Detection and quantification of Geobacter lovleyi strain SZ: implications for bioremediation at tetrachloroethene- and uranium-impacted sites

Abstract

Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 x 10(6) and 1 x 10(4) 16S rRNA gene copies per mul of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per mul of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 x 10(7) +/- 0.1 x 10(7) per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.

FIG. 1.

Melting curve analysis following qPCR for samples of G. lovleyi strain SZ (genomic DNA or plasmid DNA with a 16S rRNA gene insert of strain SZ [pSZ16S]), samples of G. thiogenes (genomic DNA), and no-template controls. The melting curves represent averages of triplicate qPCR reactions for each sample, and average T_m (±standard deviations) for each sample are listed in parentheses. −dRn/dT represents the negative derivate of the reported fluorescent signal (Rn) with respect to temperature (T). The T_m for the target amplicon is at the maximum rate of change (i.e., the greatest −dRn/dT) for each melting curve.

FIG. 2.

(A) Detection of strain SZ-like organisms via nested PCR in chlorinated ethene- and uranium-contaminated Oak Ridge IFC site groundwater taken from regions impacted by biostimulation (wells FW026 [lane 3], FW029 [lane 4], FW101-2 [lane 5], and FW102-3 [lane 6]) or not impacted by biostimulation (wells FW016 [lane 2], FW106 [lane 7], and TPB16 [lane 8]). Lane 1 is the DNA size marker (Invitrogen), and lane 9 is genomic DNA of strain SZ. (B) Average G. lovleyi strain SZ cell numbers per liter of Oak Ridge IFC site groundwater in a pilot-scale uranium bioreduction demonstration plot. Samples were taken approximately 1 month after the initial ethanol amendment (February 2004) and in May and August 2005, after 1 to 1.5 years of periodic ethanol biostimulation. The samples from August 2005 came from aquifer regions impacted or not impacted by biostimulation, as indicated. Error bars indicate standard deviations of triplicate qPCR reactions and are not shown when they are too small to depict. The asterisks indicate that the cell number estimates were extrapolated values outside the range of accurate quantification and that the melting curves did not contain the characteristic peak for strain SZ (see text for details). ND, fluorescence was not detected. (C) Melting curves following qPCR for representative samples from the Oak Ridge IFC site. The melting curves represent averages of triplicate qPCR reactions for each sample. For an explanation of −dRn/dT, see the legend for Fig. 1.