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

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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

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10

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mAU

-20

-10

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10

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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

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60

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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.