Effects of Sulfate Reduction on the Bacterial Community and


Itza Mendoza-Sanchez, Robin L. Autenrieth, Thomas J. McDonald, and Jeffrey A. Cunningham . Biological Limitations of Dechlorination of cis-Dichloroeth...
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Effects of Sulfate Reduction on the Bacterial Community and Kinetic Parameters of a Dechlorinating Culture under Chemostat Growth Conditions Dusty R. V. Berggren,†,§ Ian P. G. Marshall,‡,∥ Mohammad F. Azizian,† Alfred M. Spormann,‡,§ and Lewis Semprini†,* †

School of Chemical, Biological and Environmental Engineering, 101 Gleeson Hall, Oregon State University, Corvallis, Oregon 97331, United States ‡ Department of Civil and Environmental Engineering, Stanford University, Stanford, California, United States § Department of Chemical Engineering, Stanford University, Stanford, California, United States S Supporting Information *

ABSTRACT: Results are presented from a chemostat study where the reductive dehalogenation of PCE was evaluated in the absence and presence of sulfate. Two chemostats inoculated with the Point Mugu culture, which contains strains of Dehalococcoides mccartyi, were operated at a 50 day HRT and fed PCE (1.12 mM) and lactate (4.3 mM). The control chemostat (PM-5L, no sulfate), achieved pseudosteady-state transformation of PCE to ethene (98%) and VC (2%) at 2.4 nM of H2. Batch kinetic tests with chemostat harvested cells showed the maximum rate (kmaxX) value for each dehalogenation step remained fairly constant, while hupL clone library analyses showed maintenance of a diverse D. mccartyi community. Sulfate (1 mM) was introduced to the second chemostat, PM-2L. Effective sulfate reduction was achieved 110 days later, resulting in 600 μM of total sulfide. PCE dechlorination efficiency decreased following complete sulfate reduction, yielding ethene (25%), VC (67%), and cis-DCE (8%). VC dechlorination was most affected, with kmaxX values decreasing by a factor of 50. The decrease was associated with the enrichment of the Cornell group of D. mccartyi and decline of the Pinellas group. Long-term exposure to sulfides and/or competition for H2 may have been responsible for the community shift.



INTRODUCTION The chlorinated solvents tetra- and trichloroethene (PCE and TCE, respectively) were used extensively as solvents and degreasers during the 20th century. Due to poor disposal practices, they are among the most common groundwater contaminants.1 These, and their reductive dechlorination products, cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC), are suspected or known carcinogens, and are highly ranked on the priority list of hazardous substances to be addressed through remediation.2 The anaerobic bacterial species Dehalococcoides mccartyi can effectively dechlorinate these chlorinated aliphatic hydrocarbons (CAHs) to ethene, a nontoxic product, via organohalide respiration.3−5 Three different major phylogenetic groups (Cornell, Pinellas, and Victoria) of D. mccartyi have been identified to date.6 In situ bioremediation using these bacteria frequently requires limiting factors to be addressed to avoid accumulation of cis-DCE or VC.1 Enhancement options include the bioaugmentation of microbial cultures containing D. mccartyi and introducing an electron-donating substrate (typically fermentable organic compounds).1,7 With substrate © 2013 American Chemical Society

addition, the anaerobic reduction of ferric iron or sulfate may be stimulated.8−13 These competing acceptor reactions can hinder organohalide respiration through competition for electrons or generation of inhibitory products.9,15,16 Understanding the interactions between these anaerobic processes is key to mitigating the potential negative impacts of enhanced engineered bioremediation. This work investigated the interaction between sulfate reduction and strains of D. mccartyi under chemostat growth conditions. Thus far, studies concerning the effect of sulfate reduction on dechlorination have produced inconclusive results.9,10,12,13,17 They have shown no, partial, or complete inhibition of one or more of the dechlorination steps, and have suggested enzyme repression, H2 limitations, or sulfide toxicity as key inhibition factors.9,12,13 Received: Revised: Accepted: Published: 1879

October 17, 2012 January 2, 2013 January 14, 2013 January 14, 2013 dx.doi.org/10.1021/es304244z | Environ. Sci. Technol. 2013, 47, 1879−1886

