The mechanisms by which acidophilic bacteria such as T ferrooxidans and L ferrooxidans oxidize iron and/or sulfur is the topic of investigation by numerous researchers. In the environment, oxidation of solid mineral sulfides such as pyrite (FeS2) is greatly increased by acidophilic bacteria, and leads to production of acidic waters (known as acid mine drainage, AMD). T ferrooxidans are known to attach firmly to a solid mineral substrate such as pyrite and oxidize through direct of indirect mechanisms (Rossi, 1990), and involve accumulation of Fe 31, in the exopolymer layer, which is used as the oxidant (Blake et. al., 1992). It is believed that T ferrooxidans attach firmly to pyrite to aid in the sequestering of intermediate thiosulfate compounds, which are used as the primary energy source (Sand et al., 1995). While tremendous progress has been made on the processes involved oxidation of solid minerals such as pyrite by T ferrooxidans, and, L ferrooxidans, little has been done to show that these processes occur by these or other acidophilic organisms in nature by the same or similar mechanism. I am currently maintaining enrichment cultures of acidophilic organism, obtained by inoculating with AMD and pyritic sediment (average grain size = 100mm) from a site near Redding, CA. I would like to analyze the pyritic sediments and the microorganisms associated with them for elemental distribution by energy-filtered transmission electron microscopy (EFIEM). I am interested in using cryofixation techniques as a method for preserving the original elemental distributions within the periplasm. and the outer membrane of the organisms attached to pyrite. The reasons for this are 1) to detect the presence or absence of rusticyanin protein in the cells periplasmic space. The proteins contain copper, which should be easily detected by EFFEM; 2) to detect concentrations of iron in the outer membranes. This would allow us to confirm or disprove importance of the indirect and direct mechanisms of pyrite oxidation by these organisms. VVhile the short-term goals of this project involve cryofixation of organisms and pyrite form enrichment cultures, I would also like to apply this technique to """"""""fi-esh"""""""" samples from AMD sits. This will provide an important comparison of I)pure laboratory cultures, form which all of our current knowledge of iron and sulfur metabolism comes; 2) mixed laboratory cultures inoculated with natural sediments and water, and 3) fresh sediment sarnples from the environment. BCXZNTXFXC SUBPROJECT GRANT NUMBER: P41RR00570-27
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