Detection and Quantification of Dehalococcoides

Bioremediation Journal, 12:193–209, 2008 c 2008 Taylor and Francis Group, LLC Copyright ISSN: 1088-9868 DOI: 10.1080/10889860802477218

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Detection and Quantification of Dehalococcoides-Related Bacteria in a Chlorinated Ethene-Contaminated Aquifer Undergoing Natural Attenuation Helmut Burgmann ¨ Institute of Biogeochemistry and Pollutant Dynamics, Swiss Federal Institute of Technology Zurich (ETHZ), Zurich, Switzerland; and Centre of Ecology, Evolution and Biogeochemistry, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Kastanienbaum, Switzerland Jutta Kleikemper, Laurence Duc, Michael Bunge, Martin H. Schroth, and Josef Zeyer Institute of Biogeochemistry and Pollutant Dynamics, Swiss Federal Institute of Technology Zurich (ETHZ), Zurich, Switzerland

The present address of Jutta Kleikemper is BMG Engineering, Schlieren, Switzerland The present address of Michael Bunge is Institute of Microbiology, University of Innsbruck, Innsbruck, Austria Address correspondence to Helmut ¨ Burgmann, Centre of Ecology, Evolution and Biogeochemistry, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Seestrasse 79, CH-6047 Kastanienbaum, Switzerland. E-mail: [email protected]

ABSTRACT Detection and quantification of bacteria related to Dehalococcoides is essential for the development of effective remediation strategies for tetrachloroethene (PCE)-contaminated sites. In this study, the authors applied three methods for quantifying Dehalococcoides-like bacteria in a PCE-contaminated aquifer undergoing natural attenuation in Grenchen, Switzerland: a catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) protocol, a competitive nested polymerase chain reaction (PCR) approach, and a direct PCR end point quantification with external standards. For the investigated aquifer, multiple lines of evidence indicated that reductive dechlorination (and likely dehalorespiration) was an active process. Both PCR-based quantification methods indicated that low numbers of mostly sediment-bound Dehalococcoides were present in the contaminated zone of the Grenchen aquifer. Estimates based on the quantitative PCR methods ranged from 2.1 × 107 to 1.5 × 108 sediment-bound Dehalococcoides 16S rRNA gene copies per liter of aquifer volume. In contrast, the liquid phase only contained between 8 and 80 copies per liter aquifer volume. CARD-FISH was not sensitive enough for the quantification of Dehalococcoides cell numbers in this aquifer. Cloning and sequencing of the PCR products revealed the presence of sequences closely related to Dehalococcoides isolates such as D. ethenogenes and Dehalococcoides sp. BAV1. An apparently abundant group (termed “Grenchen Cluster”) of sequences more distantly related to Dehalococcoides was also identified, so far without cultured representatives. KEYWORDS bioremediation, CARD-FISH, dehalogenation, natural attenuation, quantitative PCR

INTRODUCTION Tetrachloroethene (PCE) and trichloroethene (TCE) are among the most common groundwater contaminants (Wiedemeier et al., 1999). Such contaminations are of concern because chlorinated ethenes may persist in the subsurface for 193


