The search for life beyond Earth is the driving force behind several future missions to Mars. An essential task in the lead-up to these missions is a critical assessment of the habitability for, and feasibility of, life. However, little research has been conducted on this issue, and our understanding of the plausibility for life on Mars remains unconstrained. Owing to the anoxic and iron-rich nature of Mars, microbial iron reduction (MIR) represents a compelling candidate metabolism to operate in the Martian subsurface, past and present. The objectives of this thesis are to address the feasibility of MIR on Mars by i) better defining the habitability of MIR on Earth, and ii) assessing the range and availability of organic electron donors in the subsurface of Earth and Mars. Samples collected from Mars-relevant environments on Earth were used to initiate MIR enrichment cultures at 4°C, 15°C and 30°C. Results indicate MIR is widespread in riverbed and subglacial sediments but not sediments from desert or recent volcanic plains. The iron-reducing microorganisms in subglacial enrichments are at least psychrotolerant and in some cases psychrophilc. Culture-independent methods highlighted the changes in diversity between temperature conditions for subglacial sediments, and indicated that members of the prolific MIR Geobacteraceae family are common. The genera Geobacter and Desulfosporosinus are responsible for MIR in the majority of enrichments. Long-term anoxia and the availability of redox constituents are the major factors controlling MIR in these environments. A MIR enrichment culture was unable to use shales and kerogens as the sole source of electron donors for MIR, despite the presence of known electron donors. Furthermore, MIR was inhibited by the presence of certain kerogens. The causes of inhibition are unknown, and are likely to be a combination of chemical and physical factors. Experiments were conducted to assess the ability of three pure strains and a MIR enrichment to use non-proteinogenic amino acids common to carbonaceous meteorites as electron donors for MIR. Results demonstrate that γ-aminobutyric acid served as an electron donor for the enrichment culture, but no other amino acids supported MIR by this or other iron-reducing cultures. The D-form of chiral amino acids was found to exert a strong inhibitory effect, which decreased in line with concentration. Theoretical calculations using published meteoritic accretion rates onto the surface of Mars indicate that the build up inhibitory amino acids may place important constrains on habitability over geologic time scales. Contamination of a pure strain of Geobacter metallireducens with a strain of Clostridium revealed a syntrophic relationship between these microorganisms. Anaerobic heterotrophs are likely to play an important role in maintaining an available supply of electron donors for MIR and similar chemoorganic metabolisms operating in the subsurface. This research indicates that MIR remains a feasible metabolism to operate on Mars providing a readily available redox couple is present. However, given the observed inhibition in the presence of bulk carbonaceous material and certain amino acids found in meteorites, the use of extraterrestrial carbonaceous material in the Martian subsurface for microbial iron reduction is questionable, and should be the focus of future research.

The bioavailability of iron is limited by the solubility of Fe(III) at circumneutral pH. In the High Nutrient-Low Chlorophyll (HNLC) zones of the ocean, the natural or anthropogenic addition of iron stimulates primary productivity and consumes carbon dioxide. As a result, iron fertilization has been proposed to mitigate anthropogenic carbon emissions and lower global temperatures. The natural sources of iron to the ocean are not fully constrained and include eolian depositions as well as inputs from continental shelf sediments, rivers, hydrothermal vents, and icebergs. Regardless of their source, the effectiveness of iron additions in promoting carbon fixation depends on the presence of organic ligands either natural or produced by microorganisms that stabilize or solubilize Fe(III) at neutral pH. For example, siderophores are well known to be expressed extracellularly by prokaryotes in the photic zones of the oceans to increase the bioavailability of iron. In this dissertation, the production of iron nanoparticles is demonstrated in vent fluids from the 90 North hydrothermal system. These iron nanoparticles may either catalyze the oxidation of sulfide to thiosulfate and produce a potential electron acceptor for microbial respiration or provide a source of iron that stimulates primary production at great distances from the hydrothermal vents. In addition, dissolved iron under the form of soluble organic-Fe(III) complexes is demonstrated to constitute a significant source of iron in estuarine sediments that receive large amounts of particulate iron from flocculation and precipitation at the salinity transition of this estuary. A novel competitive ligand equilibration absorptive cathodic stripping voltammetry (CLE-ACSV) technique reveals that the speciation of iron changes from largely colloidal or particulate in the upper estuary to truly dissolved organic-Fe(III) in the lower estuary. It is also demonstrated that organic-Fe(III) complexes are produced far below the sediment-water interface, suggesting that dissimilatory iron-reducing bacteria may play an important role in their production. These complexes then diffuse across the sediment-water interface and provide a significant source of iron to the continental shelf. The mechanism of reduction of iron oxides by iron-reducing bacteria is not fully understood and presents a unique physiological problem for the organism, as the terminal reductase has to transfer electrons to a solid electron acceptor. In this dissertation, it is demonstrated for the first time using random mutagenesis that the respiration of solid Fe(III) oxides by Shewanella oneidensis, a model iron-reducing prokaryote, first proceeds through a non-reductive dissolution step involving organic ligands that are released extracellularly by the cells. These soluble complexes are then reduced by the organism to produce Fe(II) and recycle the ligand for additional solubilization. Incubations with deletion mutants of the proteins involved in the respiration of Fe(III) revealed that the type-II secretion system, which translocates proteins on the outer membrane of gram-negative bacteria, is involved in the production of organic-Fe(III) complexes by secreting an endogenous iron-solubilizing ligand or a protein involved in the biosynthesis of this ligand on the outer membrane. In addition, periplasmic decaheme cytochromes produced by Shewanella appear to be involved in the mechanism of production of the endogenous organic ligand either directly or through a sensing mechanism that controls its production. In turn, two decaheme cytochromes positioned on the outer-membrane and hypothesized to be involved in the electron transfer to the mineral surface do not appear to be involved in the solubilization mechanism, suggesting either that the cells regulate the ligand production via periplasmic sensing systems or that these cytochromes are not involved in the solubilization mechanism. Altogether this research shows the production of organic-Fe(III) complexes in sediments generates a significant flux of dissolved iron to support primary production in continental shelf waters and that these complexes may be partly produced by iron-reducing bacteria. Indeed, experiments with a model organism demonstrate dissimilatory iron reducing bacteria produce endogenous organic ligands with high iron-binding constants to non-reductively solubilize iron oxides during the anaerobic respiration of iron oxides. The organic ligand is apparently recycled several times to minimize the energy cost associated with its biosynthesis. These findings demonstrate that the solubilization of iron oxides by organic ligands may be an important, yet underappreciated process in aquatic systems.