Electrochemically active bacteria in microbial fuel cells

Microbial Fuel Cell (MFC) technology has been heralded as a tool for energy conservation, resource recovery and valuable compound synthesis, amongst others. The MFC concept is possible due to the ability of electrochemically active bacteria (EcAB) to transfer the electrons produced from substrate degradation, out of the bacterial cell and onto the electrode surface via different mechanisms; a process called exocellular electron transfer (EET). However, despite advances and extensive studies on EET mechanisms and EcAB, like the Fe(III)-reducing <em>Geobacter sulfurreducens</em> PCA, the technology has not reached yet the stage of broad applicability. This thesis investigates characteristics and performance of EcAB in the anodic compartment of pure- and mixed-culture MFCs in an effort to shed light to processes important to MFC performance and efficiency. In order to reliably study EcAB in a microbial fuel cell environment, a gas-tight, sterile MFC setup was developed and optimized for electrochemical and microbiological studies of the anodic bacteria and consequently the electrochemically active biofilm/bioanode. In addition, a method for <em>Geobacter</em> species quantification with quantitative PCR (qPCR) was developed. Furthermore, a multiple-unit MFC setup was designed for convenient and simultaneous operation of identical MFCs ‘in-parallel’. A design for a new compact, multi-array MFC to be used as a small-scale culturing platform of EcAB based solely on their electrochemical properties is also introduced. Our research with different titanium (Ti) electrodes in the same MFC setup, suggests that the MFC electrode surface is critical as it determines attachment of EcAB and bioanode formation that leads ultimately to efficient electrochemical activity. Pt- and Ta- coated Ti electrodes performed the best while uncoated Ti with either smooth or rough surfaces performed the worst. Future MFC research could benefit greatly from enhancing the electrode interface for optimum bioanode formation and electron transfer. Our studies with flat-plate, graphite-electrode, mixed-culture microbial fuel cells (Pmax ≈ 1 W/m2) operated for several months with external load (Rext), indicated stable and reproducible characteristics including Coulombic efficiencies, average values of cell voltage, anode potentials, and current densities as well as main microbial populations of both the anolyte and the bioanode. However, transient testing of power maxima values (Pmax) that required lower Rext, or applied potential showed result variability, that might be linked to differences in electrochemical impedance factors, redox-active centers and electron-producing states of the bioanodes. Differences in the quantities of the bioanode microbial species did not seem to correlate with this variability. Since energy-conserving applications like waste-water treatment MFCs would ideally be operated with an Rext rather than applied voltage, addressing interface impedance factors, such as charge transfer resistance and electrical double layer capacitance, is important especially when MFCs are operated at lower Rext. Furthermore, mixed-culture MFCs were shown to be selective for certain bacterial consortia, including <em>Geobacter- </em>and <em>Pseudomonas</em>- related species. <em>Geobacter</em>-related species were dominant on the surface of different electrodes suggesting a pivotal role of the species in electrochemical activity and EET. This was not surprising as the original mixed-culture inoculum - used for starting up several bioelectrochemical systems at our research facilities - was amended with pure cultures of <em>G. sulfurreducens</em> PCA. However, the strain specifically selected for and present in most bioanodes was a novel <em>Geobacter,</em> strain T33 that was phylogenetically closely related (99% by 16S rRNA sequence similarity) to several strains detected in a variety of MFCs operated by other research groups under various conditions and anodic substrates. These strains formed a new phylogenetic <em>Geobacter</em> cluster, distinct from <em>G. sulfurreducens. </em>This observation suggested that strain T33 might have an ecological advantage in MFCs over <em>G. sulfurreducens</em> PCA<em>. </em>In-depth characterization of strain T33 in pure-culture experiments showed that strain T33 forms efficient bioanodes with high Pmax similar to strain PCA, but exhibits different redox-centers than strain T33. Furthermore, strain T33 has a more limited electron acceptor range, but a wider electron donor range than strain PCA, including glucose and succinate. Phylogenetic analysis indicated that strain T33 and recently described electrochemically active strains <em>G. soli</em> GSS01 and <em>G. anodireducens</em> SD-1 are closely related (99% by 16S rRNA) and form a new phylogenetic cluster within the <em>Geobacters</em>, 98% by 16S rRNA similar to <em>G. sulfurreducens</em> strains PCA and KN400. Genome-based analyses indicates that even though the two clusters share common metabolic properties, some differences exist with respect to electron donor utilization, attachment and conductive cell surface components (e-pili) production and genome rearrangement and gene acquisition. It is not sufficiently clear how the differences in the genome of strain T33 are relevant to persistence of the strain in MFCs, biofilm formation and EET, our studies overall suggest that strain T33 even though producing similar power densities as <em>G. sulfurreducens</em> PCA, might be more stable and versatile in MFCs, and therefore a better candidate for waste-water treatment if it can couple the oxidation of several organic substrates, as observed with Fe(III)-respiration, also to electrode-respiration.