Comparing On-Site to Off-Site Biomass Collection for Dehalococcoides Biomarker Gene Quantification To Predict in Situ Chlorinated Ethene Detoxification Potential

Abstract

Biostimulation and bioaugmentation have emerged as constructive remedies for chlorinated ethene-contaminated aquifers, and a link between Dehalococcoides (Dhc) bacteria and chlorinated ethene detoxification has been established. To quantify Dhc biomarker genes, groundwater samples are shipped to analytical laboratories where biomass is collected on membrane filters by vacuum filtration for DNA extraction and quantitative real-time PCR analysis. This common practice was compared with a straightforward, on-site filtration approach to Sterivex cartridges. In initial laboratory studies with groundwater amended with known amounts of Dhc target cells, Sterivex cartridges yielded one-third of the total DNA and 9-18% of the Dhc biomarker gene copies compared with vacuum filtration. Upon optimization, DNA yields increased to 94 +/- 38% (+/-SD, n = 10), and quantification of Dhc biomarker genes exceeded the values obtained with the vacuum filtration procedure up to 5-fold. Both methods generated reproducible results when volumes containing >10(4) total Dhc target gene copies were collected. Analysis of on-site and off-site biomass collection procedures corroborated the applicability of the Sterivex cartridge for Dhc biomarker quantification in groundwater. Ethene formation coincided with Dhc cell titers of >2 x 10(6) L(-1) and high (i.e., >10(5)) abundance of the vinyl chloride reductive dehalogenase genes vcrA and/or bvcA; however, high Dhc cell titers alone were insufficient to predict ethene formation. Further, ethene formation occurred at sites with high Dhc cell titers but low or no detectable vcrA or bvcA genes, suggesting that other, not yet identified vinyl chloride reductive dehalogenases contribute to ethene formation. On-site biomass collection with Sterivex cartridges avoids problems associated with shipping groundwater and has broad applicability for biomarker monitoring in aqueous samples.

Isolation of two new Dehalococcoides mccartyi strains with dissimilar dechlorination functions and their characterization by comparative genomics via microarray analysis

“…Given that Dhc strains across multiple subgroups can possess identical chlorinated ethene usage while strains within subgroups can possess divergent usage, it follows that the key biomarkers for predicting the potential to reduce halogenated compounds are the functional RDases, including the functionally characterized pceA, tceA, vcrA and bvcA genes. Previous studies have demonstrated that targeting the presence and expression of these genes can be useful for field diagnostic purposes (Behrens et al, 2008;Lee et al, 2008;Ritalahti et al, 2010;van der Zaan et al, 2010) and the results of this study further support their use in bioremediation projects. Trying to predict organohalide respiration activity from phylogenetic analysis of the 16S rRNA gene or other sequences of the core genome is not feasible because such results can be obscured by horizontal gene transfer of the functional RDases (McMurdie et al, 2009).…”

Discovery of a trans -Dichloroethene-Respiring Dehalogenimonas Species in the 1,1,2,2-Tetrachloroethane-Dechlorinating WBC-2 Consortium

“…The volume of culture harvested depended on the experiment: 50 ml of culture was harvested for the qPCR survey of the enrichment cultures, while 5 ml of culture was harvested for the time course experiments and tDCE or VC enrichment cultures. After the cultures were harvested, the Sterivex filters were frozen for at least 1 h at Ϫ80°C, and then the membrane filter in the Sterivex cartridge was excised and sliced into small (ϳ5-mm 2 ) pieces with a sterile surgical blade (24). The filter pieces were transferred to a bead-beating tube from the UltraClean soil DNA kit (MoBio Laboratories Inc., Carlsbad, CA).…”

Spatial and temporal dynamics of organohalide-respiring bacteria in a heterogeneous PCE–DNAPL source zone

“…Further, although both dechlorinating species were predominately attached to the sandy solid phase independent of the DNAPL saturation within the aquifer cell, the results suggest that the abundance of attached cells in regions containing ganglia could exceed pooldominated regions 4-5 fold, making estimates of the total microbial population (non-attached plus attached) challenging when biomass sample collection relies solely on groundwater. Variability of aqueous phase cells abundances in response to DNAPL saturation could potentially impact the interpretation of qPCR data (Lu et al, 2006;Ritalahti et al, 2010a), in particular when the porous medium has higher organic content .…”