Novel Approaches to Studying the Situ Bioremediation of Complex Mixtures

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The vast majority of Superfund sites in the U.S and the Baltimore/Chesapeake Bay area contain mixtures of organic and inorganic compounds that contaminate underlying aquifers. The environmental fate of these contaminants, and ultimately human exposure to them, is governed primarily by their interactions with microorganisms, which?individually or as a community?drive the process of in situ bioremediation. Whereas single pollutant/microorganism interactions can be determined easily in the lab, no satisfactory tools exist for predicting the fate of mixtures in the environment. Our long-term goal is to improve the success rate of bioremediation at sites containing complex chemical mixtures by using in situ microcosm array (ISMA) technology. The ISMA is a field-deployable, miniaturized laboratory consisting of a large number of small microcosms arranged in parallel. Upon deployment, incubation, and retrieval from a groundwater well, the ISMA can be analyzed to reveal the impact of mixture components on the rates of Dollutant degradation and on the structure and function of microbial communities. We hypothesize that the ISMA can aid in the design of bioremediation strategies because: (1) individual ISMA microcosms can be amended with multiple test substances to elucidate the effect of mixture components on microorganisms; (2) the response of microorganisms to presented compounds manifests itself as changes in biomass, community structure and function; and (3) these changes can be detected conveniently by biochemical, genetic and proteomic strategies. Based on the above observations, the specific aims of the project are to: 1. Determine the reproducibility and discriminatory power of the ISMA technology. We will explore how varying concentrations of inducers and co-contaminants in synthetic groundwater modulate the expression of the dioxin dioxygenase (DDase) of Sphingomonas wittichii Strain RW1, by using a large number of replicates in conjunction with semi-automated, high-throughput proteomic mass spectrometry. 2. Demonstrate in controlled laboratory conditions how the ISMA can reveal additive, synergistic, and antagonistic effects of mixture components on microbial communities. We will use defined mixtures of chemicals (e.g., dioxins, PCBs, PAHs, tolualdeyhyde, Cd, Cr, Co, Pb, Hg, Zn, Ni) and bacteria (five bioremediation agents) to study these effects in various permutations. 3. Evaluate the utility of the ISMA technology in simulated field conditions. We will conduct ISMA laboratory experiments using nonsterile groundwater, sediment, and natural microbial communities. 4. Deploy the ISMA device at a Maryland Superfund site. We will assess the utility of the new technology in a field demonstration study by examining survival of and DDase expression by Strain RW1 in situ, and by elucidating the impact of this introduced bacterium on the indigenous microbial community at the site.