J.D. Fox and T.G. Chasteen

1. Preparation for the Analysis of Selenocyanate from the Broth Cultures of Selenium-Resistant Bacteria using Solid-Phase Extraction and Capillary Electrophoresis J.D. Fox and T.G. Chasteen

2. Background (the old news) • Our interest is in selenium-resistant microorganisms. • Pseudomonas fluorescens K27 • Escherichia coli 1VH • Bacillus sp. • 130404 • These bacteria grow in the presence of toxic selenium species. • Many can even bioprocess said species.

3. Background cont. • A substantial amount of the selenium in solution is reduced to elemental selenium. • This can be seen as a blood-red precipitate.

4. Chemical Species of Interest • Oxyanions of selenium: • Selenite • Selenate • The current research focuses on selenocyante • But where did it start?

5. Relative Toxicities • Growth experiments were carried out to determine the relative toxicity of each of the selenium species • For E. coli 1VH: • 10 mM Selenate: 24.5% reduction in SGR • 10 mM Selenite: 45.8% reduction in SGR • 10 mM Selenocyanate: 31.3% reduction in SGR

6. Headspace Sampling • Part of the bioreduction process involves methylating Se to create several different volatile species that are out-gassed by the bacteria [1]. • The headspace of E. coli 1VH was sampled using solid-phase microextraction and examined via gas chromatography with fluorine-induced sulfur chemiluminescence detection.

7. Headspace Sample • MeSH – Methanethiol • DMDS – Dimethyldisulfide • DMSeS – Dimethylselenenylsulfide • DMDSe – Dimethyldiselenide • DMTS – Dimethyltrisulfide • DMSeDS - Dimethylselenodisulfide • DMDSeS - Dimethyldiselenosulfide

8. Current • The current research is focusing on the species produced between the initial amendment and the aforementioned products. • Previously, the real-time conversion of selenate to selenite was observed via the use of capillary electrophoresis [2].

9. Current cont. • Recently, analysis of the sterile-filtered broths of these bacteria were evaluated using ion chromatography with inductively coupled plasma mass spectrometry. • One important find was the presence of selenocyanate in the broth of a culture that had been amended with selenate.

10. IC-ICP/MS* Results *IC-ICP/MS analysis by Applied Speciation and Consulting, LLC, Tukwilla, WA

11. The Goal • The goal of this research is to develop a method to monitor the production of selenocyante in bacterial cultures amended with selenate. • Because of the low concentrations of selenocyanate found in previous experiments, solid-phase extraction will be employed to preconcentrate analytes.

12. CE and Standard Prep • The run buffer for the CE was a mixture of 15 mM potassium dihydrogen phosphate and 3 mM tetradecyltrimethylammonium bromide (TTAB). • The pH of this solution was adjusted to 10.5 with a 1.0 mM NaOH solution. • Selenocyanate standards were prepared in HPLC grade water. • All standards, samples and wash fluids were filtered with 0.2 micron syringe filters before being placed in the CE.

13. Solid-Phase Extraction • Aminopropyl Isolute SPE cartridges from International Sorbent Technologies were used. • The cartridges were first solvated with 10 mL of 15 mM potassium dihydrogen phosphate in a 50/50 mix of methanol and water. • The sample (10 mL of 1.0 mM selenocyanate) was then run through the cartridge at approximately 10 mL/min. • Finally, the sample was eluted with 2 mL of a solution of sodium hydroxide with a pH of 11.8.

14. CE Conditions • The capillary was kept at 25 degrees Celsius for each run. • Sample injection was accomplished with 0.5 psi pressure injection for 5 seconds. • Finally a -25 KV potential was run across the capillary for five minutes to establish and maintain the electroosmotic flow (EOF).

15. SeCN in the CE Figure 1: 5.0 mM Selenocyanate

16. Standards Figure 2: Standard Curve for Selenocyanate

17. SPE Sample Figure 3: Sample Extracted via SPE. Concentration: 2.27 mM

18. At This Point… • Using solid-phase extraction, selenocyanate concentration has been successfully raised 2.27x. • But this is just selenocyanate dissolved in water and this is a far cry from extracting selenocyanate from complex bacterial media. • So several components need to be tested: • NaCl • Yeast Extract • Peptone C

19. Testing NaCl • The next series of experiments will be designed to test whether or not the different components of the bacterial medium will be preferentially adsorbed by the SPE cartridge. • A sample containing 1.0 mM selenocyanate and 85.6 mM NaCl was extracted using SPE. • It was then run under the same conditions as the previous samples.

20. SeCN and NaCl Figure 4: NaCl and SeCN Extraction (SeCN conc. = 0.39 mM)

21. NaCl • SeCN is being retained on the SPE cartridge to the extent that the concentration of the eluted sample is increased. • NaCl is also retained by the cartridge, but more experiments are needed to determine whether or not it binds preferentially to the solid phase. • A refinement to the extraction process will be required in order to increase the overall extraction yield.

22. LB Medium with Selenium • Next, the LB medium will be prepared with 1.0 mM selenate and selenocyanate. • To test interference from selenate, NaCl will be omitted from the sample. • The same extraction technique will be used.

23. Extraction with Various Amendments

24. Conclusions So Far • Extraction of selenocyanate becomes problematic in the presence of NaCl • The presence of selenate, however, doesn’t seem to interfere with SPE • When sample is extracted in a sample medium without NaCl, selenocyanate is retained on the solid phase

25. Acknowledgements • Dr. Thomas Chasteen • Bala Krishna Pathem • SHSU Chemistry Department • The Robert A. Welch Foundation

26. References • Challenger, F (1945) Chem. Rev. 36:315-361. • Pathem BK, Pradenas GA, Castro ME, Vásquez CC, Chasteen TG (2007) Anal. Biochem. 364:138-144.