A Modeling Study and Implications of Competition between

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Environ. Sci. Technol. 2006, 40, 4473-4480

A Modeling Study and Implications of Competition between Dehalococcoides ethenogenes and Other TetrachloroetheneRespiring Bacteria JENNIFER G. BECKER* Department of Biological Resources Engineering, University of Maryland, College Park, Maryland 20742-2315

Bioaugmentation with cultures containing Dehalococcoides strains that dechlorinate cis-1,2-dichloroethene (cDCE) to ethene is often recommended at sites where indigenous populations dechlorinate tetrachloroethene (PCE) to cDCE. In these cases, Dehalococcoides populations may have to compete with other dehalorespirers for chlorinated ethenes and/or electron donors. A continuous-flow stirred tank reactor model was used to describe the substrate interactions in three conceptual models of competition between PCE-respiring populations under natural attenuation and engineered bioremediation scenarios. Model simulations were used to evaluate the effects of other chlorinated ethene respirers on substrate utilization by, and growth of, a Dehalococcoides strain (Dhc. ethenogenes strain 195) and identify the key factors influencing the outcome of competition among different dehalorespirers. The results suggest that, under natural attenuation conditions, Dhc. ethenogenes is unlikely to be the dominant population if a dehalorespirer that can compete for limiting amounts of reducing equivalents is present. Engineered bioremediation conditions resulted in greater enrichment of Dhc. ethenogenes than of competing dehalorespirers. Under several conditions, Dhc. ethenogenes coexisted with a PCE-to-cDCE (or PCE-to-TCE) dehalorespirer, primarily by functioning as a cDCE- (or TCE)-toethene dechlorinating specialist. From a bioremediation standpoint, maintenance of multiple dehalorespiring specialists appears ideal because it may result in the fastest and most extensive chlorinated ethene transformations. Thus, to improve our ability to successfully implement bioremediation, it may be helpful to characterize the indigenous PCE-to-cDCE respiring populations and the nature and distribution of electron donors used by these dehalorespirers at contaminated sites. Further understanding of these interactions requires more accurate information on the kinetics of known dehalorespiring populations.

Introduction Currently, the most promising bioremediation approaches for groundwater contaminated with tetrachloroethene (PCE) and/or trichloroethene (TCE) are based on dehalorespiration, a form of anaerobic respiration in which a chlorinated organic * Telephone: (301)405-1179; [email protected]. 10.1021/es051849o CCC: $33.50 Published on Web 06/17/2006

fax:

(301)314-9023;

 2006 American Chemical Society

e-mail:

compound serves as the terminal electron acceptor and, in the process, undergoes reductive dechlorination. Natural attenuation is a relatively low-cost bioremediation approach that relies on the intrinsic capacity of indigenous dehalorespiring microorganisms to utilize naturally occurring electron donors to reductively dehalogenate chlorinated ethenes and contain contaminant plumes. However, at many contaminated sites dechlorination of PCE and TCE is incomplete and leads to the accumulation of lightly chlorinated ethenes such as cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) (1), which also pose a health threat. The persistence of PCE daughter products in contaminant plumes is generally attributed to (1) limitation by a suitable electron donor, and/or (2) the absence of populations that possess the ability to reductively dechlorinate cDCE to VC and ethene. Therefore, engineered bioremediation approaches, including biostimulation via the addition of electron donors and nutrients or bioaugmentation with cultures that are able to efficiently transform PCE beyond cDCE, or both, are increasingly being recommended when natural attenuation does not result in complete detoxification of the parent contaminants (e.g., 1-4). Importantly, competitive interactions between microbial populations may complicate efforts to implement such engineered bioremediation approaches. For example, H2 is generally thought to be the ultimate electron donor for dehalorespiration at most chlorinated ethene-contaminated sites (5, 6), and biostimulation typically focuses on increasing H2 concentrations in chlorinated ethene plumes (7). However, other hydrogenotrophic populations, e.g., methanogens or sulfate-reducing bacteria, may also be present in contaminant plumes, compete with dehalorespiring populations for H2, and reduce the effectiveness of biostimulation (5, 6, 8). One strategy for overcoming this problem is to use syntrophic fermentation of low H2-generating organic substrates such as propionate to produce and selectively deliver H2 at low concentrations to H2-utilizing dehalorespirers, which have smaller H2 thresholds compared with many other hydrogenotrophs. H2 is used exclusively as the electron donor for reductive dechlorination by some dehalorespirers, including members of the genus Dehalococcoides. Dehalococcoides species are generally thought to play a critical role in the complete reductive dechlorination of PCE to ethene because, to date, growth of pure cultures via reductive dechlorination of ethenes with one or two chlorine substituents has only been demonstrated in closely related Dehalococcoides strains (9, 10). In addition, enrichment cultures (summarized in ref 11) and chlorinated ethene-contaminated sites (12) that exhibit dechlorination beyond the level of cDCE have generally been shown to contain sequences belonging to the Dehalococcoides phylogenetic group. Therefore, current dogma holds that bioaugmentation with an appropriate Dehalococcoidescontaining culture is needed to achieve complete reductive dechlorination of PCE at Dehalococcoides-deficient sites (11). At the same time, there is growing evidence that multiple dehalorespiring populations may inhabit at least some chlorinated ethene-contaminated sites (2, 12). Thus, in some cases, naturally occurring or bioaugmented Dehalococcoides populations may have to compete with other dehalorespiring organisms for growth substrates, including chlorinated electron acceptors and/or electron donors. Therefore, the presence of other dehalorespiring populations could conceivably affect substrate utilization by, and growth of, Dehalococcoides populations at contaminated sites. This is of practical significance because the reported kinetic conVOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Physiologies of Representative Dehalorespirers Active in Different Competition Scenarios organism

