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
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.
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 Interest
• Oxyanions of selenium:– Selenite– Selenate
• The current research focuses on selenocyante
• But where did it start?
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
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.
Headspace Sample• MeSH – Methanethiol• DMDS – Dimethyldisulfide• DMSeS – Dimethylselenenylsulfide• DMDSe – Dimethyldiselenide• DMTS – Dimethyltrisulfide• DMSeDS - Dimethylselenodisulfide• DMDSeS - Dimethyldiselenosulfide
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
IC-ICP/MS determinedconcentrations in ppm Se (SD)
SeO32- SeO4
2- SeCN-
Sterile LB growth medium 0 0 0
Sterile LB + 1 mM SeO32- 92.4 2.8 0.058
Sterile LB + 1 mM SeO42- 0.30 25.4 0.064
Bacterium 130404 + 1 mM SeO32- 24.2 (4) 2.2 (0.04) 0.037 (.003)
Bacterium 130404 + 1 mM SeO42- 0.67 (0.08) 39.4 (6.4) 0.172 (0.01)
IC-ICP/MS determinedconcentrations in ppm Se (SD)
SeO32- SeO4
2- SeCN-
Sterile LB growth medium 0 0 0
Sterile LB + 1 mM SeO32- 92.4 2.8 0.058
Sterile LB + 1 mM SeO42- 0.30 25.4 0.064
Bacterium 130404 + 1 mM SeO32- 24.2 (4) 2.2 (0.04) 0.037 (.003)
Bacterium 130404 + 1 mM SeO42- 0.67 (0.08) 39.4 (6.4) 0.172 (0.01)
*IC-ICP/MS analysis by Applied Speciation and Consulting, LLC, Tukwilla, WA
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.
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.
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.
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).
SeCN in the CE
Figure 1: 5.0 mM Selenocyanate
Minutes
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
mAU
-10
0
10
20
30
40
mAU
-10
0
10
20
30
40P/ACE MDQ-200 nm5.0 mM SeCN
Migration TimeAreaWidth
Standards
Figure 2: Standard Curve for Selenocyanate
Selenocyante Standards
y = 82415x - 9477.6
R2 = 0.9948
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
0 2 4 6 8 10 12
Conc. (mM)
Peak Area
SPE Sample
Figure 3: Sample Extracted via SPE. Concentration: 2.27 mM
Minutes
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
mAU
-20
-10
0
10
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mAU
-20
-10
0
10
20
P/ACE MDQ-200 nmElution 1
Migration TimeAreaWidth
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
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.
SeCN and NaCl
Minutes
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
mAU
-40
-20
0
20
40
60
80
100
mAU
-40
-20
0
20
40
60
80
100P/ACE MDQ-200 nm1.0 mM SeCN + NaCl Extract
Migration TimeAreaWidth
Figure 4: NaCl and SeCN Extraction (SeCN conc. = 0.39 mM)
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.
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.
Extraction with Various Amendments
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
Acknowledgements
• Dr. Thomas Chasteen
• Bala Krishna Pathem
• SHSU Chemistry Department
• The Robert A. Welch Foundation
References
1. Challenger, F (1945) Chem. Rev. 36:315-361.
2. Pathem BK, Pradenas GA, Castro ME, Vásquez CC, Chasteen TG (2007) Anal. Biochem. 364:138-144.