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respectively) fitted with Teflon caps and PEEK tubing and fittings to allow anoxic transfers (See Supporting Information (SI) Figure S1). The influent feed was a sterile basal anoxic medium described by Yang and McCarty,19 adjusted to double the buffering capacity (1 g/L K2HPO3 and 3 g/L Na2CO3) and 0.20 mM of total sulfide (HS− and H2S). Sulfide concentrations in the influent feed ranged from 0.02 to 0.03 mM due to oxidation and/or precipitation of sulfide minerals. PCE (saturated, 1.12 mM) served as the electron acceptor, and sodium lactate (4.3 mM) was added as a fermentable electron donor. Fermentation of 4.3 mM lactate to acetate can yield 8.6 mM of H2, whereas the reduction of PCE (1.12 mM) would require 4.5 mM, thus excess H2 (4.1 mM) may be produced. Sodium sulfate (1 mM) was added to the influent of the PM-2L chemostat after 733 days of operation, with no increase in lactate. Assuming the acetate produced from lactate fermentation is not used for dechlorination or sulfate reduction, competition for H2 was expected as 4 mols of H2 are required for each mole of sulfate reduced. The chemostats were continuously stirred with 2 in. Teflon stir bars to achieve complete mixing. The feed rates resulted in hydraulic residence times (HRTs) of 50 and 55.5 days for the PM-2L and PM-5L chemostats, respectively. The long HRTs were chosen to avoid cell washout of these slow-growing dechlorinating microorganisms. Aliquots of culture were periodically sampled through PEEK tubing for protein, kinetic, or microbial analysis as described in the SI. Chemical Analysis Methods. Chemical analysis included frequent monitoring of the CAHs, ethene, H2, lactate, propionate, acetate, sulfate, total sulfide, and total protein concentrations in the chemostat and effluent fluid. The sulfide concentration was determined by the methylene blue method.28 The biuret assay was used to determine cell protein concentration after digestion of cell material in 3 M NaOH (30 min at 60 °C).29 Details of anoxic sampling and chemical analysis methods are provided in the SI and work by Azizian et al.23 PCE-to-Ethene Batch Rate Tests. Sequential dechlorination of PCE to ethene was monitored in batch reactors with cells periodically harvested from the chemostats. Conducted in duplicate, the tests were carried out in 125 mL borosilicate glass reactors with screw-on caps sealed by gray chlorobutyl rubber septa (Wheaton Industries). The culture (50 mL) was transferred from the chemostat into an anoxic batch reactor containing approximately 10% H2 (balance N2). Anoxic 75:25 N2/CO2 (Airgas, Inc.) was treated in a tube furnace (model 21100, Barnstead/Thermolyne) to remove trace oxygen before being bubbled through the batch reactors for 15−20 min to purge residual CAHs, ethene, or H2 and sulfide. Measurements showed total sulfide was stripped from the reactors (as H2S) during the purging process. The reactors were amended with 82 μmol H2 (∼15 000 nM Airgas, Inc.) and 16 μmol PCE (150 μM; 99%,spectrophotometric grade, Aldrich), then incubated at 20 °C with continuous shaking at 200 rpm. The concentration of CAHs, ethene, and H2 were monitored over several days. When the aqueous H2 concentration neared 10 000 nM, 5 mL of H2 was injected to the headspace of the reactor. These tests were conducted 5 and 4 times over the course of the PM-5L and PM-2L studies, respectively. PCE-to-Ethene Batch Data Modeling. A numerical model was developed to simultaneously estimate the rate of each dehalogenation step for the PM culture by fitting data from the batch rate tests.30 Standard Monod kinetics with

Reductive dechlorination can be inhibited by alternative terminal electron acceptor processes (TEAPs) competing for dissolved hydrogen (H2) or short-chain fatty acids.13,17 Previous research indicated that sulfate is of interest since it has a similar H2 threshold (1−15 nM) as chloroethene dechlorination (0.3−2 nM).18−20 Studies of this competition have been conducted with cultures in batch reactors,13 microcosms constructed with sediment and groundwater,12,17,21 or in sediment packed column studies.22−24 While of great value to investigate competition, they do not provide for microbial growth under pseudosteady-state concentration conditions. Chemostats were used in this study to achieve controlled conditions for growth. In our study the Point Mugu (PM) dehalogenating culture was maintained under chemostat growth conditions that yielded H2 concentrations in the range of the H2 thresholds. This culture was enriched from the shallow groundwater at Point Mugu Naval Weapons Station, California (PMNWS), where sulfate concentrations range from 34 to 5500 mg/L (0.4−57 mM) due to salt water intrusion.14 Early batch studies with this enrichment found D. mccartyi strains were present, and that the culture’s VC transformation rates exhibited a halfsaturation coefficient (Ks) value of 602 μmol/L, which is in the range reported for the Cornell group of D. mccartyi.25,26 Using clone library analysis, Azizian et al.24 reported the phylogenetic composition of microbial biofilms on Bio-Sep beads obtained after the PM culture was bioaugmented to a soil column and fed TCE and fermenting substrates. D. mccartyi, Geobacter, Desulfitobacterium, and Spirochaetes phylotypes were present. The PM culture was used to inoculate chemostats to permit the microbial community to adjust to the growth conditions of low H2 concentrations (2−30 nM) in the presence of PCE and the absence of sulfate reduction. Sulfate was then introduced to one of the chemostats to promote sulfate reduction as a competing electron acceptor process. Cells were periodically harvested from the chemostats to monitor the rates of each individual step of PCE dechlorination in batch tests, or to identify the relative distribution of key genera and D. mccartyi strains through clone libraries and quantitative PCR analysis. To effectively monitor all strains of D. mccartyi with sufficient accuracy, a new method was developed based on sequencing clone libraries of the large subunit of the [NiFe] uptake hydrogenase, hupL. The hupL method differentiated D. mccartyi strains more effectively than existing methods that target the gene encoding 16S rRNA.