decades or even centuries (Johnson and Pankow, 1992). In addition, PCE is a suspected carcinogen (Ruder et al., 2001) and one of the degradation products, vinyl chloride (VC), is a known human carcinogen (Kielhorn et al., 2000). A cost-effective remediation strategy for chlorinated ethene–contaminated sites is natural attenuation, i.e., the degradation of the pollutants by intrinsic microbial populations (Lee et al., 1998). Many bacteria have been isolated that are able to re¨ ductively transform chlorinated ethenes (Loffler et al., 2003; Smidt and de Vos, 2004) in a process termed reductive dehalogenation or, if coupled to energy conservation, dehalorespiration. For example, PCE- and TCE-dehalogenating organisms include Desulfitobacterium spp., Dehalobacter restrictus, Sulfurospirillum multivorans, Desulfuromonas chloroethenica, Enterobacter spp., ¨ and Dehalococcoides ethenogenes (Loffler et al., 2003). However, most of these organisms only partially dehalogenate chlorinated ethenes to cis-dichloroethene (cDCE), Dehalococcoides ethenogenes strain 195 is the only known cultivated microbial species that is able to reductively dehalogenate PCE to the innocuous end prod´ uct ethene (Maymo-Gatell et al., 1997). Although Dehalococcoides ethenogenes degrades VC only cometaboli´ cally (Maymo-Gatell et al., 2001), other Dehalococcoides strains (e.g., VS and BAV1) have been described that use VC directly as electron acceptor (Cupples et al., 2003; He et al., 2003). The presence and activity of Dehalococcoides spp. in a certain environment seems to require strict anaerobic conditions, hydrogen as electron donor, and the presence of halogenated organic compounds because to date no other electron acceptors were found to be used ´ by these organisms (Maymo-Gatell et al., 1997; Adrian et al., 2000; Hendrickson et al., 2002; He et al., 2003), despite reports of the occurrence of the organisms at uncontaminated sites (Rahm et al., 2006). The presence of cDCE, VC, and ethene in a PCE- or TCE-contaminated aquifer is usually considered an unambiguous signal for reductive dehalogenation (Wiedemeier et al., 1999). Dehalococcoides spp. are important candidates for the remediation of chlorinated ethene-contaminated sites. However, in order to monitor natural attenuation or to follow the success of bioaugmentation and biostimulation techniques, methods are needed to quantitatively assess the presence and activity of these organisms at field sites. Over the past decades, a great variety of methods have been developed to detect and quantify microorH. Burgmann ¨ et al.

ganisms in environmental samples without cultivation, e.g., based on polymerase chain reaction (PCR) or rRNA hybridization approaches (Amann et al., 1995; Theron and Cloete, 2000). Quantitative competitive PCR (cPCR) has been widely used to quantify microorganisms in environmental samples (e.g., Bjerrum et al., 2002; Leloup et al., 2004). This approach was used previously to quantify Dehalococcoides sp. strain VS in laboratory cultures (Cupples et al., 2003; Cupples, 2008). A number of studies have attempted quantification of Dehalococcoides cell densities in aquifers, either using realtime PCR (Lendvay et al., 2003; Smits et al., 2004; Lu et al., 2006; Rahm et al., 2006), a direct, semiquantitative PCR assay based on comparison with external standards (Major et al., 2002), or catalyzed reporter deposition fluorescence in situ hybridization (CARDFISH) (Fazi et al., 2008). Ribosomal RNA–targeted oligonucleotide probes have been increasingly used in environmental microbiology to specifically detect and quantify microbial cells in natural samples (Amann et al., 1995, 2001; Sahm et al., 1999; Theron and Cloete, 2000). Recently, CARD-FISH using horseradish peroxidase (HRP)-labeled oligonucleotides was developed for sensitive detection of environmental microorganisms, even with low ribosome content (Pernthaler et al., 2004). Furthermore, novel oligonucleotide probes targeting 16S rRNA and a fluorescence in situ hybridization (FISH) protocol for the specific detection of Dehalococcoides were designed (Yang and Zeyer, 2003). The probes have been used successfully to quantify Dehalococcoides cells in a batch reactor by FISH (Aulenta et al., 2004), or by CARD-FISH in enrichment cultures (Bunge et al., 2007; Fazi et al., 2008), and in groundwater samples (Fazi et al., 2008). The objective of this study was to test for the presence of Dehalococcoides bacteria, and to quantify their partitioning between sediment and groundwater in the plume of a chlorinated ethene-contaminated aquifer undergoing natural attenuation. In contrast to previous studies on biostimulated or bioaugmented aquifers, we expected lower cell densities and activities for this site, which had a relatively low level of contamination. The geochemistry of the field site was characterized to verify that the chemical and thermodynamic conditions allow for dehalorespiration to be a viable energy generating process. To determine Dehalococcoides cell densities in water from four wells and in sediment samples, the usefulness of two PCR-based quantification methods 194

(direct PCR end point quantitation and nested competitive PCR), as well as CARD-FISH, was assessed. The presence and identity of Dehalococcoides bacteria or Dehalococcoides-related organisms were verified by cloning, sequencing, and phylogenetic analysis.


