Toxicity of selenocyanate on the

metalloid-resistant bacterium LHVE

Rebecca A. Montes & Thomas G. Chasteen

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

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)

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.

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.

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

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

96-Microwell Plate Example

Results: 96-microwell plateTop two columns=control

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

Results: 96-microwell platelast four columns=starting at 750 mM

Results: 500 mM

Results: 375 mM

Results: 425 mM

425 mM Zoomed In

Results: Comparing other metalloids

Metalloid MBC MIC

Selenate 500 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

Selenocyanate 425 mM 500 mM

0.001 mM = 1µM

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.

Acknowledgements

• Dr. Thomas G. Chasteen• Robert A. Welch Foundation

THANK YOU!

QUESTIONS??