Following electron flow: From a Gram-positive community to mechanisms of electron transfer

Research endeavors are committed to the optimization and function of microbial fuel cells (MFCs), with most efforts largely dedicated towards increasing power density by optimizing physical parameters. For MFCs to achieve their potential knowledge of the microbiological factors controlling current production is essential. The research within this dissertation characterizes the electrochemistry (Chapter 2), ecology (Chapter 3), and physiology (Chapter 4) of bacterial current production.;MFCs inoculated with thermophilic anaerobic digester were constructed and operated at 55°C for 100 days. Electrochemical performance was well replicated within the reactors (Chapter 2). Over the experimental period, current was continuous, averaging 0.57 mA (100 mA.m-2) with a mean electron recovery of 89% and maximum power output of 37 mW.m -2. Relative to similarly constructed mesophilic MFCs thermophilic MFCs may offer increased electrochemical performance with elevated current production, coulombic efficiency, and power generation. Not only does this detailed electrochemical description demonstrate that MFC technology is compatible with elevated temperature waste streams, but also functions a benchmark for future comparative studies.;To assess bacterial composition and function of anode biofilm communities we used two complementary approaches: a novel high-density oligonucleotide microarray (PhyloChip) and clone library sequencing (Chapter 3). Within the anode bacterial community, active members were distinguished from persistent members by monitoring 16S rRNA in addition to cataloging 16S rRNA gene presence. Nucleic acids from a no-acetate control (no electron donor), open circuit control (no electron acceptor), and the initial inoculum were extracted to verify community membership on current producing anodes.;To link the phylogeny with functional current production, we complemented 16S rRNA approaches with isolation of pure cultures. Several bacteria representing three genera, Thermincola, Geobacillus, and Coprothermobacter were isolated from the MFC anode (Chapter 3). These genera contain three of the five most dominant members of the anode community and collectively represent 39% of the clone library sequence diversity. Both Firmicutes isolates, Thermincola potens strain JR and Geobacillus sp. strain S2E, are of great interest given their enrichment from the initial inoculum and their ability to reduce solid phase iron, or hydrous ferric oxide (HFO). Interestingly, while both isolates reduced HFO coupled to acetate oxidation, only Thermincola potens strain JR could generate current independently with acetate as an electron donor. Strain JR generated an average of 0.42 mA in two separate experiments with a coulombic efficiency of 91%, similar to that observed for the original complex community (89%).;A combination of physiological, electrochemical and imaging methods support the hypothesis that Thermincola sp. strain JR does not produce an electron shuttling compound but requires direct contact for current production. These results, along with cryo-electron microscopy (cryo-EM), suggest that Thermincola potens strain JR directly transfers electrons from the cell membrane across the 37nm cell envelope to the cell surface. Analogous to direct electron transfer by Gram-negative organisms, physiological and genomic evidence suggests that direct extracellular electron transfer in Gram-positive bacteria is mediated by periplasmic and cell wall associated c-type cytochromes. Together, these results are the first to implicate a role for c-type cytochromes in direct extracellular electron transfer by Gram-positive bacteria (Chapter 4).;This dissertation follows electron flow in MFCs operated at 55°C to reveal a novel physiological role for Gram-positive Firmicutes within current-producing anode communities. As a result of this dissertation, an option now exists for efficient MFC current-production at elevated temperature, two novel anode-respiring bacteria have been isolated, independent electricity generation by Gram-positive bacteria has been demonstrated, the genome of one of these isolates has been sequenced, and the molecular mechanism of electron transfer by a Gram-positive anode respiring bacterium elucidated. (Abstract shortened by UMI.)...