Iron- and Sulfur-oxidizing Machinery of the bioleaching Archaeon Metallosphaera sedula

An extreme thermoacidophile Metallosphaera sedula is a metal immobilizing archaeon, which respires aerobically on iron and reduced inorganic sulfur compounds (RISCs). This microorganism has received increasing attention from the side of biomining industry as a possible candidate for the recovery of precious, base and strategic metals, trapped into metal sulfides ores, through dissimilatory iron and sulfur oxidative processes. Biological regeneration of Fe3+ from Fe2+, performed by this microorganism, is the key to chemical attack of metal sulfides. However, oxidation of sulfur and RISCs contributes to the mineral dissolution and the maintenance of acidity level required to keep the ferric iron in soluble form. Because of its inherent ability to tolerate the high temperature derived from a highly exothermic bioleaching process, M. sedula is clearly superior to mesoacidophilic biocatalysts in bioleaching kinetics, accelerating the reaction rate and shortening the leaching cycle. However, compared with the mesophiles, there is still lack of understanding of the bioleaching behavior and the processes of sulfur and iron oxidation of this extreme thermophile. The current proposal focuses on the functional identification and characterization of the Iron and Sulfur oxidizing machinery of M. sedula. Preliminary investigations in this area allowed partial functional characterization of rusticyanin-like protein. Obtained rusticyanin-like protein, in spite of being annotated as a Blue Copper Protein (BCP), exposes high iron binding capacity and thus may represent a new redox-active enzyme unique to respiratory iron oxidation in this extremely thermoacidophilic archaea. M. sedula stands out from related extremophiles by its physiological versatility, growing heterotrophically on peptides, autotrophically by fixing CO 2 , and mixotrophically on Casamino Acids and FeSO 4 or metal sulfides, thus representing an excellent model organism for basic research into bioleaching processes. Using the recently sequenced and analyzed genome of this metal mobilizing archaeon, genetic and functional genomics tools have recently been developed (targeted gene disruption, gene insertion, gene transfer), which offer the unprecedented opportunity to metabolically engineer M. sedula. The results in frames of this project will provide a strong background for a design of metabolically engineered strains with enhanced bioleaching performance, production of which will accomplish the current scientific proposal.

The extreme thermoacidophile Metallosphaera sedula is a metallophilic archaeon that lives in hot acidic conditions (73 C, pH 2) and uses various metal-containing ores to run its respiratory electron transport chain. The projects concept focused on the functional characterisation of metal oxidizing machinery of the extreme thermoacidophile M. sedula, which is a critical element in bioleaching performance of this microorganism. Metal-oxidizing properties of M. sedula have been studied utilizing an integrated suit of interdisciplinary techniques, including fine ultrastructural elemental analysis, Electron Energy Loss Spectroscopy, Scanning Electron, High-Resolution Transmission Electron Microscopy, microbiological and biochemical tools. This integrated interdisciplinary approach yielded in the establishment of a geomicrobiological toolbox, which was necessary in order to decipher metal-oxidizing machinery of M. sedula. The structural features of metal-oxidizing machinery of M. sedula grown on metal-bearing materials have been resolved down to nanometer scale. During this project, nanometer-scale microbial-mineral interfaces, bioleaching, physiology, and metal-oxidizing machinery of M. sedula grown on various terrestrial and extraterrestrial metal-bearing materials have been resolved and published in open access journals. The biooxidation capacity of M. sedula has merely been proposed as a procedure in the processing of sulfide and uranium ores, but we have shown that M. sedula can grow on non-sulfide tungsten ore scheelite and artificial tungsten-bearing compounds. We have shown that biotransformation of scheelite by M. sedula is accompanied by tungsten solubilization in the leachate solution and biomineralization of cell surface with crystalline nanoparticles containing carbide-like tungsten. This study provides information concerning the possible role of microorganisms in natural environments enriched with tungsten and corresponding microbial fingerprints, thus helping to unravel biogeochemistry of tungsten. The findings obtained during this project are useful for currently underrepresented and less studied biomining of tungsten ores, where biooxidative dissolution pre-treatment are useful. Iron/sulfur oxidation machinery and microbial-metal interface of M. sedula grown on real and synthetic extraterrestrial materials meteorite NWA 1172 and Mars regolith simulants was studied during this project. We have shown that this metal mobilizing archaeon can grow on Martian regolith simulants, actively colonizing these artificial extraterrestrial materials, releasing free soluble metals into the leachate solution and leaving specific spectral fingerprints. We also explored the physiology and metal-microbial interface of M. sedula, living on and interacting with real extraterrestrial material, H5 ordinary chondrite Northwest Africa 1172 (NWA 1172). Specific chemical analysis of the meteorite-microbial interface at nm-scale resolution allowed us to trace the trafficking of meteorite inorganic constituents into a microbial cell and investigate iron redox behavior. Our investigations provide the further step towards extending the knowledge of meteorite biogeochemistry. By furthering our knowledge of life based on extraterrestrial materials, investigation of meteorite- microbial interactions is directly relevant to both defining the habitability of extraterrestrial environments and planetary protection issues.