MEASURING THE RATE OF IRON REMOVAL BY AN ACIDOPHILIC PROTOZOAN, EUGLENA MUTABILIS, LIVING IN COAL-RELATED ACID MINE DRAINAGE

Preliminary analysis of biological activity of Euglena mutabilis, a photosynthetic, acidophilic protozoan that forms bright green benthic mats in coal-related acid mine drainage (AMD), suggests that the microorganism plays an important role in the natural attenuation of iron in these contaminated systems. Microscopic observations reveal numerous (7-10) reddish intracellular granules, which test positive for iron, suggesting that E. mutabilis has the ability to sequester iron from AMD. In addition, field data indicate that E. mutabilis contributes to high concentrations of dissolved oxygen, up to 200% saturation with the atmosphere, that may drive precipitation of iron hydroxides from AMD enriched in Fe (II). The purpose of this research is to develop a method of quantifying the rate of iron removal from AMD due to the various biological activities of E. mutabilis. An artificial, chemically-defined media resembling the chemical composition of AMD was prepared with various amounts of iron. The media was inoculated with E. mutabilis and periodically centrifuged to remove cells and precipitate, and assayed for iron concentration as compared to uninoculated controls. The concentration of iron was determined by extrapolation from a standard curve developed using a colormetric assay. Differences in iron concentration between E. mutabilis cultures and uninoculated controls were interpreted as the result of photosynthetic activity (or oxygen liberation) and the intracellular sequestering of iron. To distinguish between precipitation related to respiration and intracellular sequestering, whole cells were collected, washed extensively, lysed, and the cytoplasmic contents assayed for iron. These procedures allow for better quantification of iron removal by E. mutabilis, which can be used to assess the potential importance of the microbe in AMD remediation.