To accurately evaluate the performance of groundwater bioremediation processes, methods that can quantify the populations and the in-situ activity of relevant groups of microorganisms are needed. Molecular biological techniques that rely on DNA extraction and polymerase chain reaction (PCR) amplification are widely used to determine microbial community structure and the concentrations of specific organisms or genes in environmental samples. The objective of this project was to determine an optimal sample handling and processing strategy that yields accurate results for real-time PCR and Terminal Restriction Fragment Length Polymorphism (T-RFLP) in soil and aquifer sediments.

This project assessed the effect of DNA extraction methods and sample mass on the observed microbial community, the detection of the 16S rRNA genes for Bacteria and Dehalococcoides spp., and the tceA gene that codes for trichloroethene (TCE) reductase found in some Dehalococcoides strains. In addition, the effects of storage temperature, time, and condition on quantitative Dehalococcoides 16S rRNA and tceA genes and two vinyl chloride (VC) reductase functional genes (vcrA and bvc) in both groundwater and soil samples were investigated. Homogenized aquifer sediment was inoculated with a mixed Dehalococcoides consortium (KB-1) and an E. coli clone containing the tceA gene. DNA was extracted from three sample masses using three commercial DNA extraction methods. A new laboratory method for DNA extraction was also developed. In addition, two indirect methods that use cell separation steps prior to DNA extraction were assessed. Quantitative-PCR (Q-PCR) was used to measure concentrations of the 16S rRNA gene of Dehalococcoides spp and the tceA gene. T-RFLP was used to characterize the bacterial and archaeal community profiles.

Sample mass did not affect Q-PCR results for DNA extracted using the lab method but gave inconsistent results when the kits were used. T-RFLP profiles were less sensitive to extraction method than Q-PCR results, but the lab method resulted in more consistent reproducibility and community similarity matrices. The storage and temperature experiment showed that the storage condition had a major impact on numbers of total bacteria and the bacterial community structure. Storage of samples at 25ºC for two days and storage at 4°C for 14 days resulted in significant differences in detected concentrations of Dehalococcoides and Bacteria 16S rDNA and tceA, bvc, and vcrA genes. These storage conditions also resulted in significant shifts in the bacterial community structure as revealed by T-RFLP. None of the commercially available kits were deemed suitable for quantitative analyses. The T-RFLP analysis also showed that some commercial kits resulted in strong biases with respect to microbial community structure. The lab method was successful in eliminating the effect of extracellular DNA, with recoveries of less than 0.1% of added extracellular DNA even at high levels of addition (more than 109 copies added per sample). Below is a table of advantages and disadvantages of each DNA extraction method.

Results of initial experiments led to the development of a DNA extraction method. The new method, which uses aluminum sulfate to immobilize PCR-inhibiting compounds prior to cell lysis, gave superior results compared to the three commercial kits with respect to DNA yield, DNA purity, and gene detection. In addition, the lab method resulted in more consistent Q-PCR results and more consistent microbial community analysis. The lab method has the potential of eliminating the effect of inactive (extracellular) DNA. This method may be of value to researchers performing molecular biological techniques at Department of Defense sites. The study also showed that storage conditions will affect microbial community structure analysis. Careful assessment of DNA extraction methods and sample storage conditions are needed in bioremediation studies to allow comparisons of results in the research community.