MATERIALS AND METHODS Culture. The PM culture originated from a TCE contaminated aquifer with salt water intrusion at PMNWS, where active sulfate reduction, methanogenesis, and TCE dechlorination to cis-DCE and VC were observed.14 The PM culture was maintained for 10 years under batch growth conditions, being fed TCE and butanol as a fermenting electron donor.25,27 During this enrichment, methanogenesis ceased and the culture developed the ability to dechlorinate PCE completely to ethene.25 The PM culture was used to inoculate the first chemostat in a series of three identically operated chemostats: PM-I (inoculated April 2007), PM-2L (February 2008), and PM-5L (July 2009). Each successive chemostat was inoculated with culture from the previous chemostat. Chemostat Operation. The culture was maintained between 20 and 22 °C in GL-45 Kimax chemostat reactors (nominally 2L and 5L for the PM-2L and PM-5L chemostats, 1880

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based on CD-HIT clusters as illustrated in SI Figure S6, and dsrA assignments are presented in SI Figure S7. The abundance of major clusters (more than 10 representatives for at least one time point) is shown in Figure 5, and remaining clusters are grouped together as “other”. hupL and dsrA sequences were submitted to Genbank. hupL sequences were assigned accession numbers JX012186-JX012220 and dsrA sequences were assigned accession numbers JX012221-JX012223.

competitive inhibition were applied based on previous studies.25 The model was used to estimate the maximum reaction rate (kmaxX) for each step of the dechlorination process (assuming a fixed set of Ks values for the PM culture discussed below) to highlight when dechlorination was most affected. This was possible as the CAH mass in the batch reactors were such that the maximum concentrations observed were well above the Ks values (SI Table S1). A limitation of this method is that changes in the Ks resulting from community shifts could not be detected. This model used Ks parameters determined for the PM culture by Yu et al.:25 PCE (3.9 μM), TCE (2.8 μM), and cisDCE (1.9 μM). Yu et al. also reported a Ks for VC dechlorination of 602 μM; however, this reaction was suspected to have undergone an improvement after transitioning from batch to chemostat growth. Culture harvested from the PM-5L chemostat on day 638 was used to determine a new Ks of 12.1 μM for VC dechlorination (SI Figure S2). The appropriateness of these assumed Ks values was verified through modeling data from high- and low-PCE concentration batch rate tests, as discussed in the SI (Figures S3 and S4). Molecular Analysis Methods. Detailed procedures for molecular analysis are available in the SI. DNA extraction was performed as previously described.31 The percentage of the total bacterial community composed of Dehalococcoides (Dhc), Desulf itobacterium (Dsb), and Desulfovibrio (Dsv) was determined through qPCR measurement of relative 16S rRNA gene abundance. Monitoring the strain composition of the D. mccartyi community was performed using clone libraries of the gene encoding the putative uptake [NiFe] hydrogenase, hupL. We chose to analyze hupL as it is a marker gene that (a) is a singlecopy gene in each D. mccartyi genome sequenced to date,32 and therefore will not yield more than one sequence type per strain, (b) is an essential metabolic gene for all D. mccartyi, and therefore will likely be found in all D. mccartyi strains, and (c) as a protein-encoding gene, it shows a greater degree of sequence identity variation between D. mccartyi strains than the more commonly used 16S rRNA gene (SI Figure S5). Strains represented by D. mccartyi hupL sequence types are not necessarily physiologically identical, but the clone library approach demonstrates general trends in shifting D. mccartyi strain composition. Six hupL clone libraries were processed for the PM-2L chemostat to characterize the shifts that had occurred as the dechlorination abilities of the culture changed. The first sample corresponds to culture collection for inoculation of the PM-5L chemostat (between days 500 and 550), and the second sample on day 727, just prior to sulfate addition. The remaining four samples coincide with the batch kinetic tests performed with PM-2L chemostat culture. Two hupL clone libraries were prepared for the PM-5L chemostat to characterize a long-term steady-state observed. Clones were assigned to clusters using the CD-HIT clustering program33 at a 99.5% (for D. mccartyi hupL) or 99% (for dsrA) nucleotide identity threshold. Highly identical sequences from genomic databases were included in the CDHIT input. Representative sequences for each cluster designated by CD-HIT were aligned using the MUSCLE algorithm34 and their phylogeny reconstructed using the PHYML tree-building algorithm with 100× bootstrap35 implemented in the Geneious software package (Biomatters, Auckland, New Zealand). D. mccartyi strains were classified



