Microbial Nitrate Dependent Fe²⁺ Oxidation: A Potential Early Mars Metabolism

This thesis experimentally investigates the proposition that aqueous environments with anoxic, reducing, circumneutral conditions, inorganic electron donors and oxidants such as nitrate may have allowed chemolithotrophic microbial metabolisms, such as nitrate-dependent Fe2+ oxidation (NDFO), to thrive on early Mars. The NDFO microorganisms, Acidovorax sp. strain BoFeN1, Paracoccus sp. strain KS1 and Pseudogulbenkiania sp. strain 2002, are demonstrated to grow lithoautotrophically with a Mars-relevant olivine Fe2+ source and in martian simulant media developed from in situ and meteorite geochemical data. Additionally, mixotrophic NDFO with Fe2+ and an organic co-substrate as electron donors, was shown to increase the extent of microbial growth, which gains importance in light of the confirmation of complex organics at the martian surface. Biomineralised microbial features were discovered after culture with olivine and the oxidation of Fe2+ was measured in heterotrophic cultures. Microfossils and Fe3+ compounds in reducing contexts provide targets for biosignature detection missions. In addition, this work presents findings on the biochemical mechanisms of NDFO, quantifying the relative contributions of the Nar respiratory nitrate reductase enzyme, putative ferroxidases and nitrite accumulation to Fe2+ oxidation during nitrate reduction. Gene knockout experiments revealed that Fe2+ oxidation in the heterotrophic Salmonella enterica Serovar Typhimurium strain SL1344 is largely driven by the production of reactive nitrite ions during Nar activity. Draft genome analysis of Acidovorax sp. strain BoFeN1 and Paracoccus sp. strain KS1 revealed potential mechanisms of electron acquisition during Fe2+ oxidation, underlining that multiple mechanisms of NDFO exist. The combined findings of this thesis support the plausibility of NDFO in the deep martian past and propose mechanisms by which evidence may be preserved in the geological record of that planet