toxicity of selenocyanate on the metalloid-resistant bacterium lhve rebecca a. montes & thomas...
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Toxicity of selenocyanate on the
metalloid-resistant bacterium LHVE
Rebecca A. Montes & Thomas G. Chasteen
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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
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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)
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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.
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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.
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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
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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
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96-Microwell Plate Example
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Results: 96-microwell plateTop two columns=control
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Results: 96-microwell plateColumns 3 and 4=starting at 1 M
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Results: 96-microwell platelast four columns=starting at 750 mM
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Results: 500 mM
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Results: 375 mM
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Results: 425 mM
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425 mM Zoomed In
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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
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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.
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Acknowledgements
• Dr. Thomas G. Chasteen• Robert A. Welch Foundation
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THANK YOU!
QUESTIONS??