Dehalococcoides ethenogenes strain 195a Dehalobacter restrictus strain PER-K23d Desulfitobacterium sp. strain PCE1e Desulfuromonas michiganensis strain BB1g

chlorinated ethenes respired

electron donors used in dehalorespiration

non-chlorinated electron acceptors

PCE, TCE, cDCEb

H2c

none

PCE, TCE

H2c

none

PCE

lactate, pyruvatef, butyrate, formate, succinate, ethanol acetate, lactate, pyruvate, succinate, malatef, fumaratef

sulfite, thiosulfate, fumarate, malate S°, Fe(III), fumarate

PCE, TCE

a Refs 9 and 22. b Dhc. ethenogenes strain 195 also cometabolically transforms VC. c Acetate required for cell synthesis. 34. f Also used fermentatively. g Ref 17.

stants of different chlorinated ethene respirers vary and most of these organisms are able to respire only PCE and TCE (11). Thus, the outcome of competition between Dehalococcoides and other chlorinated ethene respirers could possibly influence the rate and extent of PCE dechlorination at a site. When evaluating the dechlorination potential of a site and deciding whether a natural attenuation or engineered bioremediation approach is most appropriate, emphasis is currently placed on testing for the presence of Dehalococcoides sequences associated with dechlorination of cDCE to VC and ethene (11, 13). It is generally assumed that PCEand TCE-respiring populations will be present. The potential impact of other dehalorespiring populations on the ability of Dehalococcoides to compete for substrates and the effectiveness of natural attenuation or an engineered bioremediation approach is not considered. The primary objective of this study was to use a mathematical model to predict the outcome of competition between a Dehalococcoides strain (Dhc. ethenogenes strain 195) and other chlorinated ethene respiring populations in an ideal model system to (1) evaluate how the presence of other chlorinated ethene respirers could affect substrate utilization by, and growth of, Dhc. ethenogenes, and (2) identify the key factors determining the outcome of the competitive interactions among different dehalorespirers.

Modeling Approach Conceptual Models of Competition. This evaluation focused on Dhc. ethenogenes strain 195, largely because of the availability of kinetic constants for this organism in pure or mixed cultures (9, 14). In addition to cDCE, Dhc. ethenogenes uses PCE and TCE as terminal electron acceptors (Table 1). Most of the other known dehalorespiring isolates can also metabolize PCE (11). Therefore, in developing the conceptual models of competition, it was assumed that two dehalorespiring populations will compete for PCE. Based on the substrate ranges of characterized PCE dehalorespirers and the availability of substrates in contaminant plumes, three different model scenarios in which Dhc. ethenogenes and another dehalorespirer compete for the electron acceptor (PCE), but differ with respect to competition for the electron donor, were identified. Additional substrate interactions could occur between PCErespiring populations, but they would represent variations on these scenarios. Examples of chlorinated ethene respiring isolates that represent the physiologies for which the three competition scenarios might arise are provided. These organisms were selected based on their substrate ranges, dechlorination products, and availability of biokinetic data. Case 1. In the first scenario (Figure 1A), Dhc. ethenogenes competes directly with a hydrogenotrophic dehalorespirer like Dehalobacter restrictus for H2. In fact, if these two populations occupy the same PCE-contaminated habitat, as they apparently did at the Pinellas Site (12), direct competition 4474