RESULTS Chemostat Performance. Trends in effluent composition within both chemostats are presented in Figure 1 . Each history

Figure 1. PM-5L (left) and PM-2L (right) Chemostats effluent chemical composition and selected time stages. CAH and ethene data (a and c) are shown on top, and organic acid and H2 data (b and d) are on bottom. In charts a and c, arrows represent when samples were taken for clone library analysis. Sulfate and sulfide concentrations in the PM-2L chemostat are included in d. A step input of 1 mM sulfate addition to the PM-2L chemostat on day 733 is indicated by a dashed vertical line in part d, and the resulting theoretical sulfate concentration in the PM-2L chemostat if no reduction reaction occurred is shown as a solid curve. Both PM-5L and PM-2L data sets are on the same scale.

is divided into distinct performance stages, denoted 5-I to 5-III, and 2-I to 2-VI for the PM-5L and PM-2L chemostats, respectively. A summary of average analyte concentrations for each stage is provided in the SI Table S4. After a 50 day startup phase, the PM-2L chemostat maintained a pseudosteady-state (not shown) where PCE was transformed to TCE (1%), cis-DCE (2%), VC (64%), and ethene (33%). A second pseudosteady-state, stage 2-II, was characterized by nearly equal concentrations of VC and ethene, trace amounts of cis-DCE, H2 concentrations near 20 nM, and 5.6 mM acetate (Figure 1c,d). The PM-5L chemostat performance during its startup phase (5-I), resembled that of the PM-2L in stage 2-II with respect to dechlorination and the H2 concentration (30 nM). The dechlorination efficiency of both chemostats improved as H2 concentrations decreased below 10 nM. Sulfate addition to the PM-2L chemostat began on day 733. The dechlorination efficiency achieved in the PM-5L (control) chemostat continued to improve through stage 5-II as the H2 concentration decreased, and by stage 5-III PCE was transformed to ethene (98%) and VC (2%). Aqueous concentrations of H2 (2.4 nM) and acetate (4.2 mM), were 1881

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observed, while propionate concentrations were below the detection limit. The total protein concentration over stages 5-II and 5-III was 23.1 ± 3.4 mg/L. The pH ranged from 7.25 to 7.33, and the total sulfide concentration remained between 20 and 30 μM. Dechlorination within the PM-2L chemostat continued to improve after sulfate addition began on day 733 (stage 2-IIIb) with the total sulfide concentrations ranging from 100 to 270 μM (Figure 1d). PCE transformation efficiency peaked at ethene (80%) and VC (20%) (Figure 1c). H2 concentrations varied between 1 and 4 nM, and acetate decreased from 5.6 to 4.3 mM (Figure 1d). During stages 2-II and 2-III, total protein was measured as 24.7 ± 3.4 mg/L, in the same range as the PM-5L chemostat in stage 5-III. Similar performance was achieved during stage 2-IV while the protein concentration increased to 47 mg/L due to the growth of sulfate-reducers. A significant increase in the rate of sulfate reduction began during stage 2-V (Figure 1d). Sulfate concentrations decreased below detection, while sulfide concentrations increased above 600 μM. As the CAH reduction efficiency declined, H2 concentrations decreased to 1.9 nM. This is in the range of H2 thresholds for sulfate reduction and dehalogenation. Pseudosteady-state CAH concentrations achieved during stage 2-VI showed PCE transformation to ethene (25%), VC (68%), and cis-DCE (7%). Acetate concentrations gradually decreased from 5.6 to 3.6 mM, indicating that acetate was likely used as an electron donor for sulfate reduction. The protein concentration increased to 60 mg/L due to the growth of sulfate-reducers. About 70% of sulfate added to the chemostat was measured as sulfide. The other 30% was likely incorporated into biomass, or precipitated as metal sulfides as indicated by the black particulates in solution following sulfate reduction. Electron balances were calculated for several of pseudosteady-state stages of both chemostats, and are detailed in the SI (Figures S8 and S9). Electron balances for stages 5-II and 5III were very similar, with 16% of electrons used for dechlorination and 62−63% associated with acetate. Biomass generation accounted for 7.5% and 8.4% of electrons in stages 5-II and 5-III, respectively. The remaining electrons (13%) that were unaccounted for may have been associated with undetected organic acids. Electron balances for stage 2-II showed 86% of electrons were associated with acetate, 13.6% for dechlorination, 8.1% in biomass, and −7.8% in excess. With effective sulfate reduction in stage VI, acetate represented 52% of the electrons, 15% were directed to sulfate reduction, 12.3% to dechlorination, and 20.3% to biomass. The increase in biomass percentage results from the growth of sulfate reducers, and the decrease in acetate likely results from utilization by sulfate reducers as both a carbon and energy source.36 Modeling PCE-to-Ethene Batch Tests. The results of PCE-to-Ethene batch kinetic tests and model simulations are shown in Figure 2. The peak shapes of each CAH mass profile were well captured by the model. Similar kinetic responses were observed with cells harvested from the PM-5L chemostat (left column of Figure 2) between days 273 (stage 5-II) and 638 (stage 5-III). TCE never accumulated above 8 μmoles in any test, and little VC dechlorination to ethene occurred until cisDCE was almost all consumed due to the inhibition of cis-DCE on VC dechlorination. The average kmaxX values estimated through model analysis of the kinetic tests are presented in Figure 3. Duplicate reactors yielded very similar results as indicated by the range of the

