Compounded Effects of Chlorinated Ethene Inhibition on Ecological

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Compounded Effects of Chlorinated Ethene Inhibition on Ecological Interactions and Population Abundance in a Dehalococcoides Dehalobacter Coculture YenJung Lai† and Jennifer G. Becker‡,* †

Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State University, Tempe, Arizona 85287-5701, United States ‡ Michigan Technological University, Houghton, Michigan 49931, United States S Supporting Information *

ABSTRACT: The development of rational and effective engineered bioremediation approaches for sites contaminated with chlorinated solvents requires a fundamental understanding of the factors limiting the in situ activity of dehalorespiring bacteria. Frequently, multiple dehalorespiring bacteria are present at contaminated sites, particularly when bioaugmentation is applied. The ecological interactions between different dehalorespiring populations canalong with hydrodynamic and other environmental factorsaffect their activity and thus the rates and extent of dehalorespiration. An integrated experimental and modeling approach was used to evaluate the ecological interactions between two hydrogenotrophic, dehalorespiring strains. A dual Monod model of dehalorespiration provided a good fit to the chlorinated ethene concentrations measured in a coculture of Dehalococcoides mccartyi 195 and Dehalobacter restrictus growing on tetrachloroethene (PCE) and excess H2 in a continuousflow reactor. Inhibition of dehalorespiration by chlorinated ethenes was previously observed in cultures containing Dehalococcoides or Dehalobacter strains. Therefore, inhibition coefficients were estimated for Dhc. mccartyi 195 and Dhb. restrictus. The inhibition effects of PCE and TCE on VC dechlorination by Dhc. mccartyi 195, and of VC on PCE and TCE dechlorination by Dhb. restrictus, were compounded when these strains were grown in coculture, and dehalorespiring population abundance and survival could be accurately predicted only by incorporating these complex interactions into the dual Monod model.

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INTRODUCTION Bioremediation is increasingly being applied to cleanup groundwater contaminated with chlorinated solvents such as tetrachloroethene (PCE) and trichloroethene (TCE). Frequently this involves the addition of enrichment cultures containing bacteria that carry out dehalorespiration, a form of anaerobic respiration in which chlorinated organic compounds serve as the terminal electron acceptor and undergo reductive dechlorination to produce progressively less chlorinated daughter products. Specifically, bioaugmentation is often needed to introduce Dehalococcoides strains because members of this genus appear to be unique in their ability to reductively dechlorinate dichloroethenes (DCEs) and vinyl chloride (VC), but are not indigenous to all contaminated sites. The in situ activity of exogenous and/or native dehalorespiring bacteria may be limited by a number of factors. A fundamental understanding of the conditions and phenomena that may affect the activity of dehalorespirers is needed so that the subsurface environment can be appropriately engineered to © 2013 American Chemical Society

overcome any limitations on the dehalorespiring populations. Some progress has been made in developing rational engineered bioremediation approaches based on phenomenological observations of the microbiology and chemistry of contaminated sites. For example, biostimulation is applied at many sites to alleviate electron donor limitation and usually involves the addition of a substrate that is fermented in situ. This leads to the formation of H2, which is used as the electron donor for dehalorespiration by Dehalococcoides and several other dehalorespiring strains. Methanogens, sulfate-reducing bacteria, and other hydrogenotrophic populations are also present at contaminated sites. Differences in the H2 thresholds of various hydrogenotrophs can be exploited to minimize the ability of other populations to compete with dehalorespiring Received: Revised: Accepted: Published: 1518

