell (7) showed that in soil microcosms PCE could be converted to TCE and c-DCE in 10-30 days with additions of lactate, propionate, crotonate, butyrate, or ethanol as electron donor. Dechlorination did not occur with the addition of acetate, methanol, and 2-propanol. These observations suggest that dechlorination is different in different microbial environments. Chloroethene-dechlorinating isolates and purified cultures have also been shown to use wide range of electron donors. Dehalospirillum multivorans, a PCE to c-DCE dechlorinating isolate was able to dechlorinate using glycerol, pyruvate, lactate, ethanol, or formate as the electron donor (8). A facultative aerobe, MS-1, is able to dechlorinate PCE to c-DCE using glucose, pyruvate, lactate, or acetate as the electron donor (9). A highly purified culture, PER-K23, was able to dechlorinate PCE to c-DCE using only H2 or formate as the electron donor (10). Even though a wide range of electron donors support dechlorination especially with mixed consortia, studies with Desulfomonile tiedjei, which reductively dechlorinates 3-chlorobenzoate, suggest that intermediates in anaerobic metabolism such as formate and H2 (11) are the actual electron donors in dechlorination. The role of H2 in reductive dechlorination has also been shown in many PCE enrichment cultures. The cultures enriched by Freedman and Gossett (3) with methanol were later shown to dechlorinate using only H2 as the electron donor (12). H2 was also the only electron donor present that supported dechlorination in a methanogenic culture enriched with PCE and lactate (4, 10). Even though H2 is not always the only electron donor, it is in many cases the electron donor used by dechlorinators. H2 is also the electron donor used by H2-utilizing methanogens. Therefore, dechlorinators and methanogens could compete for the available H2 in a mixed methanogenic dechlorinating environment. A recent study by Smatlak et al. (13) quantifies the effect of H2 partial pressure on reductive dechlorination of PCE to VC in a methanogenic consortium. Their study clearly showed that the half-velocity constant for dechlorination of PCE to VC was much lower than that for methanogenesis. The study however did not quantify the half-velocity constant of H2 utilization for conversion of VC to ethene, which is the ratelimiting step in the dechlorination of chlorinated ethenes. The purpose of this study was to investigate the role of H2 for dechlorination of all chloroethenes in a mixed methanogenic dechlorinating consortium in a fluidized bed reactor (FBR). Different steady-state H2 partial pressures were maintained in the FBR by changing the influent lactate feed rates, which allowed determining dechlorination rates at varying H2 partial pressures.

Materials and Methods A FBR was continuously fed with PCE and lactate in a reduced anaerobic mineral media (RAMM) (14) to enrich for a methanogenic dechlorinating population. The FBR was a 500-mL glass column reactor. Microorganisms were immobilized on a porous diatomaceous earth carrier, R-633 (Celite Corp., Lompoc, CA). The reactor contained 30 g of carrier material, constituting 100 mL of unexpanded bed

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volume. Total liquid volume and gas volume were 300 and 100 mL, respectively, with the remainder occupied by glass beads that sat in the bottom of the reactor. Liquid was recycled to the bottom of the reactor at a rate of 170 mL/min to ensure 50% fluidization of the carrier. All tubing including that in the recycle pump was Viton. Feed was prepared every 5 days and stored in a five-layered Teflon-lined bag (Calibrated Instruments, Hamilton, NY) that was kept in a refrigerator outside the incubator. The feed reservoir contained no headspace, thus eliminating PCE partitioning into gas phase. Less than 10% of the PCE was lost or transformed during the 5-day storage period. The FBR was operated for 14 months at 0.5-day hydraulic retention time (HRT) in a 35 °C incubation chamber (Figure 1). The concentration of PCE in the feed was increased in stepwise increments from 3.5 to 160 µmol/L over 14 months. Lactate concentration in the feed was 2.2 mmol/L. Gas was collected in a Teflon-lined bag and monitored regularly. Liquid influent and effluent were monitored for chloroethenes by withdrawing samples directly from the feed bag and the reactor, respectively. Biomass attached to the carrier was measured as protein using Bio-Rad protein assay kit (BioRad Laboratories, Hercules, CA) and ranged from 800 to 1200 mg/L during the test period. Batch vials tests were conducted to determine which of the electron donors (H2, acetate, propionate, and lactate) supported dechlorination. A 0.3-mL aliquot of carrier-bound FBR enrichment culture was transferred anaerobically to each 25-mL vial along with 10 mL of fresh RAMM. Resazurin served as the redox indicator. A small amount of lactate was initially added to all vials to allow the vials to turn fully anaerobic as indicated by color change of the indicator (blue to pink to colorless). All vials were shaken continuously in an orbital shaker during the test at 35 °C. After 6-8 h pre-incubation, chloroethene was added along with an electron donor, and the conversion of chloroethene was monitored. All electron donors were added in excess of the stoichiometric amount required for reduction of the chloroethenes. An unfed vial was also prepared without electron donor addition. For batch test with lactate, 22 µmol of lactate was added to provide a 2.2 mmol/L initial concentration. In other vials propionate, acetate, and H2 were added in amounts stoichiometrically equivalent to that expected from degradation of 2.2 mmol/L lactate. The amounts added were 14, 21, and 40 µmol of propionate, acetate, and hydrogen, respectively, based on the stoichiometry of the following four reactions.