Figure 2. Rate data (symbols) and associated the model fits (lines) using the standard Ks parameter set for the PM-5L chemostat (left) and PM-2L chemostat (right). The tests used cells harvested from the chemostats on the days indicated in the figure, that is, the results labeled “5L-638” represents a test conducted with cells harvested from the PM-5L chemostat on day 638. Batch results from a supplemental test, 5L-535, are supplied in the SI. Only one replicate is shown.

estimates. The kmaxX values were fairly constant over the pseudosteady-state periods of the PM-5L chemostat operation, with the standard deviations (as a percentage of the mean) ranging from 35% for VC to 49% for cis-DCE. There was no clear trend in changes in the kmaxX with time. The kmaxX values were also in good agreement with those reported by Yu et al. for PCE, TCE, and cis-DCE,25 while the VC values were much higher and in the range of 222 μmol/L/day measured in the Monod test (see SI) conducted with cells harvested the PM-5L chemostat on day 638 (SI Figure S2), further supporting the shift in the dehalogenating community under chemostat growth conditions. The low and high concentration tests (SI Figure S3) also yielded very similar kmaxX values (SI Table S2), showing the selected Ks values provided good fits. Results of batch kinetic tests with cells harvested from the PM-2L chemostat are presented in the right column of Figure 2. The cultures were harvested from the PM-2L chemostat on days 781, 1012, 1088, and 1172. Good model fits to the CAH mass profiles were obtained for all the tests. The rate test conducted with cells harvested 48 days after sulfate addition (day 781) shows a transformation profile similar to those in the PM-5L tests despite some active sulfate reduction. The kmaxX values were very similar to the average rates of the PM-5L tests (Figure 3) and were obtained when the most effective PCE to ethene transformation was achieved. With time, the kmaxX values decreased, especially for VC dechlorination (Figures 2 and 3). Some decrease in kmaxX 1882

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Figure 3. Average batch rate model kmaxX parameters for the PM-5L (green) and PM-2L (blue) cultures. The range of rates obtained with individual reactors shown by the error bars.

occurred for the PCE, TCE, and cis-DCE steps between days 781 and 1172 with the rates on day 1172 being 54−66% of the rates on day 781. At day 1172 (stage 2-VI), the kmaxX value of 5 μmol/L/day for VC was only 2.5% of the 197 μmol/L/day rate measured on day 781. This reduced rate of VC transformation corresponded to the period of complete sulfate reduction and PCE being transformed to ethene (25%), VC (68%), and cisDCE (7%). Bacterial Communities. Quantitative PCR (qPCR) was performed with cells harvested from the chemostat at select times. Previous analysis of the bacterial community in the PM culture showed that the organohalide-respiring community was comprised of the genera Dhc and Dsb.24 Clone library analysis of the dsrA gene from the PM-2L reactor showed that the sulfate-reducing community consisted entirely of several Dsv strains (SI Figure S7). Relative abundances of these genera in the PM-2L culture were measured using qPCR of 16S rRNA genes (Dhc, Dsb) and dsrA (Dsv) (Figure 4). Results were

might be attributed to the culture being maintained for 10 years in the absence of sulfate and washout from the chemostat prior to sulfate addition. Dsv represented approximately 20% of all bacteria in stage 2-VI when sulfate was being completely reduced. Clone Library Analysis of D. mccartyi hupL in the Chemostat Cultures. The relative distribution of D. mccartyi strains in each chemostat are displayed in Figure 5 and as a

Figure 5. Composition of the D. mccartyi community in the PM-2L (left) and PM-5L (right) chemostat cultures. Each bar indicates the number of hupL clones as a percent of the total D. mccartyi hupL clone library at the time the sample was collected. The number of clones analyzed for each sample (n) is provided.