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d

Ref 21. e Refs 15 and

FIGURE 1. Conceptual models of competition between representative PCE-respiring populations: A, Case 1; B, Case 2; and C, Case 3. Competition for TCE could occur in Cases 1 and 3, but for clarity is not included in Panels A and C. for at least one substrate appears unavoidable, given the known physiologies of these organisms. Both species are able to couple reductive dechlorination of PCE only to the oxidation of H2, and energy conservation by these organisms using non-chlorinated electron acceptors has not been observed (Table 1). Case 2. In the second scenario (Figure 1B), an organotrophic dehalorespirer like Desulfitobacterium sp. strain PCE1 (Table 1) competes directly for lactate with a lactatefermenting organism that provides the H2 needed by Dhc. ethenogenes. Indirect competition for lactate-derived electrons thus occurs between Dhc. ethenogenes and strain PCE1, which converts lactate to acetate and CO2 (15). Case 3. Several members of the genus Desulfuromonas can dehalorespire chlorinated ethenes using acetate, but not H2, as the electron donor (16, 17). Therefore, a third PCEcompetition scenario, in which the dechlorinating populations utilize exclusive electron donors (H2 and acetate), could arise because H2 and acetate are both produced from the fermentation of a wide variety of organic substrates. The interactions between Dhc. ethenogenes and Desulfuromonas michiganensis strain BB1 (Table 1) shown in Figure 1C could conceivably occur in the PCE plume at the Bachman Road Residential Wells Site (2), where a Dehalococcoides and a Dsm. michiganensis strain (BRS1) (17) were detected. Mathematical Model Development. Competition for substrates between the two dehalorespiring populations in Cases 1-3 was modeled by modifying and expanding a biokinetic model that was developed to simulate fermentative production of H2 in a mixed culture containing methanogenic and dehalorespiring populations that competed for H2 (14). The dehalorespiring population was later identified as Dhc. ethenogenes (9). Accordingly, the dehalorespirer in the model

was able to dechlorinate PCE, TCE, cDCE, and VC but did not couple growth to the transformation of VC, which is transformed cometabolically by Dhc. ethenogenes (18). The model of Fennell and Gossett (14) was expanded by adding components for the PCE-respiring populations that compete with Dhc. ethenogenes in Cases 1-3. Dechlorination was modeled using dual Monod kinetics. Inhibition terms were not included for the chlorinated ethenes for two reasons. First, despite the fact that reductive dechlorination of TCE, cDCE, and VC is catalyzed by a single reductive dehalogenase (19) in Dhc. ethenogenes, reduction of PCE to ethene by Dhc. ethenogenes in experimental studies could be described with a Monod model (14). Second, inhibition constants have not been reported for the organisms modeled in this study. Thus, the model allowed reductive dechlorination of all chlorinated ethenes to proceed simultaneously. Reductive dechlorination of TCE was assumed to produce primarily cDCE because cDCE is the most common DCE isomer formed during PCE dechlorination at contaminated sites and by most PCE-dechlorinating isolates (20) (including Dhb. restrictus (21) and Dsm. michiganensis (17)). Accumulation of 1,1-DCE and trans-1,2-DCE has been detected during the conversion of PCE to ethene by Dhc. ethenogenes, following the appearance of cDCE (22), and Dsf. sp. strain PCE1 produces trace amounts of trans-1,2-DCE (and cDCE), in addition to large quantities of TCE. However, the model did not include terms for 1,1- and trans-1,2-DCE. The electron donor Monod term was modified for the hydrogenotrophic dehalorespirers by subtracting the H2 threshold concentration from the substrate concentration in the nominator and denominator, following the approach used by Fennell and Gossett (14). Thus, H2 utilization by an organism cannot continue at H2 concentrations below its threshold. Consistent with the substrate ranges of the model organisms, only PCE and TCE dechlorination contributed to the growth of Dhb. restrictus (Case 1) and Dsm. michiganensis strain BB1 (Case 3), and only PCE dechlorination contributed to the growth of Dsf. sp. strain PCE1 (Case 2). Utilization of acetate as a carbon source by Dhc. ethenogenes and Dhb. restrictus was not included in the model, and an adequate supply of all other growth factors was assumed to be available to all dehalorespirers. Net biomass growth for all dehalorespiring populations included a decay term, as described by Fennell and Gossett (14). Several previously described models of chlorinated ethene respiration have simulated batch (23-26) or semi-continuously operated (14) cultures. However, in a batch or semicontinuous culture, a substrate delivery interval must be selected, and this can have a significant impact on the outcome of substrate competition between two populations (25). Thus, in this study, the competitive interactions between dehalorespiring populations were modeled in a continuousflow stirred tank reactor (CSTR) to avoid introducing an artifact related to the substrate delivery interval into the modeling results. Equally important, a CSTR was also chosen because the competitive interactions between populations in mixed microbial cultures are best tested under the substrate-limited growth conditions that exist in a CSTR (27). Further, a CSTR should be relatively easy to use in future experimental evaluations of the simulated competitive interactions. Nevertheless, there are several important assumptions implicit in the selection of an ideal CSTR model system that must be considered when interpreting the simulation results and their relevance to phenomena occurring in chlorinated ethene-contaminated subsurface environments. First, in a CSTR, all biomass is suspended and, along with all substrates, uniformly distributed throughout the control volume at all times. In addition, the rates of contaminant biotransforma-

