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. Knowledge of the microbial physiology intrinsic to MFCs is hindered by the limited number of current-producing isolates in pure culture and the confinement of most mechanistic studies to the model Fe(III)-reducing organisms Geobacter and Shewanella , members of the Delta- and Gamma- proteobacteria respectively. This discrepancy between known physiology and organisms evaluated in the MFC suggests that the diversity of electricity-producing organisms remains unknown. For MFCs to achieve their potential, however, 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. The thermophilic (55 o C) fermentation of solid waste has higher reaction rates, lower biomass yields, a broader range of carbohydrate utilization, and tolerance to higher loading rates when compared to mesophilic counterparts. While research has begun to examine the efficiency and microbiology of mesophilic MFCs, the equivalent electrochemical, ecological, and physiological studies have not been performed in MFCs operated under thermophilic conditions. Microbial fuel cells operated under elevated temperatures show promise due to potentially higher energy yields resulting from higher rates of metabolic activity, easier maintenance of anaerobic reducing conditions because of the lower solubility of O2 at elevated temperatures, and thermal removal of most known pathogens. Additionally, MFCs operated at 55 o C exclude the growth of Geobacter and Shewanella species and thus may enrich for novel electricity-producing communities and organisms. MFCs inoculated with thermophilic anaerobic digester were constructed and operated at 55 o 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 date, the characterization of bacterial communities in electricity production is hampered by the lack of controls, the use of low-resolution DNA fingerprinting techniques, and the failure to discriminate between active and colonizing members of the anode biofilm. 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. Research presented in this dissertation revealed that current-producing anode communities were statistically different from control reactors and the initial inoculum. PhyloChip results indicated that Firmicutes were dominant in the persistent and active bacterial community on thermophilic anodes. To identify specific Firmicutes OTUs involved in current generation, 16S rRNA clone libraries from the initial inoculum and current producing reactors were constructed. Two Gram-positive phyla, Firmicutes (11 OTUs, 229 clones, 77% of clones) and Coprothermobacteria (2 OTUs, 48 clones, 16% of clones), represented 93% of the clone sequences. Within the Firmicutes, sequences belonging to the genera Thermicanus , Alicyclobacillus , Thermincola , and Geobacillus represented 27%, 25%, 22%, and 2% of the total clones respectively. These genera could not be detected in clone libraries from the initial inoculum (482 total clones), suggesting their enrichment corresponds to current production in this system. Members of these genera have not previously been identified in MFCs operated under mesophilic conditions. 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%). In contrast to Thermincola potens strain JR, Geobacillus sp. strain S2E could not produce current in the absence of an exogenous electron shuttle (AQDS) and only produced small amounts of current in its presence (0.03 mA). While it has been shown that Gram-negative bacteria use either endogenously produced electron shuttles (contact-independent) or direct electron transfer (contact-dependent) for exporting electrons to the anode of MFCs, there is limited data about the mechanisms employed by Gram-positive bacteria for transferring electrons to insoluble electron acceptors. 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. Medium replacement experiments and cyclic voltammetry (CV) failed to detect redox active components secreted into the surrounding medium when strain JR was grown on these electron acceptors. Confocal microscopy revealed highly stratified biofilms in which cells contacting the electrode surface were primarily responsible for current generation. 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 55oC to reveal a novel physiological role for Gram-positive Firmicutes within current-producing anode communities. We confirmed the functional role for Firmicutes in this systems by demonstrating current-production from two novel bacteria from the anode surface, Thermincola potens strain JR and Geobacillus sp. strain S2E. Physiological investigation of Gram-positive extracellular respiration was carried out in strain JR demonstrating that this organism is capable of independent and direct electron transfer to insoluble electron acceptors like Fe(III) and anode surfaces. Further, genomic and physiological studies of strain JR provide evidence for multiheme c-type cytochromes in facilitating electron transfer across the Gram-positive cell envelope. 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.