phylogenetic tree in SI Figure S6. Five major clusters were identified: four clusters with a high degree of identity to D. mccartyi CBDB1, GT, and BAV1 and thus designated “Pinellas” clusters A, B, C, and D, and a cluster with high identity to D. mccartyi 195 and thus designated “Cornell”. Results from analysis of the PM-2L chemostat culture are displayed on the left, and results from the PM-5L chemostat are on the right. The Pinellas A cluster was dominant in the PM-2L chemostat before the transient stage in stage 2-IIIa (Figure 1) when H2 concentrations dropped from around 20 to 2 nM. As H2 concentrations decreased, Pinellas C and D gained dominance, and Pinellas A eventually fell below the detection limit. Following sulfate addition, the Cornell cluster slowly grew in proportion to the other strains present, making significant increases when VC dechlorination rates decreased following complete sulfate reduction around day 1100 (stage 2-V). Two hupL clone libraries were prepared for the PM-5L chemostat culture from samples collected at the beginning and end of the pseudosteady-states (stages 5-II and 5-III). The PM5L chemostat was inoculated with culture from the PM-2L chemostat having the D. mccartyi strain distribution shown in

Figure 4. Changes in the total bacteria (EUB) community in the PM2L chemostat based on 16S qPCR. Concentrations of Dehalococcoides (Dhc), Desulf itobacterium (Dsb), and Desulfovibrio (Dsv) are presented as a fraction of the total bacterial concentration. Vertical dashed lines indicate chemostat stage boundaries.

normalized to total bacteria using qPCR measurements of total bacterial 16S rRNA genes. Dechlorinating species made up more than 50% of all bacteria by the time sulfate was introduced (stage 2-IIIb). Dhc were enriched with time until the rate of sulfate reduction increased (stages 2-IV to 2-VI). The fraction of Dhc then decreased and varied between 20 and 60% of all bacteria. This coincided with the decrease in reductive dechlorination. Dsb were present in the chemostat prior to the introduction of sulfate, and represent 10−50% of all bacteria. The sulfate-reducing Dsv did not compose a significant portion of the bacterial community until the rates of sulfate reduction increased in stage 2-V. The low numbers 1883

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the first bar on the left of Figure 5. The PM-5L results show that the Pinellas A cluster was not eliminated from the chemostat with the increase in dechlorinating activity in stage 5-II, and, although the Cornell cluster increased in proportion to the other clusters in stage-5-III, this increase was not concurrent with a drop of Pinellas A below the detection limit as was observed in the PM-2L chemostat.

as ferrous iron.12 These inorganics were limited in the PM-2L chemostat; therefore, sulfide toxicity or inhibition was another possible factor inducing a shift in the D. mccartyi community. Most of the shift from the Pinellas strain cluster to the Cornell strain cluster occurred with the start of effective sulfate reduction in stages IV and V. One possibility is that the Cornell strain was able to outcompete the other strains under the growth conditions presented, including H2 competition and potential sulfide toxicity. Löffler et al.3 reviewed the typical culturing procedures applied for D. mccartyi, and sulfide concentrations of 0.2 mM were recommended for the standardized growth medium. Studies with Pinellas strain FL2 showed 5.0 mM sulfide shut down dechlorination activity, but no inhibition was observed at 1 mM.39 The maximum total sulfide concentration in the PM2L chemostat was 0.72 mM at a pH of 7.2. It is possible that the long-term exposure to sulfide under chemostat conditions was inhibiting to some strains of D. mccartyi. Further sulfide inhibition studies are needed on the specific D. mccartyi strains to determine if some are more inhibited than others. The ratio of Dhc to total bacteria did not significantly decrease with time (Figure 4), and cannot account for the 50fold decrease in VC transformation rates. However, this change did coincide with a more abrupt shift in the D. mccartyi community structure from Pinellas strains to a community more enriched in the Cornell strains (Figure 5). The fact that dechlorinating rates in the batch tests that had removed possible inhibitors and had excess H2 present well reflected microbial changes in the chemostat and indicate that the shift in the D. mccartyi community is likely responsible for the observed changes in rates. Yu et al.25 reported a very high Ks value (602 μM) for the original PM culture, which is on the same order of magnitude of that measured only for the Cornell-group D. mccartyi isolate, strain 195.25 Under the chemostat growth conditions of PM-5L chemostat, a much lower Ks value was measured (12.1 μM). The apparent 50-fold decrease in the estimated kmaxX may have resulted from a combination of kmaxX decreasing and Ks value increasing. If so, the culture may be shifting back to a dechlorinating community more like the one present when it was first isolated. Molecular methods to distinguish among strains of D. mccartyi were not available when the PM culture was initially enriched in microcosms constructed with aquifer solids and groundwater from PMNWS. The original microcosm studies showed a rapid reduction of TCE to VC, followed by very slow reduction of VC to ethene.14 Additional studies by Pang demonstrated the cometabolic nature of the PM culture when sulfate reduction was active.27 It is interesting to note that when enhanced in situ anaerobic bioremediation was applied to groundwater at PMNWS through lactate addition to stimulate the indigenous microorganisms, complete reduction of the 700 mg/L (20 mM) sulfate was rapidly achieved while the TCE and DCE present were mainly transformed to VC and trace amounts of ethene.40 Our results would support the Cornell strain being stimulated in situ at the PMNSW. This represents the first chemostat study showing shifts in the D. mccartyi community in response to sulfate reduction, competition for H2, and potential sulfide inhibition or toxicity. Pseudosteady-state dechlorination with a Dhc-containing culture was maintained for extended periods with nM H2 concentrations under chemostat growth conditions. A more diverse set of D. mccartyi strains were maintained in the PM-5L chemostat, which achieved effective rates for each of the



