Biokinetic Modeling, Laboratory Examination And Field Analysis Of Dna, Rna And Protein As Robust Molecular Biomarkers Of Chloroethene Reductive Dechlorination In Dehalococcoides Mccartyi
DNA, RNA and protein were tested as specific and robust biomarkers of anaerobic reductive dechlorination by Dehalococcoides (DMC) species in predictive modeling, with mixed microbial cultures and at a field site. A comprehensive biokinetic model of a community containing DMC strain 195 was updated to describe continuously fed reactors with specific biomass levels based on quantitative PCR (qPCR)-based population data (DNA and RNA). The model was calibrated and validated with subsets of chemical and molecular biological data from various continuous feed experiments (n=24) with different loading rates of the electron acceptor (1.5 to 482 [MICRO SIGN]eeq/L-h), types of electron acceptor (PCE, TCE, cis-DCE) and electron donor to electron acceptor ratios. Based on early model results, both competitive inhibition of chloroethene degradation and empirically derived mRNA "adjustment factors" were added to the model. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) data were taken from microcosms containing the KB-1TM consortium (SiREM Labs of Guelph, Ontario, Canada), operated under continuous, chlorinated ethene feed conditions, with the aim of clarifying relationships and creating more robust set of biomarkers that could be used at field sites bioaugmented with the KB-1TM culture. The correlation between respiration rate and mRNA transcript number was upheld for the hydrogenase HupL, and significant differences were observed for reductive dehalogenases (RDases) TceA and DET1545 when comparing the two mixed cultures studied (KB-1TM and D2). A correlation was also observed for RDase VcrA expression compared to respiration rate in the KB-1TM mixed culture. Additionally, correlation trends for HupL and VcrA were upheld when looking at proteomic ion intensities as compared to respiration rates, though protein changes were not as drastic as they were for mRNA transcripts. Additional experiments were conducted to quantify these biomarkers under stress conditions (presence of oxygen or low pH). Addition of stressors caused respiration rates to decrease significantly, whereas transcript abundances exhibited a slow decay (0.02-0.03 per hr) over the time period studied, indicating that transcript abundance alone cannot predict respiration rate in stressed conditions within hours to days following stress. A successful proteomics-based method was developed for identifying DMC and Geobacter biomarkers of reductive dechlorination at a trichloroethene-contaminated industrial site in Ft. Erie, Ontario that had been bio-augmented with the commercially-available KB-1TM microbial culture two years prior. Samples were obtained from two wells with high hydraulic connectivity to the enhanced in-situ bioremediation system, and two with low hydraulic connectivity. The DNA and RNA biomarkers detected were a set of reductive dehalogenases, and the highly-conserved hydrogenase, HupL. Proteomic biomarkers of organohalide respiration were detected in all four field samples' metaproteomes, and the key reductive dehalogenases present in the bioaugmentation culture were the most highly detected proteins overall, suggesting that deployed DMC strains maintain devotion to high RDase concentrations in the field.
Richardson, Ruth E.
Gossett, James Michael; Buckley, Daniel H
Civil and Environmental Engineering
Ph. D., Civil and Environmental Engineering
Doctor of Philosophy
dissertation or thesis