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Fox and T.G. ChasteenBackground (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.j6ZEZfZ6 f Background cont.A substantial amount of the selenium in solution is reduced to elemental selenium. This can be seen as a blood-red precipitate.Chemical Species of InterestmOxyanions of selenium: Selenite Selenate The current research focuses on selenocyante But where did it start?6EE$ ? Relative ToxicitiesGrowth 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 SGR8xzkz>{$$&Headspace Sampling7Part 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.88Z>  ^Headspace SampleMeSH  Methanethiol DMDS  Dimethyldisulfide DMSeS  Dimethylselenenylsulfide DMDSe  Dimethyldiselenide DMTS  Dimethyltrisulfide DMSeDS - Dimethylselenodisulfide DMDSeS - Dimethyldiselenosulfide P  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].   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.  IC-ICP/MS* Results$   The GoalThe 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.,N  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. P. H Solid-Phase ExtractionAminopropyl 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."C ?F,5 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).,f SeCN in the CE  Standards SPE SampleAt 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 >    Testing NaClJThe 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.KK,\ SeCN and NaCl NaCl oSeCN 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. pp$oLB Medium with SeleniumNext, the LB medium will be prepared with 1.0 mM selenate and selenocyanate. To test interference from selenate, NaCl will be omitted from the sample. The same extraction technique will be used.,.@M"Extraction with Various Amendments##(Conclusions So FarExtraction of selenocyanate becomes problematic in the presence of NaCl The presence of selenate, however, doesn t seem to interfere with SPE When sample is extracted in a sample medium without NaCl, selenocyanate is retained on the solid phase,Cz/ Acknowledgements aDr. Thomas Chasteen Bala Krishna Pathem SHSU Chemistry Department The Robert A. Welch Foundation,9 ReferencesChallenger, F (1945) Chem. Rev. 36:315-361. Pathem BK, Pradenas GA, Castro ME, Vsquez CC, Chasteen TG (2007) Anal. Biochem. 364:138-144. F"  O>, 4  ` 33` Sf3f` 33g` f` www3PP` ZXdbmo` \ғ3y`Ӣ` 3f3ff` 3f3FKf` hk]wwwfܹ` ff>>\`Y{ff` R>&- {p_/̴>?" dd@,|?" dd@   " @ ` n?" dd@   @@``PR    @ ` ` p>> f(    6n   `}  T Click to edit Master title style! !  0Pm   `  RClick to edit Master text styles Second level Third level Fourth level Fifth level!     S  0l  ^ `  >*  0o  ^   @*  0Pp  ^ `  @*H  0޽h ? 3f3FKf80___PPT10.J Default Design$ 0$(  r  S     r  S   ` @  H  0޽h ? 3380___PPT10.=J$  @$(  r  S p" `}   r  S 0" `  H  0޽h ? 3f3FKf80___PPT10."$  P($(  (r ( S 0" `}   r ( S " `  H ( 0޽h ? 3f3FKf80___PPT10.`-0$  `$(  r  S Я" `}   r  S P" `  H  0޽h ? 3f3FKf80___PPT10.q$  p$(  r  S P" `}   r  S " `  H  0޽h ? 3f3FKf80___PPT10.%p$  $(  r  S 0" `}   r  S " `  H  0޽h ? 3f3FKf80___PPT10.9  RJ (   r  S " `}   r  S P" `    vA( HS 1VH KSeCN editedZ P H  0޽h ? 3f3FKf80___PPT10.7$  ,$(  ,r , S p" `}   r , S " `  H , 0޽h ? 3f3FKf80___PPT10.`*$  4$(  4r 4 S е" `}   r 4 S ! `  H 4 0޽h ? 3f3FKf80___PPT10.0]z   8(  8r 8 S q  `}    80 TA ? ?^    X 8 0   80 ZA ? ?S" P /     8 0 & K*IC-ICP/MS analysis by Applied Speciation and Consulting, LLC, Tukwilla, WA4L 25?H 8 0޽h ? 3f3FKf80___PPT10. +:H}  D$(  Dr D S p  `}   r D S Ј  `  H D 0޽h ? 3f3FKf___PPT10i. #s+D=' = @B +$  L$(  Lr L S   `}   r L S p  `  H L 0޽h ? 3f3FKf80___PPT10.I$  H$(  Hr H S   `}   r H S 0  `  H H 0޽h ? 3f3FKf80___PPT10.0i$  P$(  Pr P S   `}   r P S 0  `  H P 0޽h ? 3f3FKf80___PPT10.p  zT(  Tr T S Ћ  `}    T 0 Pw jFigure 1: 5.0 mM Selenocyanate(2 T NA  ` H T 0޽h ? 3f3FKf80___PPT10.0k  z \(  \r \ S p  `}    \ 0Б 0 ` v*Figure 2: Standard Curve for Selenocyanate+(2+ \0 6A ?  H \ 0޽h ? 3f3FKf___PPT10i.@ͱ+D=' = @B +  0d)(  dr d S   `}    d 0P 0 ` ;Figure 3: Sample Extracted via SPE. Concentration: 2.27 mM<(2<9 d HA  ` H d 0޽h ? 3f3FKf___PPT10i. +D=' = @B +$  @l$(  lr l S v `}   r l S `v `  H l 0޽h ? 3f3FKf80___PPT10.XeL}  Pp$(  pr p S @x `}   r p S w `  H p 0޽h ? 3f3FKf___PPT10i.YPM{+D=' = @B +e  `te(  tr t S  z `}    t HA  ` # t 0y ` 9Figure 4: NaCl and SeCN Extraction (SeCN conc. = 0.39 mM):(2:P   H t 0޽h ? 3f3FKf80___PPT10.[p$  p|$(  |r | S | `}   r | S `| `  H | 0޽h ? 3f3FKf80___PPT10.`!v$  $(  r  S ~ `}   r  S @~ `  H  0޽h ? 3f3FKf80___PPT10.+ Ql  l(  r  S  `}     zA2SeCN and SeO4 Extraction 0m H  0޽h ? 3f3FKf80___PPT10.+`$  $(  r  S ` `}   r  S  `  H  0޽h ? 3f3FKf80___PPT10.+Ɉ$  $(  r  S  `}   r  S  `  H  0޽h ? 3f3FKf80___PPT10.,e   0:(  0r 0 S   `}    0 S `  `  "p`PpH 0 0޽h ? 3f3FKf___PPT10i.i-+D=' = @B +rx] pyqCBd%F ٸD " P,0q3bWRT;Oh0!O;4bjŵ_gdhc3RC1~9gݫ+ao?s?|~֩oDɠ l 4AyD_H$Nr§>d>Y@6LSyV<ȕ*@|`6Pg>EJ`!'%@1pp+r (wFXd˟yu'[1Ԍ["S?tmdHmF{,L@LvC| (-? 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