Monitoring abundance and expression of "Dehalococcoides" species chloroethene-reductive dehalogenases in a tetrachloroethene-dechlorinating flow column

Abstract

We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified "Dehalococcoides" species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 +/- 0.6) x 10(6) (mean +/- standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

FIG. 1.

RDase and 16S rRNA gene copy numbers of pure and enrichment cultures of Dehalococcoides spp. determined by real-time PCR. The graph shows the results for five Dehalococcoides sp. pure cultures (strains CBDB1, FL2, BAV1, VS, and 195) and one enrichment culture (Evanite). Each bar represents the average of the results of triplicate real-time PCRs performed on two independent DNA extractions (n = 6). Dhc, Dehalococcoides.

FIG. 2.

Chloroethene RDase gene abundance in the flow column. Gene copy numbers were determined by real-time PCR using template DNA extracted from six different column sections. Each bar represents the average of the results of triplicate real-time PCRs performed on two independent DNA extractions. Dhc, Dehalococcoides.

FIG. 3.

Transcript copy numbers of chloroethene RDase genes in the flow column. Transcript abundance was determined by real-time PCR using template RNA extracted from six different column sections. Each bar represents the average of the results of triplicate real-time PCRs performed on two independent RNA extractions.

FIG. 4.

Rarefaction curves of two 16S rRNA gene libraries from the PCE-dechlorinating continuous-flow column. The clone libraries were constructed from DNA extracted from column aquifer solids sampled 0 to 5 cm from the column inflow and 25 to 30 cm from the column inflow. Dashed lines represent the >95% confidence interval. Sequences were assigned to OTUs if they were less than 3% different from each other.