Collaborator: Indiana Energy, Department of Energy, and Exxon-Mobil

Recent scientific advances have made it possible to use molecular biological techniques for assessment of microbial communities in complex environmental systems. Molecular techniques, such as PCR amplification, cloning, and sequencing of ribosomal RNA genes, have recently been embraced by the environmental science community as important tools for predicting soil and water remediation success. The focus of this project is to evaluate the potential use of a suite of microbiological techniques for assessment of bioremediation. Numerous methods are available for evaluation of microbial biomass in soil. Traditional enrichment culture-based techniques, such as heterotrophic plate counts, are frequently used; however, biases may be introduced by media type and richness, presence or absence of oxygen, and numerous other factors. Such techniques are thought to reveal as little as ten percent of the total microbial diversity in soil. For this reason, innovative methods have been developed to more completely describe soil microbial diversity. Molecular methods, such as denaturing gradient gel electrophoresis (DGGE), rely on genetic differences to draw distinctions between microbes and microbial populations. Chemical extraction of phospholipid-fatty acids from soil can provide both a description of the diversity in that soil and an estimate of the microbial biomass present. Finally, most probable number (MPN), a specialized enrichment technique utilizing substrates of interest, gives an estimate of the number of organisms in an environment capable of degrading specific contaminants. Taken individually, DGGE, phospholipid-fatty acid analysis, and MPN are all useful tools for understanding microbial communities. In combination, however, they are likely to yield extensive information on microbial biomass and community diversity. Furthermore, they provide the capability to pinpoint dominant groups of organisms and to assess the microbial community's ability to degrade contaminants. Integration of these diverse methods represents a potentially powerful tool for characterization - and, ultimately, optimization - of bioremediation systems. To determine specifically how diversity is related to bioremediation efficiency, microbial contaminant degraders and microbial community structure in bioremediated soil will be evaluated. Contaminated soil from at least seven bioremediation/phytoremediation field trials will be collected and analyzed. Data generated from the community analyses will be compared with the MPN of contaminant degraders and percent removal of the contaminant to date. If strong correlations are identified, the protocol will be developed into a comprehensive screening methods manual to be used extensively for bioremediation projects.