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bacteria for H2 by supplying organic electron donors (e.g., propionate) for which syntrophic fermentation is thermodynamically feasible only at low H2 partial pressures. The H2 concentrations that result from fermentation of these compounds are higher than the thresholds of dehalorespirers but are thermodynamically inaccessible to most other hydrogenotrophs, and thus biostimulation with low H2-levelgenerating donors helps to selectively deliver H 2 to dehalorespiring populations.1−3 Modeling studies have shown that competitive and other interactions can also occur among different dehalorespiring populations in systems containing aqueous4 and nonaqueous phase chlorinated ethenes.5 In the current study, an integrated experimental and modeling approach was used to evaluate how the ecological interactions that could potentially arise between two hydrogenotrophic, chlorinated ethene-respiring bacterial populations affect the survival of the individual populations and the fate of the contaminants they transform. The experimental evaluations assessed population abundance and chlorinated ethene concentrations in a defined coculture maintained in a continuous-flow stirred tank reactor (CSTR) a classic microbial ecology approach uniquely applied to a bioremediation scenario. This approach allowed us to unambiguously assess the effects of the kinetic and physiological characteristics of individual populations on their interactions. In addition, the use of a continuous-flow system means that dehalorespiring populations may be simultaneously and continuously exposed to multiple chlorinated ethenes, which is also expected in contaminated groundwater environments. The working hypothesis for this study was that when the availability of the electron donor (H2) and/or an electron acceptor such as PCE or TCE limits the growth of these populations, they will have to compete for the limiting substrate(s) (Figure 1A). When H2 is provided in excess, as is frequently done in engineered bioremediation treatment approaches, the potential for a complementary interaction arises: PCE-to-DCE dechlorinators can initiate the dechlorination process, while Dehalococcoides strains can utilize the remaining H2 and transform the lesser chlorinated ethenes produced by these strains (Figure 1B). The two hydrogenotrophic, chlorinated ethene-respiring strains used in this study are Dehalobacter restrictus,6 and Dehalococcoides mccartyi 195 (formerly Dehalococcoides ethenogenes strain 195).7 Dhb. restrictus conserves energy through the dehalorespiration of both PCE and TCE.6 Dhc. mccartyi 195 is unique in its ability to completely dechlorinate PCE to ethene, but conserves energy via only the first three dechlorination reactions.8 The last step in this processtransformation of VCoccurs cometabolically in Dhc. mccartyi 195. Chlorinated ethenes have been shown to inhibit dehalorespiration in other studies conducted with Dehalococcoides-9−11 and Dehalobacter-containing cultures.12,13 Therefore, the inhibition effects of chlorinated ethenes on Dhc. mccartyi 195 and Dhb. restrictus were also evaluated in batch assays and inhibition terms and coefficients were incorporated into the mathematical model to understand how inhibition by chlorinated ethenes may affect the ecological interactions that occur when both populations are present.

Figure 1. Potential ecological interactions occurring between the hydrogenotrophic, chlorinated ethene-respiring strains Dhb. restrictus and Dhc. mccartyi 195: (A) competitive interactions involving electron donor and acceptor; (B) interactions involving electron acceptor are complementary, DCE produced by Dhb. restrictus is utilized as the electron acceptor by Dhc. mccartyi 195; and (C) compounded inhibition of dechlorination by the two populations caused by the electron acceptors and their dechlorination products. Figure 1A is reprinted with permission from Becker, J. G., A modeling study and implications of competition between Dehalococcoides ethenogenes and other tetrachloroethene-respiring bacteria. Environ. Sci. Technol. 2006, 40, 4473−4480. Copyright 2006 American Chemical Society.

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MATERIALS AND METHODS The reagents and detailed procedures used to maintain the Dhc. mccartyi 195 and Dhb. restrictus cultures are provided in the Supporting Information (SI). Inhibition Kinetic Coefficient Assays. Several previous studies observed competitive inhibition among chlorinated ethenes in mixed dehalorespiring cultures.9−11 Therefore, in the current study it was assumed that any inhibition of Dhb. restrictus or Dhc. mccartyi 195 by chlorinated ethenes would involve a competitive mechanism. A total of nine assays were conducted to evaluate different combinations of substrate and inhibitors for inhibition effects in Dhc. mccartyi 195 or Dhb. restrictus. Each inhibition kinetic assay was conducted using a set of 160 mL serum bottles containing 58 mL of media and excess hydrogen. The bottles all contained the same aqueous electron acceptor concentration, Sa [MS L−3], but different aqueous concentrations of the inhibitor, SI [MS L−3], as summarized in the SI (Table S1). Each bottle was inoculated with 3-mL of semicontinuous source culture, and the initial biomass concentration in the kinetic assay bottles, X0 [MX L−3], was estimated by measuring the protein concentration in the source culture. Competitive inhibition is described according to −qmax XSa dSa = S dt KS , a 1 + KI + Sa

(

I

)

(1)

−3

where KI [MS L ] is the inhibition coefficient, qmax [MS MX−1 T−1] is the maximum specific substrate utilization rate; X [MX 1519