3C3H6O3 f 2CH3CH2COOH + CH3COOH + CO2 + H2O (propionate) (acetate) (lactate) (1) CH3CH2COOH + 2H2O f CH3COOH + CO2 + 3H2 (2) CH3COOH f CH4 + CO2

(3)

4H2 + CO2 f CH4 + 2H2O

(4)

Lactate, propionate, and acetate were added after preincubation, but H2 was added several hours later to unfed and acetate-fed vials. During dechlorination tests in the FBR reactor, lactate, propionate and acetate concentration can be varied by changing the feed concentration into the reactor. However, initial concentrations of H2 are difficult to vary due to mass transfer limitations from gas to liquid phase and the rapid consumption of H2 by methanogens. A new technique was developed to maintain varying H2 concentrations in the FBR. The influent feed concentration of lactate was varied for a few hours while monitoring the H2 concentration continuously. Lactate conversion, producing H2, is rapid (15) and is balanced closely by H2 consumption. However, after chang-

FIGURE 1. Fluidized bed system schematic. ing lactate feed concentrations, altered H2 concentrations could be maintained for several hours till methanogenesis gradually returned the H2 partial pressure to the normal steady-state value. The lag in dynamic response was used to temporarily poise H2 at different levels. Gas was recycled rapidly (400 mL/min) to maintain gas-liquid equilibrium in the reactor. In order to study the effect of H2 partial pressure on the rate of dechlorination, a known amount of chloroethene was pulse fed while continuously feeding lactate at various feed rates. H2 partial pressures as well as the rates of dechlorination were monitored during rate tests that lasted less than 2 h. The effect of H2 partial pressure on dechlorination rates was assumed to follow Michaelis-Menten kinetics. A statistical program, SYSTAT, was used to estimate the half-velocity coefficients based on the observed relation between dechlorination rates and H2 partial pressure. Liquid samples were analyzed for chloroethenes (PCE, TCE, c-DCE) using purge-and-trap followed by a Model 8700 (Perkin Elmer, Norwalk, CT) gas chromatograph (GC) equipped with a Model 1000 Hall detector (Perkin Elmer, Norwalk, CT). Chloroethenes were separated on a wide bore 502.2 Restec capillary column that was held isothermal for 5 min at 35 °C, followed with a temperature ramp of 8 °C/min up to 155 °C. Concentrations of chloroethenes in the gas phase were determined assuming equilibrium gas-liquid partitioning. Losses due to sampling were negligible. The amount of sample withdrawn was a function of the expected concentrations in order to get response in the linear range of the instrument and varied from 0.1 to 0.4 mL. Methane, ethene, and VC were analyzed in the headspace, which was equilibrated with the liquid phase. The gas-liquid mass transfer coefficients (Kla values) were approximately 40/h as determined with tracer (propane) studies. Based on this mass transfer rate, it takes less than 2 min for chloroethenes to reach greater than 90% equilibration, and therefore mass transfer between the phases was rapid enough to maintain their concentrations close to equilibrium. The compounds were analyzed using a HP 5840 GC (Hewlett Packard, Wilmington, DE) equipped with a 6-ft HayeSep Q (Supelco, Bellefonte, PA) packed column held isothermal at 80 °C and a flame ionization detector (FID). H2 was also measured from the headspace of the reactor. H2 was analyzed using a GC with a reduction gas detector (RGD) (Trace Analytical, Menlo Park, CA). A 60/80 silica gel column at constant temperature of 150 °C was used for separating H2 from other gases. H2 partial pressure is reported as parts per million (ppm) on a volume basis in the gas phase; it can be converted to liquid concentration using Henry’s

