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Title
Nanoscale studies of metal reduction by microbial biocatalysts using in vitro biomimetic platforms
Creator
Awate, Bhushan Prabhakar
Date
2014
Collection
Electronic Theses & Dissertations
Description
NANOSCALE STUDIES OF METAL REDUCTION BY MICROBIAL BIOCATALYSTS USING IN VITRO BIOMIMETIC PLATFORMSMetal-reducing bacteria like Geobacter sulfurreducens use cytochrome proteins to reductively precipitate water-soluble uranium salts. However, the mechanism by which the cytochromes achieve multistep electron transfer to extracellular metals is not yet understood. Previous studies of cytochromes' role in electron transfer have involved a genetic approach, in which...
Show moreNANOSCALE STUDIES OF METAL REDUCTION BY MICROBIAL BIOCATALYSTS USING IN VITRO BIOMIMETIC PLATFORMSMetal-reducing bacteria like Geobacter sulfurreducens use cytochrome proteins to reductively precipitate water-soluble uranium salts. However, the mechanism by which the cytochromes achieve multistep electron transfer to extracellular metals is not yet understood. Previous studies of cytochromes' role in electron transfer have involved a genetic approach, in which specific cytochromes are either deleted or overexpressed. However, results of these genetic studies are difficult to interpret, because mutation of one gene can cause multiple phenotypic changes, resulting in complex alterations of the cell's electron-transfer machinery. These limitations can be bypassed using a biomimetic approach, in which Geobacter cytochromes are assembled into nanostructured interfaces that mimic the cell envelope and electron-carrier machinery.In this study, we heterologously expressed some of Geobacter's most abundant and conserved cytochromes in Escherichia coli. We then used these cytochromes to fabricate nanostructured biomimetic interfaces that mimicked Geobacter's double-membrane cellenvelope. A self-assembled monolayer of alkanethiols on a gold electrode mimicked the inner membrane; an aqueous layer containing PpcA (a periplasmic cytochrome) mimicked the periplasmic space; and a synthetic bilayer lipid membrane containing OmcB (an outer membrane cytochrome) mimicked the outer membrane. Cytochrome-mediated electron transfer from the gold electrode to soluble metal salts was characterized using cyclic voltammetry. The PpcA was found to transfer electrons to U(VI) more rapidly than to other soluble electron acceptors, consistent with the observation that U(VI) is reductively precipitated in Geobacter's periplasm. Spectroelectrochemical characterization of PpcA and OmcB demonstrated for the first time electron transfer between these two proteins, suggesting that they may be redox partners in Geobacter's electron transport chain.Fabrication of electrochemically active nanostructured bioelectronic interfaces that mimic Geobacter's double-layered cell envelope establishes a new experimental platform with which to characterize Geobacter's electron transfer machinery. Addition of more Geobacter components will make the interface more realistic and enable hypotheses about electron-transfer mechanisms to be systematically tested. An improved understanding of Geobacter's ability to reduce metals may lead to new technologies for in situ reductive immobilization of uranium and other toxic metals.
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