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Biological Interactions of selenium and tellurium: Bioprocessing , Detection, and Toxicity

Biological Interactions of selenium and tellurium: Bioprocessing , Detection, and Toxicity. Radhika Burra , Gonzalo A. Pradenas, Claudio C. Vásquez and Thomas G. Chasteen. Selenium. identified as an element in 1917, named from the Greek word, ‘ selene ’

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Biological Interactions of selenium and tellurium: Bioprocessing , Detection, and Toxicity

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  1. Biological Interactions of selenium and tellurium:Bioprocessing, Detection, and Toxicity Radhika Burra, Gonzalo A. Pradenas, Claudio C. Vásquez and Thomas G. Chasteen

  2. Selenium • identified as an element in 1917, named from the Greek • word, ‘selene’ • exists in different forms: • metallic, • water soluble and • gaseous. • considered as an essential trace nutrient • used in the treatment of serious deficiency diseases • used as an anti-oxidant, in glass manufacturing industry, semi- conductor materials and in electronic applications

  3. Tellurium • discovered in 1782, named after Latin word ‘tellus’ • extremely rare element • chemically related to selenium and sulfur • mildly toxic, teratogenic • used in semiconductor and electronic industry • used in the treatment of syphilis and leprosy

  4. Why we are concerned??? • exposure is fatal to living beings. • considered as a severe environmental problem. • environmental problems include, • water contamination • Kesterson Reservoir of California • Power River Basin, Wyoming • soil contamination • selenium contamination affecting plants and animals

  5. What is Bioprocessing???? • Environmental clean-up method includes: • Biological treatment-bioprocessing. • Filtration after pH adjustment • Evaporation and soil removal • Bioprocessing: also called bioremediation/bioreduction: • use of microorganisms or their enzymes for detoxification. • different microbial pathways for the metabolism of toxic • compounds. • detoxify soluble toxic ions to insoluble and other less toxic • forms. } Chemical detoxification methods

  6. Bacteria currently being used • LHVE - species of interest. • characteristics include: • gram positive, rod shaped bacteria • forms spores. • gelatinase activity. • classified as a Bacillus spp. • isolated from Huerquehue National park, Chile. • selenium (Se) resistant. • reduce Se in solution to elemental Se. • can be seen as a blood-red precipitate.

  7. Chemical species of interest • Anions of selenium: • selenite (SeO32-) • selenate (SeO42-) • selenocyanate (SeCN-) • Oxyanions of tellurium: • tellurite (TeO32-) • tellurate (TeO42-)

  8. Instrumentation • Gas chromatography with fluorine induced sulfur • chemiluminescence detector (GC-SCD) • analyze and separate volatile compounds • specific for Se, Te, and Sb compounds • detection limits are in picogram range • Gas chromatography- mass spectrometry (GC-MS) • identification of structure of the unknown compounds

  9. Sample preparation • Luria-Bertani (LB) medium: tryptone, sodium chloride, yeast • extract, water. • pH adjusted to 7. • autoclave at 1200C. • preparation of preculture. • incubation at 370C for approximately 24 hrs. • growth curve and headspace samples preparation. • amendment with different metalloid concentrations.

  10. Growth curve analysis • performed using liquid culture absorbance at 526 nm • readings are taken at regular intervals of time • log phases of growth are estimated as the linear portion of the log of absorbance versus time plot • the specific growth rate gave a clear idea about the relative toxicity of each of the amended metalloid • lower specific growth rates suggest higher toxicity

  11. Lag phase ( where the bacteria gets used to the new environment) • Log phase (growth phase of bacteria) • Stationary phase (no growth) • Death phase • www.bioc.rice.edu/.../NDL Bioreactor%20Page.htm

  12. Growth Curve Results Figure 1: Growth Curve for LHVE with 5 mM metalloid amendment.

  13. Figure 2: Growth Curve for LHVE with 10 mM metalloid amendment.

  14. Zone of Inhibition • second method of estimating the relative toxicity • it is the clear region around the paper disc saturated with metalloid • solution on the agar surface • this is an indication of the absence, or the effective inhibition, • of microbial growth by the metalloid • zone of inhibition of 52 mm was observed for tellurite amended plate • tellurite was proved to be more highly toxic than all selenium anions • these set of experiments further confirmed the growth curve results