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constant (KH) of 0.000743 mol L-1 atm-1 (16) at 35 °C. Thus, 100 ppm (10-4 atm) of H2 in the headspace is equivalent to a liquid concentration of 74.3 nmol/L. H2, methane, ethene, and VC analyses were automated to provide rapid sampling frequency. The gas from the headspace was recycled through two separate sample loops. After allowing sufficient time for equilibration, the gas in the first sample loop was injected into the GC-RGD for analysis of H2, and the gas in the second sample loop was injected into the GC-FID for analysis of methane, ethene, and VC. The automated sampling system was set up to analyze all of the above compounds at a maximum frequency of every 6 min.

Results During 14 months of FBR operation, over 80% of influent PCE was normally converted to ethene in the FBR with the rest exiting mostly as VC. After influent PCE step increases, PCE, TCE, and c-DCE were detected in small amounts for a few days. Also, the fraction of PCE converted to ethene decreased during this period, and the VC concentration was higher, accounting for up to 50% of the effluent metabolites. Eventually, the VC concentration decreased and reached steady state, accounting for approximately 20% of the influent PCE. Most of the electrons from lactate were converted to methane (approximately 95%), and the rest were used for dechlorination. Batch tests were conducted in the FBR at different initial chloroethene concentrations and a lactate feed concentration of 2.2 mmol/L (results not shown). Different chloroethenes were spiked, their removal followed with time, and the results modeled with Michaelis-Menten kinetics. The maximum volumetric rates were 49, 64, 72, and 64 µmol L-1 h-1, and the half-velocity coefficients were 2.8, 1.5, 3.0, and 360 µmol/L for PCE, TCE, c-DCE and VC, respectively (17). Thus, dechlorination rates for PCE, TCE, and c-DCE were rapid and zero order, whereas that for VC was slow and first order at concentrations up to at least 150 µmol/L. Also, the rate of dechlorination of VC was always negligible when c-DCE was present at concentrations of 3 µmol/L or more. Electron Donor Vial Tests. Cultures from the FBR were used in batch vial tests to determine which of the electron donors supported dechlorination. Lactate, propionate, and H2 supported PCE dechlorination whereas unfed and acetate fed cultures showed no (or very slow) dechlorination (Figure 2). In Figure 2A, PCE (20.4 µmol/L) was added without electron donor to the 10-mL liquid volume. During the first 9 h, only small amounts of PCE were converted, resulting in the formation of trace amounts of c-DCE (Figure 2A). At this time 40 µmol (1 mL) of H2 was added to the vial headspace, which initiated rapid transformation of PCE to c-DCE via TCE and further conversion to VC. Only traces of ethene were observed after 24 h. This result shows that reductive dechlorination of PCE did not occur without the supply of an electron donor and that H2 supported the transformation of PCE to VC. In Figure 2B, the effect of acetate on dechlorination of PCE is shown. A total of 21 µmol of acetate was added to the vial along with PCE (20 µmol/L) at the beginning of the experiment. Small amounts of PCE were converted to c-DCE (approximately 10%) during the first 9 h. TCE was not observed. After 9 h, 40 µmol of H2 was added to the vial. Transformation increased immediately, resulting in further formation of c-DCE and VC. Small amounts of ethene were formed during the 24-h test. This result suggests that acetate may be able to supply electrons at a very slow rate for dechlorination, whereas H2 is able to provide electrons for rapid dechlorination of PCE. When propionate (14 µmol) was added along with PCE (22 µmol/L) to the vials, PCE was converted immediately, resulting in the formation of TCE, c-DCE, VC, and ethene (Figure 2C). Substantial ethene was formed during the 24-h test period. Similar results (Figure

