Toxicity of selenocyanate on the metalloid-resistant bacterium LHVE

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 plateTop two columns=control

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

11. Results: 96-microwell platelast 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 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??