tion are not limited by mass transfer processes. Thus, if two populations in a CSTR are limited by the same growth substrate and wall growth is not excessive, competition results in the population with the slower substrate utilization rate decreasing in size over time and ultimately being eliminated from the system when its specific growth rate falls below the dilution rate (27). In contrast, the subsurface is dominated by attached microbial growth and inherently heterogeneous. Contaminant biotransformation rates in the subsurface are also frequently limited by mass-transfer processes such as dissolution of nonaqueous phase contaminants, sorption, or volatilization. As a result, the distribution of substrates and biomass may be spatially variable in the subsurface, and competition between two populations for the same limiting substrate may result in spatial, rather than temporal displacement of the slower growing organism. To incorporate such phenomena and predict contaminant fate and transport in the field, the biokinetic component of the CSTR model could be incorporated into an advection-dispersionreaction model of contaminant behavior, as recommended by other researchers modeling the behavior of mixed PCEdechlorinating cultures in simple laboratory systems (14, 23). Finally, in a CSTR, the concentration of a single growthlimiting substrate is controlled by the solids retention time (SRT) and the kinetic characteristics of the organism(s) transforming the growth-limiting substrate (27). In contrast, in an anaerobic batch culture, the concentration of a single growth-limiting substrate may decrease until a thermodynamically controlled threshold concentration is reached, below which metabolism is not feasible (28). The CSTR modeled in this study was assumed to have no headspace and a volume of 0.1 L. The flow rate, Q, was set at 5 × 10-3 L/d. This Q was selected because the corresponding dilution rate is slower than the maximum specific growth rate (µmax) values of the organisms examined in this study and it results in a SRT of 20 d, which is short enough to allow future experimental evaluation of steady-state competition within a reasonable timeframe. Model Implementation. The model was constructed and implemented in STELLA Research 8.0 (High Performance Systems). A Runge-Kutta 4 integration method was used with a calculation time step of 0.125 h. Further decreases in the time step resulted in a relative difference of less than 10-3. Simulations were run for 3600 h (150 d), which generally provided enough time for effluent biomass and reaction substrates and products to stabilize. Initial substrate concentrations were set equal to influent concentrations. The model was run so that, in addition to Dhc. ethenogenes, one other PCE-respiring population was active. A fermenter was also active and converted lactate, the only source of reducing equivalents supplied to the cultures, to H2 and acetate according to the following:

CH3CHOHCOO- + 2H2O f

CH3COO- + HCO3- + H+ + 2H2 (1)

The fermenter is not shown in Figure 1A and C because it does not compete for substrates in Cases 1 and 3. Dsm. michiganensis is able to utilize lactate as an electron donor for dehalorespiration (17). However, in Case 3, it was assumed to use acetate exclusively as an electron donor. Model Input. All of the kinetic parameters used in the simulations and their sources are summarized in Table 2. Maximum specific substrate utilization rate (qmax) values for the PCE-respiring populations competing with Dhc. ethenogenes were obtained directly from the literature or calculated from reported µmax values. Electron donor and electron acceptor half-saturation constants (KS) and decay coefficients (kd) were generally not available for these VOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Kinetic Constants Used in the Model Simulations constant

qmax (µmol/mg VSS/h) PCE TCE cDCE VC lactate KS,donor (µM) KS,chlorinated ethene (µM) PCE TCE cDCE VC Y (mg VSS/µmol donor)f kd (h-1)

Dhc. ethenogenesa

Dhb. restrictus

Dsf. sp. strain PCE1

Dsm. michiganensis

1.8 3 3 3

7.3b 7.3e

10.3c

8.3d 8.3e

0.1

0.1e

0.1e

0.1e

0.54 0.54 0.54 290 0.00612

0.54e 0.54e

0.54e

0.54e 0.54e

0.001

0.001e

0.00327b

0.0144c (0.0072) 0.001e

0.00190g (0.00048) 0.001e

lactate-fermentera

8.6 2.5

0.00351 0.001

a

Ref 14. b Calculated from ref 21. c Calculated from ref 34. d Calculated from ref 35. e Assumed value. f For dehalorespiring organisms that utilize organic electron donors, Y values were also calculated in terms of H2 based on the electron equivalents of the donors and are given in parentheses in terms of mg VSS/µmol H2 for comparative purposes. g Calculated from ref 17.