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FIGURE 2. Batch vial tests to determine the effect of different electron donors on dechlorination of PCE for PCE-dechlorinating FBR culture. (A) no electron donor; (B) acetate (2.1 mM); (C) propionate (1.4 mM); (D) lactate (2.2 mM). Values in parentheses correspond to initial concentrations of electron donor in the liquid phase. 2D) were observed with lactate (22 µmol). PCE (19 µmol/L) was transformed completely to VC and ethene. Batch tests were conducted with c-DCE and VC but not with TCE. Results with c-DCE and VC were strictly analogous to those with PCE. Batch test with various electron donors show that dechlorination occurred very slowly in unfed cultures and in acetate fed cultures, whereas it was rapid in cultures fed with H2, propionate, and lactate. The amount of ethene formed was greater with propionate and lactate than with H2, but the difference likely was due to less time available for degradation with H2 (15 h vs 24 h). Since propionate and lactate are converted to methane via H2, the results suggest a key role for H2 in the reductive dechlorination of PCE and the other chloroethenes. FBR Experiments: Effect of H2 Partial Pressure. Tests in the FBR to determine the effect of H2 concentration on dechlorination kinetics were preceded by tests to investigate how the H2 concentrations varied. Under normal operating conditions with a continuous lactate feed rate of 2.2 mmol/L, the steady-state concentrations of acetate and propionate were low, and the H2 partial pressure was 25-30 ppm. Stopping lactate feed resulted in a rapid drop of H2 partial pressure to below 5 ppm. A pulse addition of lactate (5.5 mmol/L of reactor volume) resulted in rapid increase of H2 partial pressure to values above 1000 ppm. These high H2 levels were maintained in the reactor for 3-6 h, followed by a rapid decline of the H2 partial pressure. Step changes in continuous addition of lactate at different concentrations were used to affect the H2 levels in the FBR (Figure 3). In the first 2 h without lactate feed, some electron donor remained from prior lactate feed, and the partial pressure of H2 was maintained at 6-8 ppm. With the lactate feed concentration at 0.83 mmol/L, the H2 concentration increased rapidly to a steady concentration, as was also seen

FIGURE 3. Response of H2 partial pressure to step changes in lactate feed concentration in the FBR. Lactate concentrations of 75, 150, 250, and 600 mg/L correspond to 0.83, 1.7, 2.78, and 6.6 mmol/L, respectively. with further increases. The steady H2 partial pressures were measured to be approximately 15, 20, 30, and 80 ppm for continuous lactate feed rates of 0.83, 1.66, 2.78, and 6.6 mmol/ L, respectively. Thus, feeding lactate at varying concentrations provided different steady-state H2 levels in the FBR. The observation that H2 levels could be elevated for a number of hours after increasing the lactate feed concentration to the FBR allowed the study of dechlorination kinetics at different H2 partial pressures. A lactate feed rate was set, and a pulse addition of PCE, TCE, or c-DCE was made. Liquid samples were taken for PCE, TCE, and c-DCE measurement. Gas recirculation and the automated gas sampling and analysis protocol allowed frequent measurement of H2, VC, and ethene with time. Reductive dechlorination rates for PCE, TCE, and c-DCE increased with higher H2 partial pressure as illustrated in Figure 4, which shows PCE dechlorination and H2 partial pressure resulting from different lactate loading rates. The lactate feed concentration in Figure 4A was 0, 0.28, 2.2, 4.4, and 8.8 mmol/L in Figure 4B-E, respectively. This resulted in H2 partial pressures of approximately 1, 3, 22, 55, and 150 ppm. A large amount of lactate (final concentration ) 5.5 mmol/L) was spiked at the beginning of the test shown in Figure 4F that resulted in a concentration of H2 that was out of range of the recorder (>1000 ppm); therefore, H2 is not plotted on this graph. Initial transformation rates were determined at different H2 partial pressures based on the slope of the line through the first three PCE measurements in each test. This method was used since initial PCE concentrations in the FBR were near the half-saturation concentration. PCE transformation was slow (3.2 µmol/L-hr) when no electron donor (H2 ) 1-2 ppm) was added to the FBR (Figure 4A). PCE degradation increased slightly (3.5 µmol L-1 h-1) with H2 partial pressure of 3-3.5 ppm (Figure 4B). The initial rates increased further with H2 partial pressure, clearly showing that PCE transformation rates were related to the H2 partial pressure in the FBR. Initial rates were 3.6, 5.9, and 6.3 µmol L-1 h-1 for H2 partial pressure of 20, 55, and 160 ppm, respectively. The initial PCE dechlorination rate was 7.2 µmol L-1 h-1 when lactate was spiked (H2 > 1000 ppm). Initial TCE and c-DCE dechlorination rates also increased with H2 partial pressure similarly to PCE. Rates of dechlorination for TCE and c-DCE were also determined based on the slope of line through the first three points and are presented in Table 1. FBR tests for VC were conducted slightly differently than those with other chloroethenes. c-DCE was added initially, and sufficient time was allowed for it to dechlorinate completely to VC. The rate of ethene production was used as a measure of VC dechlorination rates. Figure 5 shows the

