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Applied and Environmental Microbiology, May 2008, p. 2728-2739, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02199-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Quantifying Genes and Transcripts To Assess the In Situ Physiology of "Dehalococcoides" spp. in a Trichloroethene-Contaminated Groundwater Site{triangledown} ,{dagger}

Patrick K. H. Lee,1 Tamzen W. Macbeth,2 Kent S. Sorenson Jr.,3 Rula A. Deeb,4 and Lisa Alvarez-Cohen1,5*

Department of Civil and Environmental Engineering, University of California, Berkeley, California 94720-1710,1 North Wind Inc., 1425 Higham St., Idaho Falls, Idaho 83402,2 CDM, 1331 17th Street, Suite 1200, Denver, Colorado 80202,3 Malcolm Pirnie, Inc., 2000 Powell Street, Suite 1180, Emeryville, California 94608,4 Earth Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 947205

Received 26 September 2007/ Accepted 22 February 2008

Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of "Dehalococcoides" spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population, whereas the vcrA- and bvcA-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the bvcA and vcrA genes were consistently highly expressed in all samples examined, while the tceA transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the bvcA- and vcrA-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (FtL-RDase-1638) identified in the cDNA clone library tracked closely in field samples with abundance of the bvcA gene, suggesting that the FtL-RDase-1638 gene was likely colocated in genomes containing the bvcA gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of Dehalococcoides strains and their associated activity.

* Corresponding author. Mailing address: 726 Davis Hall, Department of Civil and Environmental Engineering, University of California, Berkeley, CA 94720-1710. Phone: (510) 643-5969. Fax: (510) 642-7483. E-mail: alvarez{at}ce.berkeley.edu

{triangledown} Published ahead of print on 7 March 2008.

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

Applied and Environmental Microbiology, May 2008, p. 2728-2739, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02199-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Behrens, S., Azizian, M. F., McMurdie, P. J., Sabalowsky, A., Dolan, M. E., Semprini, L., Spormann, A. M. (2008). Monitoring Abundance and Expression of "Dehalococcoides" Species Chloroethene-Reductive Dehalogenases in a Tetrachloroethene-Dechlorinating Flow Column. Appl. Environ. Microbiol. 74: 5695-5703 [Abstract] [Full Text]  


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