DISCUSSION The chemical, kinetic, and molecular analysis showed the PM5L chemostat achieved relatively steady performance throughout the experimental period. Sulfate addition to the PM-2L chemostat resulted in a gradual decline in dechlorination efficiency, a factor of 50 decrease in VC kmaxX values, and a shift in the D. mccartyi community. These changes accelerated when sulfate reduction rapidly increased and the culture was exposed to higher sulfide concentrations. Previous studies have shown that sulfate reduction primarily effects VC dechlorination rates (and cis-DCE rates to a lesser extent).10,13,17 Competition for H2 at low concentrations9,13 or toxicity/inhibition effects from products of sulfate reduction12 were indicated as being responsible for reduced rates. Our study indicates that a decline in dechlorination performance can also result from a shift in the D. mccartyi community. This shift may be related to the rate-influencing factors listed by others as explored below. Competition for H2 is frequently cited for the observed effects of sulfate reduction on dehalogenation as both processes have similar H2 thresholds.9,13 Throughout the later experimental periods, both chemostats were operating with H2 concentrations in the range of 2−4 nM, near the threshold H2 concentrations for cis-DCE, VC, and sulfate reduction. The culture that developed in the PM-5L chemostat efficiently dechlorinated PCE to ethene for over a year at H 2 concentrations between 2.0 and 2.9 nM while fed enough lactate to produce H2 in excess. It is interesting to note that about 2% of the VC remains to be transformed to ethene, thus H2 concentrations near 2.0 nM might be close to a VC threshold. The results also indicate the Ks for H2 utilization for the VC transformation step is likely in the range reported by Cupples et al.7 of 7 nM, since effective transformation to ethene was achieved at these low concentrations. A shift from the A, C, and D Pinellas strains to more of the Cornell strain occurred with long-term growth under the chemostat conditions of constant H2 concentrations. More work is needed under conditions of chemostat growth to see if this shift is a reproducible response, and if so, whether there is a competitive advantage for the growth of specific strains of D. mccartyi. The addition of sulfate to the PM-2L chemostat created conditions for competition for H2. Prior to significant sulfate reduction, H2 concentrations in stage 2-IIIb ranged from 10 to 1 nM, and around 4 nM during stage 2-IV. The results of the clone library analysis showed decreases in the Pinellas A cluster prior to significant sulfate reduction, following a similar trend as the PM-5L. Thus the conditions of the chemostat growth prior to sulfate reduction were responsible for some of the community shift observed. Products of sulfate reduction, including sulfite, thiosulfite, and sulfide, have been reported to be toxic or inhibitory to several anaerobic microbial processes, including dehalogenation.12,37,38 Hoelen and Reinhard demonstrated that abiotic sulfide was highly inhibitory to dechlorination of TCE, cis-DCE, and VC in the absence of sulfide-precipitating inorganics, such 1884