FIGURE 4. Effect of H2 partial pressure on dechlorination of PCE. PCE concentrations reported on Y-axis are total amounts in the FBR divided by liquid volume. (A) Lactate ) 0 mg/L; (B) lactate ) 25 mg/L (0.28 mmol/L); (C) lactate ) 200 mg/L (2.2 mmol/L); (D) lactate ) 400 mg/L (4.4 mmol/L); (E) lactate ) 800 mg/L (8.8 mmol/L); (F) spike addition of lactate (initial concentration in the FBR ) 5.5 mmol/L). formation of ethene at different H2 partial pressures. Rate of ethene formation (or VC dechlorination) also depended directly on the VC concentration; the average concentration of which is reported in each figure. The rate of VC dechlorination increased with H2 partial pressure just as for other chloroethenes. In order to compare the effect of H2 partial pressure on VC dechlorination, the rates from the different tests were normalized to a VC concentration of 100 µmol/L. Rates normalized to a VC concentration of 100 µmol/L are given in Table 1. In Figure 6, the rates of transformation of PCE, TCE, c-DCE, and VC normalized to the maximum initial dechlorination rates seen with lactate spike additions are shown as a function of H2 partial pressure. Each chloroethene shows a definite increase in dechlorination rate with H2 partial pressure, following a pattern typical of Michaelis-Menten kinetics. The calculated half-velocity (Ks(H2)) constants for all of the dechlorination reactions for H2 utilization varied between 12 and 28 ppm (Table 2), but the apparent differences in Ks(H2) between different chloroethenes were not significant considering the confidence intervals for the estimated values (Table 2).

Discussion Hydrogen has been shown to be a key electron donor for chloroethene dechlorination in this study and in work by others (10, 13). The mixed dechlorinating/methanogenic enrichments maintained in this study showed faster dechlorination rates with higher H2 concentrations. Though different substrates have been shown to support dechlorination, many of these are fermented in dechlorinating enrichments with production of H2. Dechlorination rates for all chloroethenes tested (PCE, c-DCE, VC) were relatively low with acetate addition to batch dechlorination tests as compared

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TABLE 1. Initial Removal Rates for PCE, TCE, c-DCE, and VC (Normalized to Concentration of 100 µmol/L) for Varying H2 Partial Pressures Resulting from Different Lactate Feed Concentrations continuous lactate feed PCE lactate (mmol/L) steady H2 (ppm) PCE rate (µmol L-1 h-1) TCE lactate (mmol/L) steady H2 (ppm) TCE rate (µmol L-1 h-1) c-DCE lactate (mmol/L) steady H2 (ppm) c-DCE rate (µmol L-1 h-1) VC lactate (mmol/L) steady H2 (ppm) normalized VC rate (µmol L-1 h-1)