MATERIALS AND METHODS Field Site Samples for this study were taken from four existing groundwater-monitoring wells (Kb97.10, Kb97.08, Kb97.02, and Kb97.04) in a PCE-contaminated aquifer in Grenchen, Switzerland (Figure 1). Several sources of contamination have been found, probably originating from the clock maker and machine industry in Grenchen (Steiner, 1999; Kontar, 2001). Continuous monitoring of the site over several years revealed that PCE, TCE, cDCE, and VC are the main contaminants (see Balsiger (2004) and the diploma theses of Kontar (2001) and Steiner (1999)). Wells Kb97.02 and Kb97.08 were selected for this study because they contained different suites of chlorinated ethenes, VC or PCE, TCE, and cDCE, respectively. Well Kb97.04 was chosen to examine the occurrence and survival of Dehalococcoides spp. downstream of the contaminated zone and Kb97.10 as uncontami-

nated control well. The monitoring wells consist of an 11.5 cm inner diameter polyvinyl chloride casing that partially penetrates the aquifer to a depth of 10 to 13 m (Kb97.02, Kb97.04, and Kb97.08) or ∼3 m (Kb97.10) below the potentiometric surface. All wells were screened in the saturated zone (Kontar, 2001). The potentiometric surface of the confined aquifer is located at 2 to 5 m and the aquitard at 15 to 25 m below ground surface (Kontar, 2001). The sandy (fine to medium grain size) sediment consists mostly of till, alluvial fans, and aggradation sediments with disseminations of sandstone and peat (Figure 1). Hydraulic conductivity in the aquifer ranged from 1.35 × 10−4 to 5.3 × 10−4 m s−1 (single-well pumping tests), the porosity was estimated at 0.12, and the linear groundwater flow velocity at 2 m day−1 to the north of Kb97.08 and 0.18 m day−1 to the south of this well (Kontar, 2001).

Sampling All water samples were retrieved on August 11 or September 02, 2003, from all wells and on July 28, 2004 (from Kb97.08 only). Water samples were taken from well Kb97.10 (Figure 1) at a depth of 4.5 m, and from wells Kb97.02, Kb97.04, and Kb97.08 at a depth of 12 m below ground surface using a Grundfos MP-1 pump (Grundfos Pumpen AG, F¨allanden, Switzerland).

FIGURE 1 Map of the study site near Grenchen, Switzerland (redrawn from a site map by Geotest, Zollikofen, Switzerland). 195

Dehalococcoides Quantification in Aquifer


Before sampling, at least two well volumes (240 L) of water were purged from the wells. For the analysis of chlorinated ethenes, water samples were collected in 40-ml glass vials, immediately acidified with HCl to pH 2, and closed without headspace with a Teflonlined screw cap. Samples for anions were filtered in the field using 0.45-µm polyvinylidenefluoride filters (Millipore, Bedford, USA) and stored in 12-ml plastic vials. Samples for alkalinity, pH, dissolved organic carbon (DOC), CH4 , ethene, and H2 analyses were collected without headspace in 117-ml serum bottles using butyl rubber stoppers. Samples for H2 analysis were poisoned with 250 µl of 10 M anoxic NaOH to achieve a final pH of ∼13. All samples were stored at 4◦ C prior to analysis. Dissolved O2 , S(−II), and ferrous iron (Fe(II)) concentrations were determined immediately in the field (see below), as was groundwater temperature using an appropriate sensor (Checktemp, Eurotronik, Germany) fitted to a flow cell. For total cell counts, FISH, CARD-FISH, DNA extraction, and enrichment culture preparation, unfiltered water was collected in sterile 117-ml or 1-L serum bottles without headspace using sterile butyl rubber stoppers. All samples for biological analyses were placed on ice for transport until further processing in the laboratory within a few hours. Sediment samples were collected on July 28, 2004, and November 11, 2004, from the contaminated, saturated zone close to well Kb97.08 (Figure 1) at depths of 3.3 and 3.5 m below ground surface with a handheld hollow-stem auger (Humax, Lucerne, Switzerland). Samples were stored under N2 atmosphere on ice during transport until immediate further processing in the laboratory within a few hours.

