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Title:Long-range extracellular electron transport by dissimilatory metal-reducing bacteria across a physical separation
Author(s):Michelson, Kyle
Director of Research:Werth, Charles
Doctoral Committee Chair(s):Valocchi, Albert
Doctoral Committee Member(s):Sanford, Robert; Guest, Jeremy
Department / Program:Civil & Environmental Eng
Discipline:Environ Engr in Civil Engr
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Geobacter, Shewanella, nanowires, electron shuttling
Abstract:Nanopores in anaerobic sediments are stores of secondary minerals like Fe and Mn oxides that are often the most abundant electron acceptors for microbial respiration. The reduction of these minerals is catalyzed almost exclusively by dissimilatory metal-reducing bacteria (DMRB) that are defined by their ability to gain energy by coupling the oxidation of organic compounds and hydrogen to the reduction of metals. While DMRB are known to use a variety of electron transport mechanisms to reduce Fe and Mn minerals in contact with their outer membrane, little is known about their capacity to reduce sequestered minerals deep within pore spaces that are too small for cell passage. To study long-range extracellular electron transfer (LR-EET) to sequestered minerals, microfluidic reactors with the novel ability to separate DMRB from the insoluble Mn(IV) mineral birnessite were etched into silicon using photolithography or electron beam lithography. The central feature of the microfluidic reactors was a 1.5 μm thick wall containing an array of <200 nm deep pores that allowed the diffusion of solutes but not the passage of cells. The nanoporous wall bifurcated two parallel flow channels; one containing cells, and the other containing a mineral. Bacteria were completely prevented from crossing the wall as demonstrated using brightfield microscopy, fluorescent staining, and scanning electron microscopy (SEM). At the completion of an experiment, a novel method was used to debond the anodically bonded reactors for high-resolution imaging using SEM. Microfluidic reactors were reused by cleaning between experiments using a newly developed protocol. The first mechanism studied was reduction of birnessite by microbial nanowires using Geobacter sulfurreducens KN400. The nanoporous wall in these experiments was composed of an array of pillars separated by <200 gaps (i.e. nanopores) to provide nanowires access to the entire depth of deposited birnessite. Using optical microscopy and Raman spectroscopy, it was demonstrated that birnessite can be reduced up to 15 μm away from cell bodies, similar to the reported length of Geobacter nanowires. Inhibition of nanowire production showed that nanowires were essential for reducing birnessite across the nanoporous wall by LR-EET, but not for reducing birnessite by direct contact. In the latter case, birnessite reduction was likely the result of electron transfer from outer membrane c-Cyts. Reduction across the wall required reducing conditions, provided by Escherichia coli, and an exogenous supply of riboflavin. Riboflavin was found to act not as a diffusible electron shuttle, but as a bound redox cofactor. The high binding affinity of riboflavin reported for outer membrane c-type cytochromes (c-Cyts) suggests that riboflavin was bound to OmcS, a c-Cyt that decorates the nanowire surface. Upon addition of a soluble electron shuttle (i.e., AQDS), it was also demonstrated that reduction extends up to 40 μm into a layer of birnessite, well beyond the reported nanowire length of 15 μm. The second mechanism studied was the reduction of birnessite by electron shuttling using Shewanella oneidensis MR-1. The nanoporous wall in these experiments was composed of a series of highly uniform <200 nm slits along the top of a continuous wall in response to the increased ability of these bacteria to penetrate narrow pore spaces. It was demonstrated that birnessite reduction was driven by the endogenous production of riboflavin (RF) and flavin mononucleotide (FMN), which mediate redox reactions as diffusion-based shuttles or c-Cyt bound cofactors. Experiments with mutants that lacked flavin exporters showed that birnessite reduction is controlled by the concentration of flavin in the system. Addition of exogenous flavin to cells lacking flavin exporters restored birnessite reduction to wild-type rates in the microfluidic reactors. Experiments with mutants lacking conductive nanowires showed that nanowires were not responsible for birnessite reduction by LR-EET, and that the metal-reducing (Mtr) pathway, currently believed to be critical for efficient reduction of insoluble metal oxides, is not required for high rates of reduction by LR-EET. These results suggest the existence of alternative electron pathways for metal reduction, which may be investigated in future work. It was also demonstrated that S. oneidensis can decouple growth from metabolism, potentially expanding the conditions under which metal reduction can be possible in the natural environment. The results presented in this dissertation may lead to more accurate estimations of mineral redox cycling in anaerobic sediments, improved models of contaminant transport, and broadened understanding of carbon exchange between atmospheric and terrestrial ecosystems. Shewanella and Geobacter spp. are widely used in other applications such as energy production and wastewater treatment, and the results in my dissertation may aid in the design of more efficient bioelectrical systems.
Issue Date:2018-04-18
Rights Information:Copyright 2018 Kyle Michelson
Date Available in IDEALS:2018-09-04
Date Deposited:2018-05

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