Quantifying genes and transcripts to assess the in situ physiology of "Dehalococcoides" spp. in a trichloroethene-contaminated groundwater site

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 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.

FIG. 1.

Molar concentrations of chlorinated ethenes and ethene in groundwater samples over the 1-year monitoring period for treatment plots 1 and 2. Concentrations are reported as averages for eight sampling locations within each treatment plot, and error bars represent analytical error. The date and concentration of whey injected into the respective treatment plots are indicated on the top axis, and manipulations implemented at the site are indicated by arrows on the graphs. For clarity, only one of the two sampling events performed within 2 weeks of each other during March 2005 is plotted on the graph.

FIG. 2.

Dynamics of the Dehalococcoides (Dhc) 16S rRNA gene and RDase gene concentrations over the 1-year monitoring period for treatment plots 1 and 2. Manipulations implemented at the site are indicated by arrows on the graphs. Data at each time point are averages for samples from the eight monitoring wells in the respective plot, and each error bar represents one standard deviation for the qPCR method.

FIG. 3.

The sums of RDase gene concentrations are compared against the Dehalococcoides (Dhc) 16S rRNA gene concentrations on a log-log scale. Each data point represents a sample collected at a monitoring well from treatment plots 1 and 2 during each sampling event. Each error bar represents one standard deviation for the qPCR method. The dashed line represents the hypothetical 1:1 correlation between the two variables.

FIG. 4.

(A) Expression profile of the Dehalococcoides (Dhc) 16S rRNA gene and the RDase genes during the February 2006 and April 2006 sampling events. Data were calculated from triplicate RT-qPCRs, and each error bar represents one standard deviation. Labels on the x axis represent monitoring well designations. (B) Gene concentrations at the corresponding monitoring wells. Data were calculated from triplicate qPCRs, and each error bar represents one standard deviation.

FIG. 5.

Concentrations of the FtL-RDase-1638 gene are compared against Dehalococcoides (Dhc) 16S rRNA gene and RDase gene concentrations. The data represent two sampling events for monitoring well 2B4. FtL-RDase-1638I and FtL-RDase-1638II represent regions from bp 381 to 449 and from bp 1056 to 1129, respectively, on the sequenced clone. Data were calculated from triplicate qPCRs, and each error bar represents one standard deviation.