organisms and were set equal to the KS and kd values reported for Dhc. ethenogenes in a mixed culture (14). Yield values (Y) were obtained by converting values reported in the literature to the appropriate units or calculated theoretically based on the thermodynamics and stoichiometry of the half-reactions involved in energy generation and cell synthesis (29). Competition in each of the three cases described above was simulated for remediation of a PCE-plume using natural attenuation and engineered bioremediation (biostimulation). The influent substrate concentrations were based on conditions at the Bachman Road Residential Wells Site (2). For both sets of conditions, the aqueous PCE concentration was 9 µM (∼1500 µg/L), which falls within the range of PCE concentrations at the Bachman Road Residential Wells Site (0-1620 µg/L). Under natural attenuation conditions, the electron donor is typically the limiting substrate. According to Wiedemeier et al. (30), natural attenuation of chlorinated solvents is likely to be successful if, among other factors, the concentration of volatile fatty acids exceeds 0.1 mg/L. If present entirely in the form of the carboxylate lactate, this concentration corresponds to approximately 1 µM. A slightly higher concentration of lactate (5 µM) was used for the natural attenuation scenario. The fermentation of 5 µM lactate (eq 1) yields 20 electron microequivalents (e- µeq)/L as H2 and 40 e- µeq/L as acetate, respectively. Complete dechlorination of 9 µM PCE requires 72 e- µeq/L and, therefore, was limited by electron donor availability under natural attenuation conditions. Electron acceptor-limited conditions were achieved in the engineered bioremediation simulations by providing lactate at 100 µM, the concentration used in field studies of biostimulation at the Bachman Road Residential Wells Site. For both sets of bioremediation conditions, the initial concentration of each population, X, was set to 0.015 mg volatile suspended solids (VSS)/L, based on the work by Lee et al. (25). All other initial physical and chemical characteristics were modeled as described by Fennell and Gossett (14).

Results and Discussion Case 1. The simulated results of competition between Dhc. ethenogenes and Dhb. restrictus for H2 and chlorinated ethenes under natural attenuation conditions are shown in Figure 2. Under electron donor-limited conditions, the growth of Dhb. restrictus was favored over that of Dhc. ethenogenes, which, it appears, would have been gradually eliminated from the CSTR if a longer simulation period had been used (Figure 2A). Dhb. restrictus became the dominant population because its faster substrate utilization kinetics (Table 2) allowed Dhb. 4476

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FIGURE 2. Model simulation results for Case 1 under natural attenuation (A and B) and engineered bioremediation (C and D) conditions. A and C: Concentrations of Dehalobacter restrictus and Dehalococcoides ethenogenes. B and D: Rates of dechlorination of chlorinated ethenes by the two populations. restrictus to out compete Dhc. ethenogenes for most of the PCE and TCE (Figure 2B), and more importantly, a significant portion of the limiting H2 (data not shown). A different outcome of competition between Dhc. ethenogenes and Dhb. restrictus for growth substrates was observed under engineered bioremediation conditions. In this case, Dhc. ethenogenes was the dominant population, and the Dhb. restrictus concentration decreased continuously, suggesting it might ultimately have been displaced from the CSTR during a longer simulation period (Figure 2C). As under the natural attenuation conditions, Dhb. restrictus initially out competed Dhc. ethenogenes for more than half of the PCE and TCE because of its faster substrate utilization kinetics (Figure 2D). However, Dhc. ethenogenes was able to quickly build up a high biomass concentration by growing on cDCE and excess H2 and thereafter also began transforming increasingly larger fractions of PCE and TCE at the expense of Dhb. restrictus. Preliminary model simulations were performed with a calculation time step of 0.0625 h to evaluate the effect of a hydrogenotrophic methanogen with previously described kinetic characteristics (14) on the outcome of

