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Applied and Environmental Microbiology, November 2007, p. 6898-6904, Vol. 73, No. 21
0099-2240/07/$08.00+0     doi:10.1128/AEM.01218-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites{triangledown} ,{dagger}

Benjamin K. Amos,1,§ Youlboong Sung,2,3,§,¶ Kelly E. Fletcher,1 Terry J. Gentry,2,{ddagger} Wei-Min Wu,4 Craig S. Criddle,4 Jizhong Zhou,2,3 and Frank E. Löffler1,5*

School of Civil and Environmental Engineering,1 School of Biology, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, Georgia 30332-0512,5 Environmental Sciences Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, MS-6038, Oak Ridge, Tennessee 37831,2 Institute of Environmental Genomics and Department of Botany and Microbiology, University of Oklahoma, 101 David L. Boren Boulevard, Norman, Oklahoma 73019,3 Department of Civil and Environmental Engineering, Stanford University, 380 Panama Mall, Stanford, California 94305-40204

Received 31 May 2007/ Accepted 29 August 2007

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 106 and 1 x 104 16S rRNA gene copies per µl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per µl 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 107 ± 0.1 x 107 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.

* Corresponding author. Mailing address: School of Civil and Environmental Engineering, Georgia Institute of Technology, 311 Ferst Drive, 3228 ES&T Building, Atlanta, GA 30332-0512. Phone: (404) 894-0279. Fax: (404) 894-8266. E-mail: frank.loeffler{at}ce.gatech.edu

{triangledown} Published ahead of print on 7 September 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

§ B.K.A. and Y.S. contributed equally to this work.

Present address: Environment and Energy Research Center, Gwangyang Environmental Research Department, Research Institute of Industrial Science and Technology (RIST), 699 Kumho-Dong, Gwangyang-Si, Chollanam-Do 545-090, South Korea.

{ddagger} Present address: Department of Soil and Crop Sciences, Texas A&M University, 550A Heep Center, College Station, TX 77843-2474.

Applied and Environmental Microbiology, November 2007, p. 6898-6904, Vol. 73, No. 21
0099-2240/07/$08.00+0     doi:10.1128/AEM.01218-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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