Enrichment Cultures A basal, anoxic medium (Yang and McCarty, 1998) was prepared and supplemented with 4 mg/L yeast extract and 0.5 mg/L resazurine as redox indicator. The final pH was ∼7.2. Anoxic medium (∼20 ml) was dispensed into 117-ml serum bottles, which were closed using butyl rubber stoppers. Ten grams of sediment (from either 3.3 or 3.5 m depth) was transferred to the bottles containing medium under N2 atmosphere in an anaerobic chamber. A second enrichment culture series was constructed using water from Kb97.08 amended with minerals, trace metals, vitamins, sodium sulfide (Yang and McCarty, 1998), yeast extract, and H. Burgmann ¨ et al.

resazurine (see above). Finally, all bottles were crimpsealed and briefly flushed with a 80% N2 /20% CO2 gas mixture. Thereafter, the cultures were amended with sterile, anoxic solutions that contained either acetate or pyruvate to achieve final concentrations of 5 mM or with 10 ml of sterilized H2 gas to achieve a final concentration of ∼10% in the gas phase. PCE was added at a final concentration of ∼160 µM. All treatments and controls with autoclaved inoculum were performed in duplicates and incubated statically at 25◦ C in the dark for 70 or up to 172 days.

Analytical Methods For the analysis of chlorinated ethenes in water samples, a 6-ml N2 headspace was introduced into the sampling vials, which were then shaken for 2 h at room temperature. Chlorinated ethenes were quantified by injecting 0.5 ml of headspace gas into a Fisons HRGC Mega 2 Series gas chromatograph (Fisons, Milan, Italy) equipped with a flame ionization detector and a GS-Q fused-silica capillary column (length, 30 m; inside diameter, 0.53 mm; Agilent Technologies, Basel, Switzerland). Analyses were performed by using a temperature program (2 min at 45◦ C, 20◦ C/min from 45◦ C to 180◦ C, 7 min at 180◦ C). Methane and ethene concentrations were determined by gas chromatography (GC Carlo Erba Model 8000, Rodano, Italy) on a HayeSep N column with N2 as carrier gas and a flame ionization detector, using a headspace method (Bolliger et al., 1999). Chlo2− ride, NO− 3 , SO4 , acetate, and formate concentrations were determined using a DX-320 ion chromatograph (Dionex, Sunnyvale, CA) (Kleikemper et al., 2002). Alkalinity was measured by potentiometric titration using Gran plots for graphical determination of the end point (Stumm and Morgan, 1981), and pH was measured in the laboratory with a MP 225 pH meter equipped with an InLab407 electrode (both MettlerToledo, Schwerzenbach, Switzerland). Dissolved inorganic carbon (DIC, sum of H2 CO3 , HCO− 3 , and 2− CO3 ) concentrations were calculated from alkalinity and pH (Stumm and Morgan, 1981). Dissolved O2 , S(−II), and Fe(II) were measured colorimetrically using a DR/890 colorimeter (Hach, Loveland, CO, USA) following standard protocols. The concentrations of DOC were determined commercially following a standard method (DIN EN 1484/H3) using a TOC-analyzer (Shimadzu, Tokyo, Japan). Hydrogen measurements 196

were conducted using a gas chromatograph equipped with a reduction gas detector (GC-RGD2; Trace Analytical, Stanford, CA, USA) (Conrad et al., 1989).


