The contamination of soil with 2,4,6-trinitrotoluene (TNT) is a worldwide concern, due mainly from activities in the munitions and defense industries. Current methods to treat contaminated soil are costly and environmentally disruptive. Recently, plants and their associated microorganisms have been shown to reduce TNT in the soil. This process can be enhanced through the introduction of bacteria with known TNT reducing enzymes to the site.
The ability to effectively monitor a microbial community is necessary to design and implement remediation strategies for contaminated soil. Single-strand conformation polymorphism (SSCP), a technique which separates DNA fragments based on their sequence, was used to analyze PCR amplified 16S ribosomal RNA gene fragments of common soil bacteria. Separation was performed using capillary electrophoresis (CE), as opposed to other common gel techniques, to eliminate the need for band analysis on gel matrices.
CE-SSCP was utilized for analysis of bacteria in pure culture, soil, and to monitor an introduced species. By combining multiple universal primer sets, twelve common soil bacteria in pure culture could be separated. Studies to determine microbial community fingerprints and track species of interest in soil were also performed, and the DNA extracted from the soil needed to be diluted before amplification to reduce the inhibiting effects of humic acids that co-extract with the soil DNA. Results of the bacteria spiking studies revealed that high concentrations of the introduced species were necessary for identification. When monitoring inoculated bacteria for phytoremediation studies, a shift in the native profile was observed in contaminated soil. Furthermore, it is possible that the introduced species with TNT-remediation capabilities were observed in the explosive containing samples, although additional studies need to be performed to confirm these results.
The final step of this dissertation was to design a primer set specific for the TNT-reducing bacteria used for the phytoremediation study. Preliminary results with agarose gel electrophoresis showed that the designed primer set was selective for the genus, but not the species of interest. Additional experiments using soil collected from contaminated sites need to be performed to determine the effectiveness of the plant/bacteria combination for phytoremediation strategies.