1. Examination of Lead Uptake in Three Yeast strains Proposed thesis statement for Graduation with Distinction Timothy Capps December 2004

2. Lead Mining in Missouri East central Missouri is known as the lead-belt region. Chat is the byproduct of mining lead ore. Large mounds of the material dot the landscape in the lead belt. Although the lead concentration is considered hazardous, little has been done to contain it. Original Hypothesis: Chat represents a natural enrichment for lead-resistant microorganisms. These organisms may provide insight into: –the physiological effects of lead on the cell –possible remediation strategies involving bioaccumulation

3. Rhodosporidium paludiginum Pb3 Pb3 TSA Roane Media

4. Effect of Lead Concentration on Growth Rate

6. Project proposal Compare Pb3 – now known to be Rhodosporidium paludiginum with type strain ATCC 46517 Include Rhodotorula rubra as additional control Measure growth rates and lead uptake in 3 organisms

7. Growth Rate Experiments Defined minimal media called “Roane media” Try 1, 0.1, 0.01, and 0 mM bulk Pb concentrations Measure A 600 over the course of growth Done at 25 o C without shaking

8. Lead Uptake Centrifuge culture at end of growth rate experiment Sample supernatant for Pb Acidify and filter sample Measure Pb by ICS AES (AA) Uptake measured as [Pb]uninoculated control - [Pb]culture Uptake will be normalized to cell number (OD)

9. Example of Growth Rate Data

10. Correlation of Optical Density with Measures of Cell Growth Measured A 600 in Pb3 Carried out dilution and plating to determine CFU/ml/OD – Value Determined dry cell weight – Value Now can estimate population by simply measuring A 600

11. Questions to be Answered Is Pb-tolerance a unique feature to Pb3? –If growth rate pattern varies between strains then yes –Two-factor ANOVA comparing growth rates of organisms at various [Pb] Is Pb-uptake a unique feature to Pb3? –Does Pb uptake vary by organism? –Compare strains by ANOVA at each [Pb]

12. Timeline

13. Dissemination