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L−3] is the concentration of a dehalorespiring population; and KS,a [MS L−3] is the half-saturation constant for the electron acceptor. The kinetic assays were completed within 10 h. During this relatively short time period, biomass did not increase significantly. Therefore, X was treated as a constant and equal to X0, and the initial dechlorination rate, VT = dSa/ Xdt [MS MX−1 T−1] was also treated as a constant. Electron acceptor concentrations were monitored and used to calculate dSa/dt. Using previously obtained estimates of KS,a and qmax,14 KI was fit to linearized form of eq 1 given by qmax Sa VT

⎛ KS,a ⎞ = Sa + KS,a + ⎜ ⎟SI ⎝ KI ⎠

⎛ ⎜ dSa Sa = −qmax X ⎜ dt ⎜ KS ,a 1 + SI KI ⎝

(

)

⎞ ⎟ ⎟ + Sa ⎟ ⎠

⎛ ⎞ S H2 − S H2,threshold ⎜ ⎟ ⎜K ⎟ ( S S ) + − S ,H H H ⎝ 2 2 2,threshold ⎠

(4)

H2 utilization by the dehalorespirers was modeled by multiplying the dehalorespiration rate (eq 3 or 4) by the stoichiometric coefficient for H2 utilization and accounting for the fraction of electron donor equivalents consumed in cell synthesis. Net biomass growth included terms that account for cell synthesis resulting from dehalorespiration of chlorinated ethenes that serve as terminal electron acceptors (PCE, TCE, and, in the case of Dhc. mccartyi 195, DCE) and decay terms. The full set of substrate utilization and growth equations and information on the Monod kinetic parameter estimates used in the model simulations is given in the SI. Analytical Methods. Aqueous samples (1-mL) were obtained from reactor sampling ports and transferred to 11.84 mL amber glass vials sealed with Teflon-lined caps. Samples were incubated at 30 °C for 30 min, before removing headspace samples for analysis of chlorinated ethenes and ethene (0.5 mL). Chlorinated ethenes were analyzed using a Hewlett-Packard 5890 Series II Plus gas chromatograph (GC) equipped with a flame ionization detector, as previously described.15 The concentrations of chlorinated ethenes and ethene in samples were determined by comparison with calibration curves that were prepared using standards of known concentrations. Partitioning of the volatile compounds into the gas-phase was accounted for using Henry’s Law and the following dimensionless Henry’s constants at 30 °C: 0.917 (PCE), 0.491 (TCE), 0.190 (DCE), and 1.26 (VC).15 Protein concentration in the semicontinuous source cultures was measured using the Bradford assay (Sigma-Aldrich, St. Louis, MO). Aqueous samples (1 mL) were combined with 0.05 mL of 4.4 N NaOH in glass centrifuge tubes and incubated at 85 °C for 20 min to achieve cell lysis. The cooled samples were neutralized by adding 0.05 mL of 4.4 N HCl. Protein standards were prepared using bovine serum albumin (Lyophilized powder; Acros Organics). DNA Extraction and PCR. Population abundance in the anaerobic CSTRs was quantified using quantitative real-time PCR amplification (q-PCR) of 16S rRNA gene sequences. DNA was extracted from 10 mL samples using the DNeasy Blood & Tissue Kit (QIAGEN; Valencia, CA), as previously described.16 q-PCR was performed using the specific 16S rRNA gene primers shown in Table S1 (SI) and a Roche LightCycler 480 System (Roche Diagnostics Corporation, Indianapolis, IN) following the methods of Huang and Becker.16 Calibration curves (log 16S rRNA gene copy concentration versus an arbitrarily set cycle threshold value [CT]) for each strain were obtained by using serial dilutions of pure culture genomic DNA. The gene copy number in standards containing known DNA concentration was calculated according to Ritalahti et al.17 assuming an average molecular weight of 660 g/mol per base pair in double-stranded DNA and one gene copy per genome. The size of the Dhc. mccartyi 195 genome is assumed to be 1.5 Mbp,18 while the size of the Dhb. restrictus genome was assumed to be equal to that of Desulfitobacterium haf niense strain Y51 (5.7 Mbp).19

(2)