Geochemical Calculations In situ free energy yields (G ) per moles of H2 for dehalorespiration, iron and sulfate reduction, and methanogenesis in groundwater of wells Kb97.02 and Kb97.08 were calculated from ambient concentrations of the involved reactants and products (Table 1) according to Jakobsen et al. (1998) using Gibbs free energies of formation as reported (Stumm and Morgan, 1981; Dolfing and Janssen, 1994). We calculated the number of Dehalococcoides cells that can theoretically be supported by the observed geochemical conditions assuming that dehalorespiration was ascribed solely to Dehalococcoides sp.: we used the calculated G values for dehalorespiration and the chlorinated ethene concentrations in wells Kb97.08 and Kb97.02. These wells are approximately on one groundwater flowline. Furthermore, the following TABLE 1 Concentrations of Chlorinated Ethenes, Potential Dechlorination Products, Oxidants, Reduced Compounds, Potential Electron Donors, and other Parameters Measured in Four Wells of a Chloroethene-Contaminated Aquifer

Wells KB97.10 KB97.08 KB97.02 KB97.04 Chlorinated ethenes/potential dechlorination products PCE [µM] bda 0.240 bd bd TCE [µM] bd 0.072 bd bd cDCE [µM] bd 0.166 bd bd VC [µM] bd bd 0.099 bd ETH [µM] bd trb 0.046 0.0084 Potential terminal electron acceptors O2 [µ M] 14.94 4.22 6.06 3.84 NO− [µM] 24 94 50%. Distances were estimated using the maximum composite likelihood method in Mega4, and branch lengths are reestimated by linearization (Takezaki et al., 1995). Sequences were derived from the sequences cloned in this study (bold) and selected published 16S rRNA gene sequences of isolates and environmental clones. GenBank accession numbers are given in parentheses. Near-full-length clones are labeled with clone origins: S: Sediment 3.3 m near Kb97.08; W: Kb97.08 well water; M: enrichment cultures from Grenchen aquifer material with H2 and PCE. Short-fragment clones are numbered and originate from Kb97.02, Kb97.04, and Kb97.08 well water.

spp. in enrichment cultures prepared from Grenchen sediment samples (Table 3), with the PCR methods generally underestimating the CARD-FISH numbers by a factor of up to 3.5. The PCR reactions for both direct and competitive PCR approaches resulted in linear relationships between log (template concentration) and PCR product intensity on the agarose gel over at least 203

four orders of magnitude. For cPCR, equality of competitor and target amplification efficiency was shown by quantification of Dehalococcoides ethenogenes DNA. For the direct PCR protocol, use of a high-cycle PCR and end-point quantification requires all samples to be within the exponential amplification phase. The fulfillment of these requirements could be deduced from Dehalococcoides Quantification in Aquifer

the linear standard curve (between ca. 5 and 5 × 103 target copies per PCR reaction) and preliminary experiments with different cycle numbers (data not shown).

Total Cell Numbers and CARD-FISH in Environmental Samples Total cell numbers (DAPI) on a per aquifer volume basis in well Kb97.10 were approximately one order of magnitude higher than in the other wells. The lowest cell numbers were detected in well Kb97.08 (Table 3). In sediment, total cell numbers were three to four orders of magnitude higher than in water samples on a per aquifer volume basis (Table 3). In well water and sediment samples, cell numbers stained with Dehalococcoides specific probe Dhe1259c were in the range of 0.1% to 0.8% of total cells and thus fell below the 1% false positive rate detected with probe non-338.