competition between Dhc. ethenogenes and Dhc. restrictus under engineered bioremediation conditions. Because chlorinated ethenes were limiting, consumption of the excess H2 by the methanogen did not have a substantial effect on Dhc. ethenogenes and Dhc. restrictus biomass levels (data not shown). Case 2. The model predicted that, under natural attenuation conditions, the relatively fast PCE utilization kinetics of Dsf. sp. strain PCE1 (Table 2) allowed it to rapidly divert all of the lactate away from the fermenter and build up a high biomass concentration (Figure 3A and B). Without fermentative production of H2, elimination of Dhc. ethenogenes from the CSTR was also predicted. Under engineered bioremediation conditions, strain PCE1 also utilized lactate at a much higher rate compared with the fermenter. However, because the electron donor was provided in excess, the fermenter was still able to produce a relatively large amount of H2 (not shown). As a result, Dhc. ethenogenes became dominant primarily by utilizing the H2 to dechlorinate TCE and cDCE (Figure 3C and D), for which it had no competition. Utilization of nearly two-thirds of the PCE by strain PCE1 enabled it to maintain a stable population that was approximately one-third the size of the Dhc. ethenogenes population. The results of the Case 1 and Case 2 simulations for a CSTR could have several practical implications for the application of natural attenuation. The results suggest that under electron donor-limited conditions, it is unlikely that Dhc. ethenogenes will be the dominant dehalorespirer if a PCE-respiring population that can compete with it for reducing equivalents is present. If the organism competing with Dhc. ethenogenes cannot mediate more than one or two dechlorination reactions, then the major end-product of PCE biodegradation will be TCE or cDCE, unacceptable bioremediation end-points. If downgradient natural or exogenous electron donor sources are available in a contaminant groundwater plume, it is conceivable that Dehalococcoides strains could be spatially separated from populations with faster PCE utilization kinetics and thus survive by growing on lesser chlorinated ethenes. This scenario could not be tested with the simple CSTR model. Nevertheless, the Case 1 and 2 modeling results suggest that the detection (or addition) of a Dehalococcoides strain alone may not guarantee that complete dechlorination can be sustained at a site. If other dehalorespiring populations are present, it may also be important to characterize the distribution of the different chlorinated ethene respirers and the availability of electron donors to these populations when assessing the potential for complete dechlorination at a site. Interestingly, a survey of chlorinated ethene-contaminated sites for the presence of Dehalococcoides 16S rRNA gene sequences (12) found that Dehalococcoides 16S rRNA gene sequences could not be detected at sites where cDCE accumulated but were present at locations where complete dechlorination of PCE was observed. There are a number of potential explanations for these observations. For example, it is possible that key growth factors were lacking or contaminants were present at levels that were toxic for Dehalococcoides species at the sites where cDCE accumulated. Another potential explanation that arises from the results of this study is that other dehalorespiring populations may be present and able to out compete Dehalococcoides populations for limiting amounts of electron donor at the sites where daughter products accumulate. In an engineered bioremediation scenario, electron donor is generally provided in excess. Under these conditions, the presence of another dehalorespirer that relied on the same source of reducing equivalents as Dhc. ethenogenes did not prevent Dhc. ethenogenes from becoming dominant by growing on the lesser chlorinated ethenes and electron donor

FIGURE 3. Model simulation results for Case 2 under natural attenuation (A and B) and engineered bioremediation (C and D) conditions. A and C: Concentrations of Desulfitobacterium sp. strain PCE1 and Dehalococcoides ethenogenes. B and D: Rates of dechlorination of chlorinated ethenes by the two dehalorespiring populations and rates of lactate utilization by the fermenter and strain PCE1. cDCE utilization by Dhc. ethenogenes is not shown in D because it overlaps the plot of TCE transformation by this population. Note the differences in the X-axes scales. not utilized by the other dehalorespirer. This could explain why the application of bioaugmentation coupled with biostimulation successfully established new Dehalococcoides populations and dechlorination of cDCE to ethene within test areas at some sites that intrinsically dechlorinated PCE to cDCE (e.g., 3). Case 3. Under natural attenuation conditions, Dsm. michiganensis out competed Dhc. ethenogenes for most of the PCE and TCE due to its faster substrate utilization kinetics, but was the less abundant population in the CSTR due to the relatively low Y reported for this organism (Figure 4A and B, Table 2). The low Y may have been due to uncoupling of dechlorination and growth (17). A Y value of 0.0154 mg VSS/ µmol acetate (0.00385 mg VSS/µmol H2), which is much closer to the YH2 values of the other dehalorespirers, was calculated from a fs° value (29) reported for Dsm. michiganensis (31), where fs° (or fs(max)) represents the fraction of electron donor equivalents that are used in cell synthesis without considering decay. When the theoretical Y for Dsm. michiganensis was used in the Case 3 natural attenuation simulation, the trend shown in Figure 4A was reversed and Dsm. michiganensis became the dominant population (Figure 4C). Regardless of which Y was used for Dsm. michiganensis, Dhc. ethenogenes survived largely on H2 and cDCE, because neither substrate could be used by Dsm. michiganensis. The behavior of Dhc. ethenogenes under engineered bioremediation conditions when the experimentally determined Y for Dsm. michiganensis (Table 2) was used was similar to that observed in Case 1. That is, after rapidly building up a high biomass concentration by growing on cDCE and abundant levels of H2, Dhc. ethenogenes was able to out compete Dsm. michiganensis for all of the limiting electron acceptors (Figure 4D and E). Consequently, Dsm. michiganensis was eliminated from the CSTR. These results could explain population changes that occurred in an enrichment culture developed from contaminated aquifer materials at the Bachman Road Residential Wells Site (2). The culture initially contained at least one Dehalococcoides population, as well as a Dsm. michiganensis strain. After being VOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Model simulation results for Case 3 under natural attenuation (A-C) and engineered bioremediation conditions (DF). A, C, D, and F: Concentrations of Desulfuromonas michiganensis and Dehalococcoides ethenogenes. B and E: Rates of dechlorination of chlorinated ethenes by the two populations. An experimentally determined Y was used for Dsm. michiganensis (0.0019 mg VSS/ µmol acetate, Table 2) to obtain the simulation results in A, B, D, and E. A theoretical Y was used for Dsm. michiganensis (0.0154 mg VSS/µmol acetate) to obtain the simulation results in C and F. fed with high concentrations of lactate for one year, the Dsm. michiganensis strain was lost from the culture, but the Dehalococcoides population was retained. Conceivably, Dsm. michiganensis might be able to survive with a low Y under these conditions in contaminated subsurface environments by respiring alternative electron acceptors such as ferric iron or sulfur (Table 1). When the theoretical Y for Dsm. michiganensis was used in the Case 3 engineered bioremediation simulation, the acetotrophic dehalorespirer was not eliminated from the CSTR (Figure 4F). However, the concentration of Dhc. ethenogenes was nearly 2.5 times greater than that of Dsm. michiganensis at the end of the simulation period. It is interesting that the model predicted that two dehalorespiring populations could coexist in the CSTR under several conditions. When two dehalorespirers coexisted, each organism functioned primarily as either a PCE-to-cDCE (PCEto-TCE in the case of strain PCE1) or cDCE- (or TCE)-toethene dechlorinating specialist, even though Dhc. ethenogenes alone is capable of completely dechlorinating PCE to ethene. Specialization occurred in large part because the qmax constants for Dhc. ethenogenes growing on PCE and TCE are lower than the reported qmax,PCE constants of the PCEto-cDCE and PCE-to-TCE dechlorinators listed in Table 2. Thus, if two dehalorespiring populations have similar or the same KS values, as was assumed here, then it seems likely that Dhc. ethenogenes will have difficulty competing with other dehalorespirers for highly chlorinated ethenes, unless an abundant electron donor supply is available to Dhc. ethenogenes. 4478

