1. Toxicity of selenocyanate on the metalloid-resistant bacterium LHVE Rebecca A. Montes & Thomas G. Chasteen

2. SeCN - Background Information Selenocyanate (SeCN - ) – Produced by the petrochemical refining of crude oils (in wastewaters) – Also present in mining wastewaters – Toxic levels of SeCN - present in soils and waters all over the world – Can be converted to elemental Se (red color) – Extremely water soluble Makes it through chemical remediation processes

3. About LHVE Characteristics of LHVE gram positive, rod shaped bacteria forms spores. gelatinase activity. classified as a Bacillus sp. isolated from Huerquehue National park, Chile selenium (Se) resistant converts Se in solution to less toxic insoluble Se (red)

4. Purpose The purpose of these experiments is to investigate how toxic the anion selenocyanate (SeCN - ) is to the metalloid-resistant bacterium LHVE. Finding the toxicity of SeCN - will help with biodegradation and bioremediation processes which are used to clean up the environment.

5. Bioremediation and Biodegradation – The use of microorganisms, such as bacteria, to remove pollutants or breakdown waste. – The breakdown of toxic chemicals, by bacteria, into their less toxic forms.

6. MIC and MBC MIC –Minimum Inhibitory Concentration – The lowest concentration of an anti-microbial substance that will inhibit visible growth of a microorganism after incubation overnight. – Inhibits bacterial growth MBC-Minimum Bactericidal Concentration – The lowest concentration of an anti-microbial substance that will prevent the growth of an organism after subculture on to media without antibiotic. – Kills the bacteria

7. Basic Procedure Prepare pre-culture Distribute pre-culture in all wells Add desired amount of toxicant to first row of wells 2-fold serial dilution across width of plate Incubate 24 hours at 37°C Add resazurin dye to each well Incubate 24 hours at 37°C Evaluate microwell plate for MIC results On LB plus agar plates, distribute contents of wells surrounding the MIC Incubate plates 24 hours at 37°C Evaluate LB plus agar plates for MBC results

8. 96-Microwell Plate Example

9. Results: 96-microwell plate Top two columns=control

10. Results: 96-microwell plate Columns 3 and 4=starting at 1 M

11. Results: 96-microwell plate last four columns=starting at 750 mM

12. Results: 500 mM

13. Results: 375 mM

14. Results: 425 mM

15. 425 mM Zoomed In

16. Results: Comparing other metalloids MetalloidMBCMIC Selenate500 mM-1.5M 500 mM Selenite 150 mM -250 mM 150 mM Tellurate 350 μM- 650 μM 350 μM Tellurite 9.78 μM-11.7 μM 9.78 μM Selenocyanate425 mM 500 mM 0.001 mM = 1µM

17. Conclusions The toxicity of SeCN - is comparable to that of selenate and only slightly less toxic than selenite. Tellurite and tellurate were extremely more toxic SeCN -. SeCN - important because not as well known as other metalloids MIC and MBC values useful because they can be used to determine maximum concentrations at which LHVE will be useful in bioremediation and biodegradation processes.

18. Acknowledgements Dr. Thomas G. Chasteen Robert A. Welch Foundation

19. THANK YOU! QUESTIONS??