DESCRIPTION (provided by applicant): Cr(VI) contamination of soil and groundwater is a significant problem worldwide. In the United States, chromate is the third most common contaminant of hazardous waste sites and the second most common inorganic contaminant found in the environment. In situ and ex situ bioremediation processes that exploit the intrinsic metabolic capabilities of dissimilatory metal ion-reducing bacteria (DMRB) remain potent, potentially cost-effective approaches to the reductive immobilization or detoxification of environmental contaminants. The microbial catalysis of Cr(VI) reduction to sparingly soluble, less bioavailable Cr(III), for example, is a promising remediation strategy for Cr(VI)-contaminated subsurface soil and groundwater environments. The genus Shewanella represents one of the few groups of microorganisms that have received intensive investigation because of their wide ecological distribution, diverse respiratory capacities, and environmental relevance. Despite several advances made in elucidating Shewanella biology as it relates to chromate transformation, fundamental questions about the specific chromate reduction mechanism remain unclear. This information gap includes (i) the identity of dedicated chromate reductase(s), (ii) the cellular localization of chromate transformation (e.g., distal appendages, outer cell surface, periplasm, cytoplasmic membrane, cytosol), and (iii) the environmental parameters under which microbial populations have the greatest specific chromate reduction rates. The problem in predicting and assessing bioremediation performance is compounded by the lack of fundamental knowledge of the molecular basis, regulatory mechanisms, and biochemistry enabling bacterial metal-reducing capabilities. We propose to engineer nanoscale methodologies, comprising of chromate-tagged nanoparticles and intracellularly grown gold nanoislands to function as enhancers for Surface Enhanced resonance Raman scattering probing to generate chemical maps of chromate reduction sites as well as to monitor the reduction dynamics in exquisite molecular and single-organism detail. Objectives of this study are to 1) assess the impact of gold nanoparticle composition, geometry, and functionality on cell viability, growth, and efficacy of microbial chromate reduction using S. oneidensis as a model system; 2) track the localization of chromate transformation at single-cell resolution using functionalized gold nanostructures as well as using intracellularly grown gold nanoislands by Raman chemical imaging; and 3) evaluate the influence of bioremediation-relevant environmental factors on chromate transport, localization, and reduction rates. The development of passive and active nanoprobes in conjunction with confocal Raman chemical imaging will constitute a significant step in enabling a platform for dynamic monitoring of intracellular events and compartmentalization of metal reduction sites at single-cell resolution. The knowledge gained from this novel study will contribute to the development of scientifically grounded strategies for improving bioremediation efficacy.

Project Narrative The objective of this study is to develop a highly sensitive single-cell chemical imaging platform using surface enhanced confocal Raman microscopy to study the compartmentalization of chromate reduction sites in the metal-reducing bacterium Shewanella oneidensis. We will use multifunctional nanoparticles as well as intracellularly grown gold nanoislands to reveal metal reduction sites in single cells. Success in this proposed study will provide us with a novel single-cell Raman imaging platform and lifetime imaging techniques for dynamic monitoring of bioremediation pockets in microorganisms to address a variety of mechanistic questions related to biological reduction of heavy metals, bioremediation efficacy, and the fate of metal contaminants in the environment. 1