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These findings are supported by the results of a recent study in which it was shown that one or more members of the genera Dehalococcoides and Desulfitobacterium were present in a PCE-to-ethene dechlorinating culture maintained in a CSTR on PCE, fermented yeast extract, and high concentrations of benzoate (32). The Dehalococcoides-like bacteria were capable of completely dechlorinating PCE to ethene using H2 as the electron donor in a subculture supplied with H2 and PCE. The role of the Desulfitobacterium-like bacteria in the CSTR was not clear, but based on the modeling results obtained in this study (in particular for Case 2 under engineered bioremediation conditions) it seems likely that at least a portion of the PCE (and TCE) was transformed in the CSTR by Desulfitobacterium-like bacteria. The electron donor utilized by the Desulfitobacterium-like bacteria in the CSTR and acetate-enriched subcultures may have been acetate, as suggested by the authors, or other volatile fatty acids, e.g., butyric acid, produced by the fermentation of yeast extract supplied to the CSTR and/or endogenous bacterial decay (5, 14). From a practical standpoint, conditions that stimulate and sustain multiple dehalorespiring populations in situ or in enrichment cultures developed for bioaugmentation applications may result in the most effective bioremediation treatments by taking advantage of the relatively fast substrate utilization kinetics of the PCE-to-cDCE (or TCE)-respiring specialists, as well as the ability of organisms such as Dhc. ethenogenes to “clean up” the partially dechlorinated compounds produced by other dehalorespirers. If the PCE-tocDCE dechlorinating organism happens to be an acetotroph like Dsm. michiganensis, then an added benefit of maintaining multiple dehalorespiring specialists may be more efficient utilization of the reducing equivalents provided by fermentable organic substrates. In this study, the benefits of utilizing both H2 and acetate derived from lactate fermentation in dehalorespiration processes are clear from the results of the natural attenuation condition simulations. As shown in Figure 5, only 20 e- µeq/L were available for dehalorespiration processes in Cases 1 and 2; therefore, at steady-state, dechlorination of PCE did not proceed beyond the level of cDCE (Case 1) or TCE (Case 2), despite the presence of an active Dhc. ethenogenes population in Case 1. In contrast, a total of 60 e- µeq/L could be used by Dsm. michiganensis and Dhc. ethenogenes in the form of acetate and H2, respectively, for dehalorespiration processes in Case 3, and PCE dechlorination proceeded beyond cDCE to VC and ethene. These observations support the idea that when characterizing chlorinated ethene-contaminated sites, it would be beneficial to assess any indigenous PCE-to-cDCE dechlorinating organisms and the nature and distribution of electron donors used by these dehalorespirers, in addition to testing for the presence of cDCE-to-ethene-dechlorinating Dehalococcoides populations (13). In this study, population interactions among two dehalorespirers in three competition scenarios were simulated for two bioremediation approaches. Clearly, additional substrate and population interactions could conceivably arise. For example, inhibition among chlorinated ethenes has been shown to be important in some mixed batch cultures, as previously summarized (26, 33), and appropriate inhibition kinetic constants could be added for different PCErespiring populations if necessary to fit the model to experimental data. The model could also be modified to describe the utilization of acetate for biosynthesis by Dhc. ethenogenes (9) and Dhb. restrictus (21). It also would be interesting to substitute other organisms that can grow on cDCE and/or VC for Dhc. ethenogenes in the model simulations. Evaluation of competitive interactions within more complex cultures containing three or more dehalorespiring populations might also be instructive.