In experiments evaluating the inhibition of VC dechlorination by higher chlorinated ethenes, the inhibitor was added after PCE had been completely transformed to VC. Experimental System. The experimental system, including duplicate anaerobic continuous-flow stirred tank reactors (CSTRs), is described in detail in the SI. Components of the reactor system were autoclaved and assembled aseptically prior to initiating an experiment. Sterile, anaerobic media was pumped from the reservoir at a flow rate of 80 μL/min, which resulted in a 20 day SRT, using a syringe pump (Harvard Bioscience Inc.; Holliston, MA) equipped with two 100 mL gastight syringes (Hamilton, Reno, NV). The media contained 5000 μM acetate as the carbon source. The media reservoir was maintained under positive pressure using H2/CO2 (80%:20%), resulting in an aqueous concentration of approximately 645 μM H2. The H2/CO2 gas mixture was passed through oxygen traps (Trigon Technologies, Shingle Springs, CA) and a sterile polytetrafluoroethylene filter (0.2 μm, Millipore, Billerica, MA). The PCE stock solution (3.6 mM) was pumped at a flow rate of 0.4 μL/min using a syringe pump (Sage Instruments; Freedom, CA) and combined with the media feed to achieve an influent concentration 15 μM PCE. The experiment was initiated by inoculating each CSTR with Dhc. mccartyi 195 and Dhb. restrictus at concentrations that resulted in approximately 109 16S rRNA gene copies/L. Modeling Approach. The model used by Becker4 to describe theoretical interactions between dehalorespiring populations in continuous-flow systems without headspace was adapted for use in the current study. The multiple-step reductive dehalogenation process was described using one of two models. In one set of simulations, a modified dual Monod model of the form: ⎞ ⎛ ⎞⎛ SH2 − SH2,threshold dSa Sa ⎟ ⎟⎟⎜⎜ = −qmax X ⎜⎜ ⎟ dt ⎝ KS ,a + Sa ⎠⎝ KS ,H2 + (SH2 − SH2,threshold) ⎠ (3)

was used to account for the ability of both electron donor concentration (SH2) and electron acceptor concentration (Sa) to limit dechlorination rates and the complete cessation of dechlorination at H2 concentrations at or below an organism’s H2 threshold (SH2,threshold). Importantly, the modified dual Monod model assumes that there is no interaction among chlorinated ethenes. In the second set of simulations, eqs 2 and 3 were combined to account for the inhibitory affects of some chlorinated ethenes on the rate of dechlorination of other chlorinated ethenes. The inhibition model of dehalorespiration had the general form given in eq 4: 1520

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RESULTS AND DISCUSSION Using a modified dual Monod dehalorespiration model and kinetic parameter values assumed based on literature values, Becker4 predicted a shift from a complementary to a competitive electron acceptor interaction between Dhc. mccartyi 195 and Dhb. restrictus when excess H2 is available. The simulations showed that initially Dhb. restrictus was predominantly responsible for dechlorination of PCE to DCE, which supported the growth of Dhc. mccartyi 195. As Dhc. mccartyi 195 accrued biomass, it increasingly outcompeted Dhb. restrictus for PCE and TCE. Consequently, after an initial period of accumulation, the Dhb. restrictus biomass concentration decreased continuously, and Dhc. mccartyi 195 was established as the dominant population. Population abundance was experimentally measured in a coculture of Dhc. mccartyi 195 and Dhb. restrictus using qPCR in the current study (Figure 2) and followed the general biomass trends predicted by the earlier model simulations.4 16S rRNA gene copy numbers in both populations increased rapidly within the first three days of the experiment. After day 3, Dhc. mccartyi 195 kept growing until it leveled off on day 10 at a steady state concentration of approximately 109.84 gene copies/

