Quantifying Genes and Transcripts to Assess the In Situ Physiology of Dehalococcoides spp. in a Trichloroethene-Contaminated Groundwater Site

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

^* To whom correspondence should be addressed. Email: alvarez{at}ce.berkeley.edu.

	Abstract

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 TCE-DNAPL site at Ft. Lewis (WA) that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA and bvcA genes were indigenous to the site. Identified RDase gene copy numbers closely summed 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 three orders of magnitude in the total Dehalococcoides population through the one-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 five percent 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 towards 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 co-located 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.