0 1.5 3.2

0.28 3.0 3.5

2.2 20 3.6

4.4 55 5.9

8.8 160 6.3

5.5 >1000 7.2

0 2 2.4

0.28 4 2.5

2.2 18 5.3

4.4 45 5.8

8.8 150 9.6

5.5 >1000 10.2

0 1 1.3

0.28 3.5 1.6

2.2 18 5.3

4.4 55 8.7

8.8 170 13.3

5.5 >1000 13.8

2.8 28 0.098

6.6 80 0.16

11 150 0.18

5.5 >1000 0.19

0 1 0.025

0.83 22 0.073

to much higher rates with the addition of H2, propionate, or lactate. The novel methodology developed in this study provided a means of determining the effect of H2 on dechlorination rates in the methanogenic enrichment culture. High lactate fermentation kinetics provided an instantaneous source of H2 that was consumed predominantly by methanogens in the FBR. Under normal steady-state conditions, H2 concentrations were low due to the long cell age and the development of a high methanogenic population. Lactate feed variations to the reactor changed the H2 production rate. Since methanogens were the primary consumers of H2, the FBR H2 concentration adjusted to a different value so that the H2 consumption rate equaled the production rate. A pseudosteady-state condition was maintained for several hours following a lactate feed change until corresponding changes in methanogenic population brought the H2 level back to the normal steady-state value. In the FBR studies, steady H2 partial pressures were maintained for approximately 3 h, which was sufficient time to study dechlorination kinetics. The dechlorination rates of all the chloroethenes tested (PCE, TCE, c-DCE, and VC) had a similar dependence on H2 concentration. The transformation kinetics followed a Michaelis-Menten relationship between dechlorination rate and H2 partial pressure with half-velocity constants (Ks(H2)) in the range of 12-28 ppm. The only other report quantifying dechlorination kinetics as a function of H2 partial pressure is by Smatlak et al. (13), which showed a Ks(H2) of 140 ( 70 ppm for conversion of PCE to VC. They did not study the role of H2 in the transformation of VC to ethene. The Ks(H2) for PCE to VC by Smatlak et al. (13) is almost an order of magnitude higher than our results. This difference could be due to differences in the enrichment cultures or the methodology used to obtain the Ks(H2). Dechlorination of PCE to ethene was carried out by one dechlorinating bacteria in their culture, whereas PCE dechlorination to ethene in our FBR was carried out by at least two dechlorinating microorganisms (17). The Ks(H2) values for dechlorination appear to be much lower than reported Ks(H2) values reported for methanogenesis by at least 1 or 2 orders of magnitude. Smatlak et al. (13) have reported Ks(H2) for both the methanogenic and dechlorinating bacteria in the same mixed enrichment. Their methanogenic Ks(H2) was 1300 ( 250 ppm in a butyrate degrading PCE/methanogenic enrichment. Ks(H2) values cited by Robinson and Tiedje (18) for H2 utilization by methanogens were much higher, ranging from 3000 to 17000 ppm. An even wider range of Ks(H2) was cited by GiraldoGomez et al. (19) with most values ranging from 1300 to 18500 ppm. In their study, they measured a Ks(H2) value of 11 ppm for methanogenesis, which is considerably lower than that

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reported in other studies. Dechlorinators may have difficulty competing for H2 in cultures where methanogens have such low Ks(H2) values. More research is needed to determine methanogenic Ks(H2) values in dechlorinating/methanogenic enrichments. The results thus far show that the chloroethene dechlorinators are better than methanogens at scavenging H2 at low concentrations. This has significant implications for the competition for H2 between dechlorinating and methanogenic bacteria in substrate-limited anaerobic environments. The ability of dechlorinators to compete with methanogens depends on three factors: relative cell yields, affinity for a common electron donor, and maximum specific utilization rates. The energy yield for H2 use in dechlorination reactions is theoretically four times higher than for methanogenesis and can potentially result in higher cell yields than for methanogens (17). The Ks(H2) values for dechlorinators are significantly lower than those for methanogens. Clearly, the dechlorinators seem to have an advantage over methanogens based on the first two factors. However, the maximum specific utilization rates (K) for dechlorinators (20 µmol H2 mg of protein-1 day-1) estimated in the FBR (17) are much lower than those reported for H2-utilizing methanogens (100-2600 µmol mg of protein-1 day-1) (20). Maximum specific utilization rates for dechlorinators were determined by dividing the maximum dechlorination rate by the estimated dechlorinator population. The dechlorinating bacteria population in the FBR was estimated by using the loading rate, bacterial decay rate, and a theoretical yield coefficient of 3 g of protein/mol of Cl- reduced. Using the above values (K ) 600 µmol mg of protein-1 day-1 for methanogens and 20 µmol mg of protein-1 day-1 for dechlorinators and Ks(H2) ) 1000 ppm for methanogens and 20 ppm for dechlorinators), the relative specific H2 utilization rates can be estimated for different H2 partial pressures. The ratio of specific H2 utilization rates of dechlorinating bacteria to that for methanogens is calculated as 1.12, 0.85, 0.5, 0.3, and 0.1 for H2 partial pressures of 10, 20, 50, 100, and 500 ppm, respectively. At a 10 ppm H2 partial pressure, the specific utilization rate of dechlorinators is higher than methanogens, but at a partial pressure of 20 ppm, the methanogens hold a slight advantage. Assuming that the yield coefficient for dechlorinators is approximately four times that for methanogens, the specific growth rate of dechlorinators is higher than that of methanogens up to approximately 100 ppm H2 partial pressure. Thus, dechlorinators can compete successfully with methanogens up to a H2 partial pressure of 100 ppm, and the competitive advantage is larger at lower H2 partial pressures. H2 seldom exceeds 100 ppm in methanogenic environments, so this analysis indicates that dechlorinators should normally have an advantage.