L. In contrast, the concentration of Dhb. restrictus decreased at a relatively high rate between days 3 and 10 and then continued to decline gradually for the remainder of the experiment. When compared to the chlorinated ethene levels, which did not change substantially after five days (Figure 2B), the population shifts that occurred between days 5 and 10 demonstrate that the structure of dechlorinating cultures was dynamic, even during periods of consistent chlorinated ethene removal. These results highlight one of the advantages of maintaining multiple dehalorespiring bacteria within a dechlorinating culture. If one population becomes inhibited or limited by substrate availability, dechlorination may be sustained by a population with different characteristics. Consistent with the earlier model predictions, these experimental data suggest that Dhb. restrictus initially grew rapidly on PCE and TCE, but Dhc. mccartyi 195 transformed increasing fractions of the PCE and TCE as the Dhb. restrictus population declined in the CSTRs. The steady-state concentrations of PCE, TCE, and DCE were all less than 1 μM. VC was the major dechlorination product throughout the experiment, and the average aqueous VC concentration during the steady-state period was 15 μM. Ethene was not detected until day 20, and never exceeded 0.9 μM (data not shown). The model simulations performed by Becker4 were useful for evaluating the significance of key ecological interactions between dehalorespiring populations under typical bioremediation scenarios. However, kinetic parameter estimates are strongly influenced by culture history and kinetic assay methodology,16,20 and thus the model results obtained using kinetic parameter inputs from the literature could not be directly compared to the experimental data obtained in this study. Therefore, the model simulations were repeated using kinetic parameter estimates that were carefully obtained for Dhc. mccartyi 195 and Dhb. restrictus under conditions that are directly relevant to the experimental system used to evaluate the ecological interactions within the coculture.14 The dual Monod dehalorespiration model simulations performed with the parameter estimates specifically estimated for Dhc. mccartyi 195 and Dhb. restrictus provided an excellent fit to the measured concentrations of the chlorinated ethenes during the start-up and steady-state phases, respectively, of CSTR operation (Figure 3B). Qualitatively, the model simulations also captured several general trends in the biomass data including the initial increase in both populations and the dominance of Dhc. mccartyi 195 and gradual decline of Dhb. restrictus after the initial start-up phase (Figure 3A). However, the dual Monod model clearly did not provide a good quantitative fit to the experimental biomass measurements. For example, the steady-state Dhc. mccartyi 195 biomass concentration was approximately four times greater than that of Dhb. restrictus during this period. In contrast, the simulated Dhc. mccartyi 195 concentrations were only 1.1−1.8 times greater than the Dhb. restrictus concentrations from day 5 to day 30. These results suggest that the dual Monod model of dehalorespiration does not capture some phenomenon that limits the ability of Dhb. restrictus to grow via dehalorespiration of PCE and TCE in the presence of Dhc. mccartyi 195. While dual Monod kinetics provided adequate description of dehalorespiration of chlorinated ethenes in several studies,1,21,22 in other studies conducted using cultures containing Dehalococcoides strains, dechlorination rates were inhibited by other chlorinated ethenes, which compete for key enzymes.9−11 Therefore, the importance of competitive inhibition effects in

Figure 2. Experimental measured (A) 16S rRNA gene copy numbers and (B) chlorinated ethene concentrations in a Dhb. restrictus-Dhc. mccartyi 195 coculture growing on PCE and excess H2 in duplicate CSTRs. Symbols represent the mean concentration in the two reactors and error bars represent 95% confidence intervals. 1521

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Figure 3. (A) 16S rRNA gene copy numbers and (B) chlorinated ethene concentrations in a Dhb. restrictus-Dhc. mccartyi 195 coculture growing on PCE and excess H2 in duplicate CSTRs. Lines represent model predictions made using the modified dual Monod dehalorespiration model. Symbols represent the mean concentrations in duplicate reactors and are also shown in Figure 2.

Figure 4. Inhibition effects on initial rates of dehalorespiration by Dhb. restrictus, including inhibition of: (A) TCE dechlorination by PCE; (B) PCE dechlorination by VC; and (C) TCE dechlorination by VC.

Dhc. mccartyi 195 and Dhb. restrictus was evaluated in the current study. The initial rate of dechlorination of TCE by Dhb. restrictus decreased with increasing PCE concentrations, and PCE concentrations as low as 50 μM exhibited an effect (Figure 4A). The KI value (0.23 μM) estimated using eq 2 is more than an order of magnitude smaller than the KI value reported for PCE inhibition of TCE dechlorination in a mixed culture containing a Dehalococcoides strain (Table 1) and suggests that PCE strongly inhibits TCE respiration by Dhb. restrictus. In contrast, PCE concentrations of up to 100 μM did not noticeably impact TCE or DCE dechlorination by Dhc. mccartyi 195 (data not shown). The rates of DCE dechlorination by Dhc. mccartyi 195 decreased slightly with increasing TCE concentrations (up to approximately 75 μM; Figure 5D). The effects of PCE on VC dechlorination were more dramatic (Figure 5A). VC dechlorination was strongly inhibited, even at PCE concentrations 30 μM. Dechlorination of VC by Dhc. mccartyi 195 was also inhibited by TCE and DCE (Figure 5B,C). VC dechlorination rates were negligible at TCE concentrations >80

