Transcript Identification of Organoselenium and Organotellurium Compounds

Identification of organoselenium and
organotellurium compounds in the
headspace of genetically-modified
organisms using GC/SCD
James D. Fox, Bala Krishna Pathem, and Thomas G. Chasteen
Sam Houston State University
Russell Gerads and Hakan Gürleyük
Applied Speciation and Consulting, LLC, Tukwilla, WA
Abstract

The genetically modified bacteria Escherichia coli 1VH carry a plasmid containing
genes conferring resistance to the antibiotic ampicillin as well as resistance to
common, yet highly toxic oxyanions of selenium and tellurium. For instance,
selenate and tellurite are two common biospheric forms of these metalloids. In
addition to this resistance, volatile sulfur, selenium and tellurium compounds are
observed in the headspace of cultures amended with the corresponding metal salts.
The growth rates of the bacteria were examined with amendments of various
concentrations of sodium selenite, sodium selenate, and potassium selenocyanate
using the optical density of each culture as a measure of culture population. In
addition to this the headspace of the cultures were examined using gas
chromatography coupled with a fluorine-induced chemiluminescence detector.
Analysis of Se-containing oxyanions such as SeCN- were carried out using ion
chromatography with inductively coupled plasma/mass spectrometry. Experiments
involving bacterial amendments with or bacterial production of SeCN- are important
because of the interest in this anion's determination in industrial waste streams
contaminated with selenium.
Bacterial and Sample Preparation

E. coli 1VH – Escherichia coli JM109 cells were modified to express the genes
encoded in a 3.8-kb chromosomal DNA fragment from a metalloid-resistant
thermophile, Geobacillus stearothermophilus V.This metalloid resistant organism,
1VH, was cloned in Dr. Claudio Vásquez’s laboratory at University of Santiago,
Chile.

LB Medium – The nutrient broth for the bacteria was made by combining
tryptone, NaCl and yeast extract in water. The pH of the media was adjusted to 7
and the media sterilized. Before inoculation, ampicillin was added to the medium.
Pre-cultures of 1VH were prepared by adding a single colony from a plated
culture to 100-200 mL of LB medium. These were incubated at 37° C for
approximately
24 hours to allow the bacteria to reach stationary phase.

To prepare the metalloid solutions, 100 mM solutions were made for sodium
selenite, sodium selenate and potassium selenocyanate. A 1.0 mM solution of
sodium tellurite was also made. Each solution was sterile-filtered.
Sample Preparation cont.
(for growth curves)

To prepare samples for growth curve analyses, LB medium was
distributed into test tubes (9 mL for the control samples and 8 mL for the
samples to be tested with metalloid amendments). The batch was
sterilized and, once cooled, 0.1 mL of ampicillin solution was added to
each test tube. To the control was added 1.0 mL of stationary 1VH from
the pre-culture broth. For the amended solutions, 1.0 mL of the sterile
metalloid solution and 1.0 mL of stationary 1VH were added.

All samples were sealed with screw-caps and incubated at 37° C between
readings in a constant-temperature water bath.
Sample Preparation cont.
(for headspace analysis)

Headspace samples were prepared by distributing LB medium in 9.0 mL
aliquots into test tubes. After sterilization, 0.1 mL of ampicillin solution
was added to each test tube. To the control was added 1.0 mL of
stationary 1VH. To each of the amended solution was added 1.0 mL of
stationary 1VH and 0.1 mL of a 100 mM metalloid solution. Each test
tube was capped with an open-ended screw cap equipped with a
Teflon®-lined septum.