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(2) CERCLA 2007 Priority List Support Document. http://www. atsdr.cdc.gov/cercla/supportdocs/appendix-a.pdf (accessed May 16, 2012). (3) Löffler, F. E.; Yan, J.; Ritalahti, K. M.; Adrian, L.; Edwards, E. A.; Konstantinidis, K. T.; Müller, J. A.; Fullerton, H.; Zinder, S. H.; Spormann, A. M. Dehalococcoides mccartyi gen. nov., sp. nov., obligate organohalide-respiring anaerobic bacteria, relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidetes classis nov., within the phylum Chlorof lexi. Int. J. Syst. Evol. Microbiol. 2012, DOI: 10.1099/ijs.0.034926-0. (4) MaymoGatell, X.; Chien, Y. T.; Gossett, J. M.; Zinder, S. H. Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 1997, 276 (5318), 1568−1571. (5) He, J. Z.; Ritalahti, K. M.; Yang, K. L.; Koenigsberg, S. S.; Löffler, F. E. Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium. Nature 2003, 424 (6944), 62−65. (6) Hendrickson, E. R.; Payne, J. A.; Young, R. M.; Starr, M. G.; Perry, M. P.; Fahnestock, S.; Ellis, D. E.; Ebersole, R. C. Molecular analysis of Dehalococcoides 16S ribosomal DNA from chloroethenecontaminated sites throughout North America and Europe. Appl. Environ. Microbiol. 2002, 68 (2), 485−495. (7) Cupples, A. M.; Spormann, A. M.; McCarty, P. L. Vinyl chloride and cis-dichloroethene dechlorination kinetics and microorganism growth under substrate limiting conditions. Environ. Sci. Technol. 2004, 38 (4), 1102−1107. (8) Gerritse, J.; Drzyzga, O.; Kloetstra, G.; Keijmel, M.; Wiersum, L. P.; Hutson, R.; Collins, M. D.; Gottschal, J. C. Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium f rappieri TCE1. Appl. Environ. Microbiol. 1999, 65 (12), 5212−5221. (9) Aulenta, F.; Pera, A.; Rossetti, S.; Papini, M. P.; Majone, M. Relevance of side reactions in anaerobic reductive dechlorination microcosms amended with different electron donors. Water Res. 2007, 41 (1), 27−38. (10) Boopathy, R.; Peters, R. Enhanced biotransformation of trichloroethylene under mixed electron acceptor conditions. Curr. Microbiol. 2001, 42 (2), 134−138. (11) Drzyzga, O.; Gerritse, J.; Dijk, J. A.; Elissen, H.; Gottschal, J. C. Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulf itobacterium f rappieri TCE1 in defined chemostat cultures grown with various combinations of sulphate and tetrachloroethene. Environ. Microbiol. 2001, 3 (2), 92−99. (12) Hoelen, T. P.; Reinhard, M. Complete biological dehalogenation of chlorinated ethylenes in sulfate containing groundwater. Biodegradation 2004, 15 (6), 395−403. (13) Heimann, A. C.; Friis, A. K.; Jakobsen, R. Effects of sulfate on anaerobic chloroethene degradation by an enriched culture under transient and steady-state hydrogen supply. Water Res. 2005, 39 (15), 3579−3586. (14) Keeling, M. T. Bench-scale study for the bioremediation of chlorinated ethylenes at Point Mugu Naval Air Weapons Station, Point Mugu California, IRP Site 24. Master of Science Thesis, Oregon State University, Corvallis, 1998. (15) Fennell, D. E.; Gossett, J. M.; Zinder, S. H. Comparison of butyric kid, ethanol, lactic acid, and propionic acid as hydrogen donors for the reductive dechlorination of tetrachloroethene. Environ. Sci. Technol. 1997, 31 (3), 918−926. (16) Aulenta, F.; Majone, M.; Tandoi, V. Enhanced anaerobic bioremediation of chlorinated solvents: Environmental factors influencing microbial activity and their relevance under field conditions. J. Chem. Technol. Biotechnol. 2006, 81 (9), 1463−1474. (17) Aulenta, F.; Beccari, M.; Majone, M.; Papini, M. P.; Tandoi, V. Competition for H2 between sulfate reduction and dechlorination in butyrate-fed anaerobic cultures. Process Biochem. 2008, 43 (2), 161− 168. (18) Löffler, F. E.; Tiedje, J. M.; Sanford, R. A. Fraction of electrons consumed in electron acceptor reduction and hydrogen thresholds as indicators of halorespiratory physiology. Appl. Environ. Microbiol. 1999, 65 (9), 4049−4056.

transformation steps. When dechlorination in the PM-2L chemostat was challenged with sulfate, a decrease in extent of transformation of VC to ethene coincided with accumulating sulfide, and a shift in the D. mccartyi community to one dominated by a representative of the Cornell group. Multiple stressors likely contributed to the community shift, including competition for donor and sulfide inhibition, or potentially toxicity. More studies are needed to determine if such shifts are reproducible under controlled chemostat growth conditions. Inhibition studies should be conducted to determine if the Cornell strain of D. mccartyi is more tolerant of sulfide than the other strains present in the PM culture. Measurements of the kmax and Ks values for VC would help further support molecular based tests. Increases in the lactate concentration might also show if donor limitations were also responsible for the decrease in dechorination performance. It might also be possible to conduct studies where sulfate is added to the chemostat as FeSO4 so that the sulfide formed is precipitated as FeS in an attempt distinguish between sulfide toxicity and substrate limitations.



ASSOCIATED CONTENT

S Supporting Information *

Details of molecular methods, batch kinetic tests, phylogenetic trees, electron balances, and model development included. This material is available free of charge via the Internet at http:// pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Phone: 541-737-6895, e-mail: [email protected] edu. Present Addresses

§ CH2M HILL, 1100 NE Circle Blvd. Suite 300, Corvallis, Oregon 97330 ∥ Center for Geomicrobiology, Department of Bioscience, Aarhus University, Ny Munkegade 116, 8000 Aarhus C, Denmark

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This study was supported by the Department of Defense Strategic Environmental Research and Development Program (SERDP) through the grant ER 1588. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. D.B. performed the rates tests and the modeling analysis, interpreted the data, and drafted the manuscript; I.M. performed the molecular analysis and contributed to writing of the manuscript; M.A. performed the chemostat analysis; A.S. directed the molecular analysis and contributed to the interpretation of the results; L.S. directed the chemostat tests, batch rate tests, and modeling analysis, and also contributed to the interpretation of the results.



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