A Functional Genomic Approach to Chlorinated Ethenes Bioremediation

With the recent advances in genomic sciences, a knowledge-based approach can now be taken to optimize the bioremediation of trichloroethene (TCE). During the bioremediation of a heterogeneous subsurface, it is vital to identify and quantify the functionally important microorganisms present, characterize the microbial community and measure their physiological activity. In our field experiments, quantitative PCR (qPCR) was coupled with reverse-transcription (RT) to analyze both copy numbers and transcripts expressed by the 16S rRNA gene and three reductive dehalogenase (RDase) genes as biomarkers of Dehalococcoides spp. in the groundwater of a TCE-DNAPL site at Ft. Lewis (WA) that was serially subjected to biostimulation and bioaugmentation. Genes in the Dehalococcoides genus were targeted as they are the only known organisms that can completely dechlorinate TCE to the innocuous product ethene. Biomarker quantification revealed an overall increase of more than three orders of magnitude in the total Dehalococcoides population and quantification of the more liable and stringently regulated mRNAs confirmed that Dehalococcoides spp. were active. Parallel with our field experiments, laboratory studies were conducted to explore the physiology of Dehalococcoides isolates in order to develop relevant biomarkers that are indicative of the metabolic state of cells. Recently, we verified the function of the nitrogenase operon in Dehalococcoides sp. strain 195 and nitrogenase-encoding genes are ideal biomarker targets to assess cellular nitrogen requirement. To characterize the microbial community, we applied a high-density phylogenetic microarray (16S PhyloChip) that simultaneous monitors over 8,700 unique taxa to track the bacterial and archaeal populations through different phases of treatment. As a measure of species richness, 1,300 to 1,520 taxa were detected in groundwater samples extracted during different stages of treatment as well as in the bioaugmentation culture. We found that the community structure was sensitive to manipulation such as the injection of whey. In addition to Dehalococcoides spp., the PhyloChip also detected dechlorinating bacteria from other phyla such as Sulfurospirillum multivorans and Dehalobacter restrictus. Although these organisms only dechlorinate TCE to dichloroethene, their populations increase at the site over time suggested they also played an important role. Over 600 subfamilies were also found to be active in the microbial community with many of those being important players in geochemical processes. Overall, through the use of high throughput molecular techniques, a comprehensive view of the functionally important organisms and the microbial community was obtained, providing knowledge that can be used to guide the manipulation of the bioremediation processes to achieve the most efficient treatment.