This Small Business Innovation Research Phase I project proposes to develop and test new methods to label bacteria with 13C, and to detect the labeled bacteria using modified gas chromatography-mass spectrometry. Bioaugmentation requires that bacterial transport be monitored to assure that injected bacteria have contacted all areas of the contaminant plume. This has usually been determined by plate counts. However, this leads to an underestimation of bacterial concentrations due to the inability of plate counts to detect organisms, which are still viable and active, but will no longer grow on bacteriological media. The incorporation of stable isotopes into cells before injection, with subsequent detection of the isotopically labeled cells would allow all injected organisms to be enumerated, regardless of culturability. Methods will be developed to yield high incorporation of 13C into bacterial fatty acids. Analysis 13C-labeled fatty acid extracts using gas chromatography-chemical reaction interface-mass spectrometry (GC-CRIMS) will be optimized with respect to the lower detection limits and determination of isotopic enrichment. Sediment microcosm and intact sediment core experiments will be conducted to evaluate the labeling and detection of 13C-labeled fatty acids. It is anticipated that this methodology will enhance the ability to detect and track injected organisms during bioaugmentation. Bioremediation of contaminated soil and groundwater using bioaugmentation is expected to become increasingly widespread, especially for organic and inorganic pollutants that are not amenable to other physical/chemical or biological remediation schemes, or at sites where other remediation strategies are not effective. The development and application of the proposed methods will allow the bacterial transport component of bioaugmentation to be effectively monitored and validated. Since more accurate measurements of viable bacterial cell densities will be possible, expenditures for additional injections will only occur when they are absolutely necessary, thereby reducing the overall cost of remediation. Additionally, these methods have potential use in other basic and applied research in the areas of microbial ecology, microbial pathogenicity and microbial physiology, as well as in non-microbiological fields of study, since stable isotopes can be used as tracers in much the same way as radioactive compounds, but without the risk to human and environmental health.