μM and DCE concentrations >170 μM during the first 5 h of the assay. Previous studies have shown that VC inhibited Dehalobacter strains growing on chlorinated ethanes.12,13 In the current study, VC inhibited dechlorination of PCE and, to a greater extent, TCE by Dhb. restrictus (Figure 4B and C), as reflected in the KI estimates (Table 1). In addition, when the VC concentration exceeded 30 μM, TCE was only partially transformed by Dhb. restrictus to DCE within a 48 h period (data not shown). The KI estimates obtained in the current study are generally higher than those estimated in previous studies conducted with cultures containing Dehalococcoides strains (Table 1). In some cases, this variability may be due to differences in the way the inhibition constants were estimated. For example, KI values are sometimes simply assumed to be equal to the corresponding KS values.9 In the current study, the most important observations are (1) VC dechlorination by Dhc. mccartyi 195 was inhibited by PCE and TCE; and (2) VC inhibited PCE and TCE dechlorination by Dhb. restrictus. In a batch system, these interactions between chlorinated ethenes may not be significant because the dehalorespiring populations generally experience sequential exposure to chlorinated ethenes as the parent 1522

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Table 1. Summary of Inhibition Constants (KI) for Cultures That Dehalorespire Chlorinated Ethenes inhibitor

electron acceptor

model parameter

PCE PCE TCE TCE DCE VC VC VC

TCE VCd DCE VC VC DCE PCE TCE

KI,PCE/TCE KI,PCE/VC KI,TCE/DCE KI,TCE/VC KI,DCE/VC KI,VC/DCE KI,VC/PCE KI,VC/TCE

Dhc. mccartyi 195a

Dhb. restrictusa

Dhc. mccartyi VSb

0.23 9.6 10.7 13 74.7

mixed culture containing Dehalococcidesc 3.9

3.6 7.8

2.8 2.8 1.9

3.74 0.56

a

This study. bCupples et al., 2004. cYu et al., 2005, KI values were assumed equal to estimated KS values. dVC is transformed cometabolically by Dhc. mccartyi 195.

Figure 5. Inhibition effects on initial rates of dehalorespiration by Dhc. mccartyi 195, including inhibition of: (A) VC dechlorination by PCE; (B) VC dechlorination by TCE; (C) VC dechlorination by DCE; and (D) DCE dechlorination by TCE.

model that incorporated inhibition effects (eq 4, Figure 6A). The inhibition model provided a much better fit to the experimentally measured concentrations of Dhb. restrictus and Dhc. mccartyi 195 biomass. These data provide strong evidence that the inhibitory effects of VC on TCE dechlorination by Dhb. restrictus resulted in much poorer growth of Dhb. restrictus than would be expected in the absence of VC. It is significant that the modeling and experimental results both suggest that an active Dhb. restrictus population cannot be sustained when VC concentrations are elevated. Interestingly, the incorporation of inhibition effects into the dehalorespiration model had little impact on the predicted chlorinated ethene concentrations (Figure 6B). The inhibition model provides a good fit to the experimental chlorinated ethene data, but the dual Monod model also describes the data well (Figure 3B). The ability of the two models to provide a good fit to the effluent chlorinated ethene concentrations can be understood by comparing the total rates of PCE and TCE dechlorination in the coculture predicted by the modified dual Monod model (Figure 7A) and the inhibition model (Figure

compound is converted to progressively less chlorinated daughter products. In contrast, dehalorespiring populations may be simultaneously and continuously exposed to multiple chlorinated ethenes in flow-through systems due to the constant introduction of the parent compound(s) and their ongoing transformation into daughter products. When multiple dehalorespiring populations are present, the potential for compound inhibition effects exists. For example, in the current study, the production of VC by Dhc. mccartyi 195 significantly slows TCE transformation by Dhb. restrictus, and the higher concentrations of TCE that result from VC inhibition will slow the rate of dechlorination of VC by Dhc. mccartyi 195, resulting in more VC accumulation. The revised conceptual model of the ecological interactions occurring between Dhb. restrictus and Dhc. mccartyi 195 that incorporates the compounded inhibition effects is shown in Figure 1C. Evidence of the compounded effects of inhibition in the Dehalobacter-Dehalococcoides coculture growing on PCE can be clearly seen by comparing the biomass concentrations predicted by the modified dual Monod model (eq 3, Figure 3A) and the 1523

