Transcript Determination if Selenate and Selenite species in bacterial culture

Capillary Electrophoretic
Determination of Selenium and
Tellurium Oxyanions in Bacterial
Cultures
by
Bala Krishna Pathem
and Thomas G. Chasteen
Department of Chemistry
Sam Houston State University
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Instrument configuration
High voltage supply
Computer
Detector
Capillary
Rinse Buffer
vial
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Buffer vial
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Sample vial
Source: ceandcec.com
Buffer vial
Empty vial
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Flow reversal

For Anions:
– In order to speed up the separation, the EOF
can be reversed by addition of cationic
surfactants.
– A negative voltage is applied.
– We have used TTAB and HTAB in our work.
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Source: ceandcec.com
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Method development

We developed a method for the
identification of selenium species in
presence of TSB or LB bacterial growth
medium, complex and well-defined media.

We applied the method for simultaneous
estimation of selenate and selenite or
tellurite in live cultures during their
biological reduction by bacteria.
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Capillary

Fused silica capillaries were used for all the
studies done in this research.
Dimensions:
50 cm effective length, 75 µm i.d, 375 µm
O.D.

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Method
Buffer: 15 mM KH2PO4 + 1 mM TTAB
 pH - 10.5
 Injection Pressure: 0.5 psi
Time: 5 sec
 Separation Voltage: – 25.0 KV
 Capillary temp: 25oC
 Run time: 5 min
 Detection: 190 nm for selenium oxyanions
220 nm for tellurium oxyanions

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Bacterial growth conditions

Two bacterial species were used in our research.
1. Precultures of Bacillus sp. were grown in TSB
(pH 7.0) at 30 oC and then amended with
1.0 mM selenate.
2. Precultures of genetically modified clone of E.coli K12 (1VH) were grown in LB medium with Ampicillin
(pH 7.0) at 37 oC and then amended with 0.05 mM
tellurite.
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Bacillus sp. Growth rate in 1.0 mM selenate and
selenite
Selenate
Selenite
0
-0.5
ln (abs)
-1
-1.5
-2
-2.5
-3
-3.5
0
1
2
3
4
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Time (Hrs)
Figure 1
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Bioreduction of selenate by Bacillus sp.
Figure 2
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Figure 3
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Growth Curve
Selenate Conc (mM)
1.2
-0.5
1
ln (abs)
-1
0.8
-1.5
0.6
-2
0.4
-2.5
0.2
-3
-3.5
Selenate Conc (mM)
0
0
0
2
4
6
8
10
12
Time (Hrs)
Figure 4
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Growth curve
Selenite Conc (mM)
0
0.045
0.035
ln (abs)
-1
0.03
-1.5
0.025
0.02
-2
0.015
-2.5
0.01
-3
Selenite conc (mM)
0.04
-0.5
0.005
-3.5
0
0
2
4
6
8
10
12
Time (Hrs)
Figure 5
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Bioreduction of 0.05 mM tellurite by 1VH
mAU
TeO32-
0 h 5 h 24 h
after inoculation
Figure 6
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Tellurite reduction by 1VH
0.06
Tellurite conc (mM)
0.05
0.04
0.03
0.02
0.01
0
0
1
5
8
10
15
20
24
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Time (Hrs)
Figure 7
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Conclusions

Once bacteria enters stationary phase (4 h):
– Rate of selenate reduction decreases
– Selenite reduction process begins or increases
significantly, 25% drop in SeO32- in 1 h

Varied bacterial response to toxic salts
– Selenite is more toxic than selenate for both these
organisms, based on specific growth rates
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Conclusions

Method offers excellent LOD (3S/N)
– Selenate : 1.0 ppm or 0.007 mM
– Selenite : 0.25 ppm or 0.002 mM

Linearity
– Selenate: 0.9996 (0.1 mM to 1.0 mM)
– Selenite : 0.9998 (0.1 mM to 1.0 mM)
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Conclusions
This method affords high sample throughput and minimal or no sample preparation
for biological samples.
 Perfect tool for the Qualitative or
Quantitative analysis of selenium and
tellurium oxyanions in solution.
 Applicable for analysis in environmental or
agricultural samples.

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Acknowledgements
This work was supported by
Sam Houston State University’s
Faculty Enhancement Research Fund
The Robert A. Welch Foundation
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QUESTIONS??
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