Competitive PCR and Direct PCR For water samples, Dehalococcoides-specific cPCR and dPCR detected Dehalococcoides spp. abundances in all wells except Kb97.10, with calculated abundances of less than 0.0031% or cell densities of approximately 8 to 80 cells per liter (Table 3). In sediment, both quantitative PCR methods detected about two orders of magnitude higher Dehalococcoides spp. abundances than in water samples, ranging from 0.13% to 0.73%. Due to this and the much higher total cell number in sediment, the calculated Dehalococcoides cell densities were more than five orders of magnitude higher in sediment than in water on a per-volume basis (Table 3). For all samples, Dehalococcoides concentrations determined with cPCR were slightly higher than with dPCR, but the results of both methods were in the same order of magnitude, followed the same trend, and were significantly correlated. The quantitative PCR approaches confirmed the observation that Dehalococcoides abundances in the aquifer were too low (10% sequence deviation, it is questionable whether the “Grenchen Cluster” bacteria belong to the genus Dehalococcoides. They are not targeted by the CARDFISH probes Dhe1259c and Dhe1259c, and the long sequences retrieved from this cluster as well as the Lahn cluster sequences exhibit mismatches to the primers used in the quantitative PCR approaches (Dhe728f and Dhe1155r). Consequently, for future studies new primer sets may have to be designed that either target the “Grenchen Cluster” and other groups in their entirety or exclude them, respectively. Dehalococcoides Quantification in Aquifer


Both PCR-based methods (and indirectly, by its failure, CARD-FISH) indicated that Dehalococcoides-like sequences were generally of low abundance. The much higher abundance of Dehalococcoides-related bacteria in sediment versus well water samples indicated that the preferred habitat is on the surface of sediment particles. This poses a difficulty for the assessment of the Dehalococcoides population at sites undergoing natural attenuation. Sediment sampling is considerably more labor intensive and costly than well sampling. New sampling procedures for attached bacteria in aquifers should accordingly be investigated, e.g., approaches to dislodge and remove bacteria with suitable solutions injected into the aquifer. On the other hand, the occurrence of Dehalococcoides-related bacteria primarily bound to sediment may have considerable importance for understanding and improving remediation strategies for contaminations with chlorinated ethenes. The localization of Dehalococcoides spp. in biofilms attached to particles may facilitate maintenance of anaerobic microniches in aquifers, which may allow the occurrence of anaerobic dehalogenation processes in partially aerobic systems (Field et al., 1995). It may also facilitate the formation of effective syntrophic consortia, enabling complete dechlorination of PCE (Bunge et al., 2007). CARD-FISH performed well for the quantification of Dehalococcoides spp. in enrichment cultures and could also detect sparse cells in the aquifer samples. Therefore, it became clear that not the sensitivity of the staining procedure (e.g., constrained by low ribosomal contents or the small cell size) was the reason for the failure of CARD-FISH, but the small number of target cells in the Grenchen aquifer. Absolute Dehalococcoides cell numbers in Grenchen water samples were clearly below the detection limit given by the false-positive detection rate of 1%, allowing no reliable quantification in this aquifer. This indicates that the observed problems could probably not be solved by filtration of a higher sample volume. In addition, the probes used for CARD-FISH did not target the apparently abundant organisms of the “Grenchen Cluster” that were targeted by the PCR approaches. Quantification of Dehalococcoides in PCEcontaminated aquifers that undergo natural attenuation has previously been reported by Lu et al. (2006), who reported between 0.2 to 2.6 × 106 cells L−1 in groundwater using a quantitative PCR assay (Grenchen: 1.7 to 6.7 × 102 cells per liter groundwater). H. Burgmann ¨ et al.