FIGURE 5. Steady-state concentrations of ethenes and total e- µeq available for dehalorespiration processes in Cases 1-3 under natural attenuation conditions. Steady-state concentrations of ethenes were reached within approximately 1800 h. The Case 3 results were obtained using the theoretical Y for Desulfuromonas michiganensis (0.0154 mg VSS/µmol acetate; Table 2). Similar results were obtained for Case 3 when the experimentally determined Y was used for Dsm. michiganensis (0.0019 mg VSS/µmol acetate). The following values were used in calculating the e- µeq available for dehalorespiration: H2, 2 e- eq/mol; acetate, 8 e- eq/mol; oxidation of lactate to acetate, 4 e- eq/mol. Case 1, 10 µM H2 is available to the two dehalorespiring populations from the fermentation of 5 µM lactate (eq 1). Case 2, 5 µM lactate is available to Desulfitobacterium sp. strain PCE1 for oxidation to acetate after the lactate fermenter is eliminated from the CSTR. Case 3, 10 µM H2 and 5 µM acetate are available to Dehalococcoides ethenogenes and Dsm. michiganensis, respectively, from the fermentation of 5 µM lactate. Nevertheless, the model results presented here represent a first step in understanding how the presence of other dehalorespiring populations could affect substrate utilization by, and growth of, Dehalococcoides populations. In particular, the results suggest that the domination or even survival of Dehalococcoides populations could be jeopardized in a CSTR in which growth is limited by electron donor availability, if dehalorespiring populations that can compete for low levels of reducing equivalents are present. Although the outcome of competitive interactions in a homogeneous CSTR may be manifested differently than in a heterogeneous environment (27) such as a groundwater contaminant plume, the modeling results could help explain why Dehalococcoides sequences often cannot be detected in systems that do not dechlorinate PCE beyond cDCE. In fact, detection of Dehalococcoides sequences or bioaugmentation with Dehalococcoidescontaining cultures may not ensure that complete dechlorination of PCE can be sustained in the presence of PCEto-cDCE dechlorinating specialists unless adequate electron donor can be specifically delivered to Dehalococcoides populations. An approach for minimizing competition for H2 from other hydrogenotrophic populations based on fermentation of low-H2-generating substrates such as butyrate and propionate has previously been recommended (5, 6, 14). Because soluble low-H2-generating organic substrates are degraded slowly and thus persist for relatively long distances downgradient from their point of application, it is interesting to speculate that biostimulation with soluble low-H2-generating fermentable substrates also could be an effective approach for specifically delivering electron donor to Dehalococcoides populations, even if competition for substrates in a contaminant plume forces Dehalococcoides populations downgradient where lesser chlorinated ethenes produced by PCE-to-cDCE degrading specialists are abun-

dant. Based on the CSTR results, it seems likely that if Dhc. ethenogenes is sustained along with another PCE-respiring population, it will likely function as a cDCE- (or TCE)-toethene dechlorinating specialist, which may promote both rapid removal and complete dechlorination of the parent contaminants. PCE-degrading enrichment cultures are sometimes maintained on high concentrations of readily fermentable substrates (2, 32). In the CSTR model, these conditions resulted in greater enrichment of Dhc. ethenogenes than of competing dehalorespirers. Several simulations highlighted the uncertainty about some kinetic constants and, along with the sensitivity of the model predictions to certain parameters (Supporting Information), underscore the need for a more complete and accurate characterization of the kinetics of known dehalorespiring organisms. This information is needed to further our understanding of how competitive interactions can affect the growth and substrate utilization patterns of key chlorinated ethene respiring populations and make predictions about contaminant behavior during site cleanup using natural attenuation or engineered bioremediation approaches.

Acknowledgments This material is based upon work supported by a PECASE award made through the National Science Foundation under Grant No. 0134433. Dr. Donna E. Fennell (Rutgers University) graciously provided the model of H2 production and consumption and related information.

Supporting Information Available Sensitivity of the model predictions to the input parameters for Cases 1-3 under natural attenuation and engineered bioremediation conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review September 19, 2005. Revised manuscript received May 4, 2006. Accepted May 8, 2006. ES051849O