  15. Zone of Inhibition of LHVEat 25 mM tellurite & 100 mM selenium anions tellurite control selenite selenate selenocyanate

  16. Headspace Analysis • part of the bioreduction process involves methylating Se • the headspace of the bacteria is sampled using solid-phase • microextraction fiber (SPME) • fiber thickness is 75 µm (larger the surface area, the greater the adsorption) • fiber exposure time is about 20-45 minutes. • splitless injection of sample in 2750C injector. • temperature Program: 300C for 2 minutes, ramped 150/min and • held at 2750C for 5 minutes.

  17. What do you mean by headspace? G = the gas phase (headspace)The gas phase referred to as the headspace and lies above the condensed sample phase S = the sample phaseThe sample phase contains the compound(s) of interest which are volatile in nature that diffuse into the gas phase until equilibrium is attained Ref:duiblog.arizonaduicenter.com/tags/defense/ 

  18. Solid Phase MicroExtraction • rapid, simple, sensitive, • solvent-free extraction technique • works on adsorption and • desorption principle • concentrate the headspace • gases Ref: www.chem.sc.edu/.../lab/images/RGFig1.JPG

  19. Headspace Results MeSH- methanethiol DMeDS- dimethyl disulfide DMeTS- dimethyl trisulfide Figure 3: Chromatogram of LHVE control after 48 h.

  20. MeSH- methanethiol, 2.63 DMeSe- dimethyl selenide, 5.58 DMeDS- dimethyl disufide, 8.78 DMeSeS- dimethyl selenenyl sulfide, 10.09 DMeDSe- dimethyl diselenide, 11.29 DMeTS- dimethyl trisulfide, 12.67 DMeSeDS- dimethyl selenenyl disulfide, 13.68 DMeDSeS- dimethyl diselenenyl disulfide, 15.64 DMeTSe- dimethyl triselenide, 17.34 Figure 4: Chromatogram of LHVE amended with 1.0 mM selenite, after 48 h.

  21. MeSH- methanethiol, 2.60 DMeDS- dimethyl disufide, 8.76 DMeTS- dimethyl trisulfide, 12.66 Figure 5: Chromatogram of LHVE amended with 1 mM tellurite, after 48 h.

  22. Table of Retention Times of Headspace compounds in GC-SCD

  23. GC-MS Results Figure 6: Total ion chromatogram of an empty SPME fiber.

  24. From the SPME fiber Figure 7: Total ion chromatogram of LHVE control after 72 h.

  25. DMeSeS- dimethyl selenenyl sulfide, 6.3 DMeDSe- dimethyl diselenide, 7.32 DMeSeDS- dimethyl selenenyl disulfide, 9.47 *DMeDSeS- dimethyl diselenenyl disulfide, 10.38 *DMeTSe- dimethyl triselenide, 11.17 DMeSeDS, 9.47 * TWO NEW COMPOUNDS Figure 8: Total ion chromatogram of LHVE amended with selenite after 72 h.

  26. Figure 9: Mass spectrum of dimethyl diselenenyl sulfide at 10.38 min.

  27. Figure 14: Mass spectrum of dimethyl triselenide at 11.17 min.

  28. Conclusions • amendments had pronounced effect on the specific growth rate (SGR) • of LHVE • TeO32- > > SeO32- > SeO42- = SeCN- • zone of inhibition experiments, further confirmed the SGR results • headspace analysis showed a diverse production of organo-sulfur • and -selenium containing volatiles, but no organo-tellurium • identification of two new compounds: DMDSeS, DMTSe

  29. Acknowledgements • Department of Chemistry, Sam Houston State University • Ms. Rachelle Smith, • Analytical Laboratory Manager, TRIES Lab • Funding from Robert A. Welch Foundation • Rekha Raghavendra, for guiding in toxicity experiments • Dr. Stacey Edmonson, UWGRE

  30. Thank You…

  31. Questions????

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