A number of studies have estimated Dehalococcoides cell numbers in bioaugmented and biostimulated PCE-contaminated aquifers by using quantitative PCR assays (Major et al., 2002; Lendvay et al., 2003; Rahm et al., 2006). Fazi et al. (2008) report 2.6% to 6.5% Dehalococcoides sp. using CARD-FISH in well water from a bioremediated aquifer. Compared to all of these studies, we found several orders of magnitude less Dehalococcoides–related bacteria in groundwater samples from the Grenchen aquifer. The numbers reported for sediment by Lendvay et al. (2003) for biostimulated and bioaugmented sites (8.6 × 103 to 2.5 × 106 cells g−1 ) are likewise only partly in the same order of magnitude as those found in the Grenchen aquifer (4.6 × 103 to 1.9 × 104 cells g−1 by dPCR). The higher numbers reported in these studies may be mainly due to the much higher chlorinated ethene concentrations and are likewise affected by the installed biostimulation and bioaugmentation systems. Hence, although the density of Dehalococcoides-like bacteria in the aquifers is different, overall PCE degradation rates per cell could be similar. To verify if the determined cell densities for Dehalococcoides-related bacteria could explain the PCE degradation observed in the field, we calculated, based on thermodynamic data (Bach and Edwards, 2003), Dehalococcoides-like cell numbers that can be supported by the chlorinated ethene concentrations moving through a defined aquifer volume between wells Kb97.08 and Kb97.02. Computed numbers for Dehalococcoides-related cells in groundwater and aquifer material spanned two orders of magnitude (Table 5), mostly due to the wide range of estimates on carbon contents per cell given in the literature. Using Norland’s (Norland et al., 1987) assumption for typical bacterial carbon content or Duhamel’s (Duhamel et al., 2004) assumptions for Dehalococcoides cells, the predicted numbers agreed well (within a factor of two) with those determined by our quantitative PCR methods. These calculations indicated that the measured cell densities could indeed be sufficient to explain the reductive dehalogenation observed at the Grenchen site. Aqueous PCE concentrations as low as in this aquifer (0.2 µM) seem to be actively transformed by Dehalococcoides-related bacteria, in contrast to the findings of Cupples et al. (2004), who determined net cell decay below 0.7 µM VC plus cDCE. 206

TABLE 5 Calculated Cell Numbers in Sediment and Groundwatera Cell number, (L aquifer)−1

% of total cells

Duhamel Norland Balkwill

Sediment

Water

Sediment

0.79 ± 0.32 0.21 ± 0.084 0.010 ± 0.0039

0.0079 ± 0.0033 0.0021 ± 0.00088 0.000097 ± 0.000041

1.5 × 10 ± 0.58 × 10 3.8 × 107 ± 1.5 × 107 1.8 × 106 ± 0.72 × 106 8

Water 8

84.7 ± 35.3 22.4 ± 9.3 1.0 ± 0.4

a Calculated based on thermodynamic data, assuming three different cell carbon contents (Norland et al., 1987; Balkwill et al., 1988; Duhamel et al., 2004) (see text).


CONCLUSIONS This study successfully used quantitative PCR methods to quantify low numbers of Dehalococcoides and close relatives that are involved in the reductive dechlorination at the Grenchen natural attenuation site and likely to be active in dehalorespiration. The measured numbers are in agreement with geochemical data and thermodynamic considerations. The association of Dehalococcoides-related bacteria with sediment particles poses a challenge to monitoring, but may be an important factor for dehalogenation in aquifers that warrants further study.

ACKNOWLEDGEMENTS The authors wish to thank Daniel Hunkeler (University of Neuchatel, Switzerland) for help with sampling and providing access to the site. They thank the Canton of Solothurn, Switzerland, for providing access to the site, Ralf Conrad (MPI Marburg, Germany) for the hydrogen measurements, Bachema (Schlieren, Switzerland) for the DOC analyses, Dr. Steve Zinder for providing the authors with a culture of Dehalococcoides ethenogenes strain 195, and members of the MPI Bremen (especially J. Wulf) for initiating the authors into the secrets of CARD-FISH. The authors are thankful to Max H¨aggblom and several anonymous reviewers who helped them to improve earlier versions of the manuscript.

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