FIGURE 6. Relative rate of PCE, TCE, c-DCE, and VC as a function of H2 partial pressure in the FBR. Continuous line represents predicted relative rates assuming Ks(H2) values of 12, 19, 28, and 23 for PCE, TCE, c-DCE, and VC, respectively.

TABLE 2. Half-Velocity Coefficient of H2 Utilization for Different Chloroethenes

FIGURE 5. Effect of H2 partial pressure on dechlorination of VC. Ethene production was used as a measure of VC dechlorination rate. Average concentration of VC is reported for each test. Ethene concentrations reported on Y-axis are total amounts in the FBR divided by the liquid volume. Lactate feed concentrations in different figures were (A) lactate ) 0 mg/L; (B) lactate ) 25 mg/L (0.28 mmol/ L); (C) lactate ) 75 mg/L (0.83 mmol/L); (D) lactate ) 250 mg/L (2.78 mmol/L); (E) lactate ) 400 mg/L (4.4 mmol/L); (F) lactate ) 600 mg/L (6.6 mmol/L); (G) lactate ) 1000 mg/L (11 mmol/L); (H) spike lactate addition so that initial concentration ) 5.5 mmol/L. If the amount of electron donor is limited, the dechlorinating bacteria should out-compete the methanogenic bacteria. In this case, dechlorination would proceed satisfactorily with less production of methane. Thus, for stimulating in-situ bioremediation, less substrate feeding may allow dechlorination to occur with a decreased chance of plugging the subsurface soil from excess methanogenic bacterial growth. This analysis is hypothetical, since H2 utilization kinetics have been determined for only two dechlorinating

compd

half-velocity coeff (KsH2), ppm (nmol/L)

95% confidence interval, ppm (nmol/L)

PCE TCE c-DCE VC

12 (9) 19 (14) 28 (21) 23 (17)

0 to 26 (0 to 20) 3 to 40 (2.2 to 26) 17 to 38 (13 to 29) 15 to 31 (11 to 23)

enrichments, but it does point to a strong possibility that the amount of electron donor needed to promote chloroethene dechlorination can be minimized if the H2 partial pressure can be maintained at low levels. This would favor continuous low level electron donor addition in reactors or in biostimulation of contaminated aquifers versus batch feeding. Operation at higher H2 partial pressures would require much more electron donor than that needed for dechlorination, and a larger portion of H2 would be used by methanogenic bacteria. H2 partial pressures in different anaerobic subsurface environments are typically lower than half-velocity constants for dechlorinators. Lovley et al. (21) measured H2 partial pressures to be 9-13, 1.3-2.0, 0.25, and 0.07 ppm for methanogenic, sulfate-reducing, iron-reducing, and nitratereducing environments, respectively. These H2 partial pressures correspond almost to the lower thermodynamic limits for use of the respective electron acceptors. While the ability of dechlorinators to compete with methanogens has been discussed earlier, the significantly lower H2 partial pressures may make the dechlorinators much less competitive in other anaerobic environments.

Acknowledgments We thank the Water Environment Research Foundation (Grant 91-TFT-3), the King County Department of Natural Resources, and the National Institute for Environmental Health Sciences (Grant ESO-4696) for funding this research.

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Received for review July 29, 1996. Revised manuscript received February 10, 1997. Accepted February 21, 1997.X ES9606539 X

Abstract published in Advance ACS Abstracts, April 15, 1997.