Comparing on-site to off-site biomass collection for Dehalococcoides biomarker gene quantification to predict in situ...

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.