1. 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.  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 Why we are concerned???

5.  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. What is Bioprocessing???? } 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 (SeO 3 2- ) selenate (SeO 4 2- ) selenocyanate (SeCN - )  Oxyanions of tellurium: tellurite (TeO 3 2- ) tellurate (TeO 4 2- )

8.  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 Instrumentation

9. Sample preparation  Luria-Bertani (LB) medium: tryptone, sodium chloride, yeast extract, water.  pH adjusted to 7.  autoclave at 120 0 C.  preparation of preculture.  incubation at 37 0 C 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. control tellurite selenite selenate selenocyanate Zone of Inhibition of LHVE at 25 mM tellurite & 100 mM selenium anions

16.  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 275 0 C injector.  temperature Program: 30 0 C for 2 minutes, ramped 15 0 /min and held at 275 0 C for 5 minutes. Headspace Analysis

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 phase The 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 Ref: www.chem.sc.edu/.../lab/images/RGFig1.JPG  rapid, simple, sensitive, solvent-free extraction technique  works on adsorption and desorption principle  concentrate the headspace gases

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

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

22. CompoundFormula Boiling Point ( 0 C) Retention Time (min) MethanethiolCH 3 SH62.63 Dimethyl selenideCH 3 SeCH 3 585.58 Dimethyl disulfideCH 3 SSCH 3 1108.78 Dimethyl selenenyl sulfideCH 3 SeSCH 3 13110.09 Dimethyl diselenideCH 3 SeSeCH 3 15311.29 Dimethyl trisulfideCH 3 SSSCH 3 17012.67 Dimethyl selenenyl disulfideCH 3 SeSSCH 3 19013.68 Dimethyl diselenenyl sulfide CH 3 SeSeSCH 3 217*15.64 Dimethyl triselenideCH 3 SeSeSeCH 3 236*17.34 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. Figure 7: Total ion chromatogram of LHVE control after 72 h. From the SPME fiber

25. Figure 8: Total ion chromatogram of LHVE amended with selenite after 72 h. 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

26. Figure 9: Mass spectrum of dimethyl diselenenyl sulfide at 10.38 min. m/zFragment 80Se 93CH 3 -Se- 110CH 3 -Se-CH 3 127CH 3 -Se-S- 142CH 3 -Se-S-CH 3 160-Se-Se- 175CH 3 -Se-Se- 190CH 3 -Se-Se-CH 3 207CH 3 -Se-Se-S- 216CH 3 -Se-Se-S-CH 3 222CH 3 -Se-Se-S-CH 3

27. Figure 14: Mass spectrum of dimethyl triselenide at 11.17 min. m/zFragment 80Se 95CH 3 -Se- 160-Se-Se- 175CH 3 -Se-Se- 190CH 3 -Se-Se-CH 3 255CH 3 -Se-Se-Se- 270CH 3 -Se-Se-Se-CH 3

28. Conclusions  amendments had pronounced effect on the specific growth rate (SGR) of LHVE TeO 3 2- > > SeO 3 2- > SeO 4 2- = 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????