Detection and Quantification of Dehalococcoides-Related Bacteria in a Chlorinated Ethene-Contaminated Aquifer Undergoing Natural Attenuation

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

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 Häggblom and several anonymous reviewers who helped them to improve earlier versions of the manuscript.

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

Notes

^a Below detection;

^b Trace amount;

^c Not analyzed.

^a Based on bacterial PCR product and DNA concentration, respectively. See Material and Methods for details.

^b n.d. = No stained cells observed.

^c b.d. = Some stained cells observed, but below detection limit (1% false positive rate with probe non-338).

^d n.p. = No PCR product observed.

^a Δ G′ values were calculated according to the Nernst equation, e.g., for the reduction of PCE to TCE: ΔG′ = Δ G ⁰ + RTln c(PCE)× c(H ₂)/c(TCE)× c(Cl ⁻)× c(H ⁺).Δ G⁰ values were taken from (Dolfing and Janssen, 1994) and (Stumm and Morgan, 1981), and concentrations of reactants and products from Table 1.

^b Values for chlorinated ethenes are given for the reduction to the next lesser chlorinated compound.

^c Because the composition of Fe-oxides in the Grenchen aquifer is unknown, we assumed a more or less stable Fe(III) oxide (FeOOH (lepidocrocite)) with Δ G° _f values (free energy of formation) of −473 and −494 kJ mol⁻¹, respectively (Jakobsen and Postma, 1999).

^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).