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Figure 7. Total PCE and TCE dechlorination rates mediated by the coculture and the ratio of the PCE (or TCE) dechlorination rate mediated by Dhc. mccartyi 195 relative to that mediated by Dhb. restrictus, as predicted by (A) the modified dual Monod model, and (B) the inhibition model.

lactate fermentation products, but Dehalobacter was not detected during the period when PCE was dechlorinated. Based on the results of the current study, it is possible that inhibition of the Dehalobacter strain by VC contributed to its loss from the experimental packed column. The results of this study also have important implications for bioremediation practice, particularly for treatment scenarios in which maintenance of a stable Dehalobacter population is beneficial. For example, the high PCE and TCE qmax values characteristic of Dhb. restrictus are highly desirable at sites where chlorinated ethenes are present as nonaqueous phase liquids (NAPLs), because rapid utilization of aqueous-phase chlorinated ethenes enhances the dissolution and speeds up removal of the NAPL-phase contaminant sources.5 Importantly, it appears that a Dehalococcoides strain that metabolizes VC at high rate must be present to prevent VC accumulation in these and other treatment scenarios where active Dehalobacter populations would be beneficial. More generally, the results of this study highlight the need to characterize the inhibition effects that may impact the activity of important dehalorespirers and include inhibition terms in mathematical models of chlorinated ethene biodegradation and transport in order to accurately describe chlorinated ethene degradation in contaminated aquifers.

Figure 6. (A) 16S rRNA gene copy numbers and (B) chlorinated ethene concentrations in a Dhb. restrictus-Dhc. mccartyi 195 coculture growing on PCE and excess H2 in duplicate CSTRs. Lines represent model predictions made using the inhibition model. Symbols represent the mean concentrations in duplicate reactors and are also shown in Figure 2.

7B). They are very similar. However, the model outputs exhibit dramatic differences with respect to the amounts of PCE transformed by each population. The modified dual Monod model predicts that after four weeks, Dhc. mccartyi 195 will convert approximately two times more PCE and one-half as much TCE in the experimental reactors, compared to Dhb. restrictus. In contrast, the inhibition model predicts that within four weeks Dhc. mccartyi 195 will convert approximately 70 times more PCE and TCE compared to Dhb. restrictus (Figure 7B), and by day 40, the ratio of PCE or TCE dechlorinated by Dhc. mccartyi 195 relative to the amount dechlorinated by Dhb. restrictus increases to approximately 150. The ability of Dhc. mccartyi 195 to completely outcompete the VC-inhibited Dhb. restrictus strain for both PCE and TCE within a relatively short time period explains why the Dhb. restrictus biomass levels declined so rapidly in the continuous flow reactors. Interestingly, VC accumulated in a lactate-fed packed column that contained nonaqueous-phase PCE and was inoculated with an enrichment culture containing several dehalorespiring populations, including Dehalococcoides and Dehalobacter strains and Geobacter lovleyi strain SZ.23 The Dehalococcoides and Geobacter strains colonized the reactor and grew on PCE and

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ASSOCIATED CONTENT

S Supporting Information *

The reagents, cultures, and 16S rRNA primers used for qPCR; the initial conditions in the inhibition constant assays; a 1524

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description and schematic of the experimental reactor system; information on the equations and Monod kinetic parameter estimates used in modeling. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (906)487-2942; fax: (906)487-2943; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by a PECASE award made through the National Science Foundation under Grant No. 0134433. Deyang Huang constructed the experimental reactor system and his help with various aspects of the experimental and modeling work is gratefully acknowledged.

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REFERENCES

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dx.doi.org/10.1021/es3034582 | Environ. Sci. Technol. 2013, 47, 1518−1525