Coupled biogeochemical evaluations: Determining rates for TCE cometabolism

Chlorinated solvent wastes, primarily TCE, often occur as diffuse plumes within complex geologic environments where coupled processes must be understood in order to implement effective remediation strategies. This study evaluated the biogeochemical processes that dictate the rate of TCE cometabolism in a contaminated aquifer using multiple innovative technologies. Specifically, TCE cometabolism via the soluble methane monooxygenase (sMMO) pathway was evaluated in a natural aquifer with TCE concentrations that range from 100 to 500 μg L^-1 groundwater. TCE cometabolic rates were interpreted in the context of enzyme activity, the numbers of cells likely to have cometabolic capability by fluorescent in situ hybridization (FISH), and gene expression. Initial studies were focused on Methylosinus trichospoihiurn OB3b grown under controlled conditions, (methane as sole carbon source, thus active enzymes). In order to obtain rates of degradation over short periods of time and in small volume reactors (2 mL), a Kros-Flo system was used to concentrate the biomass from the batch culture. The rate of degradation was determined by adding concentrated cells in the form of a pellet to ampoules in the presence of un-amended groundwater with ambient concentrations of TCE and oxygen. In general, higher biomass correlated with increased rates of TCE degradation. Inhibition studies with acetylene, an irreversible inhibitor of sMMO, provided clear evidence that metabolism of the contaminant was cometabolic and based on the sMMO enzyme. Separately, microcosm studies with natural, un-concentrated populations from the aerobic portion of the TCE contaminated plume at the Test Area North (TAN) site at the Idaho National Lab demonstrated similar responses with increased rates of TCE degradation (0.12 E^-4 L D^-1, 0.15E^-5 L D^-1) when there is higher cell densities of TCE cometabolizing microorganisms (2.3E⁺⁵, 4.2E⁺⁴ respectively). A followon study is currently being carried out at a TAN well using natural microbial populations. Microbial cells from approximately 600 gallons of groundwater were concentrated and used as the inocula for a series of TCE degradation assays. Preliminary results show TCE degradation concomitant with cometabolic activity, determined by enzyme probes and FISH analysis. In general, increased biomass was related to increased rates of TCE degradation with both cultured cells and cells indigenous to the TCE plume at TAN. The value of using an integrated suite of tests including direct contaminant degradation measurement in microcosms, enzyme activity probes, and molecular approaches was well established. This coupled approach provides direct evidence in support of natural attenuation for a site or critical information for tracking active bioremediation and will lead to estimates of the rate at which indigenous microbial communities cometabolize TCE.