These samples were incubated at 37° C for approximately 72 hours in
order to allow headspace gasses to accumulate.
Growth Curve Analysis
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The growth curve analysis was performed using liquid culture
absorbance/scatter at 526 nm.
Absorbance readings were taken at regular intervals, using
sterile solutions of each of the different media for blanks.
The readings continued until cultures had reached the
stationary growth phase.
Log phases of growth were estimated as the linear portion of
the log absorbance versus time plot. A short lag phase was
assumed.
The slope of the linear least squares fit, the specific growth
rate, gave a clear idea about the relative toxicity of each of
the amendments. Lower specific growth rates suggest higher
toxicity.
Headspace Analysis
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Headspace sampling was accomplished using a solid-phase
microextraction (SPME) fiber (carboxen-polydimethysiloxane with
75 micrometer thickness) exposed to the headspace of the bacteria for
30 minutes.
The fiber was then inserted into the injection port (275° C) of the GC
using a temperature program that held 30 ° for 2 minutes, ramped
15 °/min and held 275° C for five minutes.
A Sievers® 300 fluorine-induced sulfur chemiluminescence detector was
coupled with the GC in order to detect organo-sulfur, -selenium and –
tellurium compounds in the sample.
Compounds were identified based on retention times of commercial
standards or via GC/MS.
Fluorine-Induced Chemiluminescence Detect
The Fluorine-Induced
Chemiluminescence Detector
1100 V AC
Electrical Discharge
SF
F2
in
out
6
PMT
Photon Counting
Electronics
Integrator
Heated Transfer Line
F2 into
reaction cell
Capillary Column from
Gas Chromatograph
SF
6
Reagent
h
To Fluorine Trap and Vacuum Pump
(reaction cell pressure ~ 1 torr)
Growth Curve Results
Growth Data for 1VH in LB Medium (Control)
ln Absorbance at 526 nm
0.000
-0.500
-1.000
-1.500
y = 1.4236x - 2.9997
R2 = 0.9974
-2.000
-2.500
-3.000
0.00
0.50
1.00
1.50
2.00
Time (hours)
2.50
3.00
3.50
4.00
Growth Curve Results cont.
Growth Data for 1VH in 10 mM Selenite
ln Absorbance at 526 nm
0.000
-0.500
-1.000
-1.500
y = 0.7722x - 2.5714
-2.000
R2 = 0.996
-2.500
-3.000
0.00
0.50
1.00
1.50
2.00
Time (hours)
2.50
3.00
3.50
4.00
Growth Curve Results cont.
Growth Data for 1VH in 10 mM Selenate
ln Absorbance at 526 nm
0.000
-0.500
-1.000
-1.500
y = 1.0747x - 2.7066
R2 = 0.9931
-2.000
-2.500
-3.000
0.00
0.50
1.00
1.50
2.00
Time (hours)
2.50
3.00
3.50
4.00
Growth Curve Results cont.
Growth Data for 1VH in 10 mM KSeCN
ln Absorbance at 526 nm
0.000
-0.500
-1.000
-1.500
y = 0.9781x - 2.6206
R2 = 0.996
-2.000
-2.500
-3.000
0.00
0.50
1.00
1.50
2.00
Time (hours)
2.50
3.00
3.50
4.00
Growth Curve Results cont.
Growth Data for 1VH in 0.1mM Tellurite
ln Absorbance at 526 nm
0.000
-0.500
-1.000
-1.500
y = 0.203x - 1.9543
R2 = 0.9569
-2.000
-2.500
0.00
1.00
2.00
3.00
Time (hours)
4.00
5.00
6.00
Headspace Results for Control Culture
MeSH – Methanethiol
DMDS – Dimethyldisulfide
DMTS – Dimethyltrisulfide
Headspace Results for 10 mM Selenite
Culture
MeSH – Methanethiol
DMDS – Dimethyldisulfide
DMSeS – Dimethylselenenylsulfide
DMDSe – Dimethyldiselenide
DMTS – Dimethyltrisulfide
DMSeDS - Dimethylselenodisulfide
Headspace Results for 10 mM Selenate
Culture
MeSH – Methanethiol
DMDS – Dimethyldisulfide
DMSeS – Dimethylselenenylsulfide
DMDSe – Dimethyldiselenide
DMTS – Dimethyltrisulfide
Headspace Results for 1.0 mM KSeCN
Culture
MeSH – Methanethiol
DMDS – Dimethyldisulfide
DMSeS – Dimethylselenenylsulfide
DMDSe – Dimethyldiselenide
DMTS – Dimethyltrisulfide
DMSeDS - Dimethylselenodisulfide
DMDSeS - Dimethyldiselenosulfide
Ion ChromatographyInductively Coupled Plasma/Mass Spectrometry Analysis of
Se-Amended Bacterial Cultures*
Sterile LB growth medium
IC-ICP/MS determined
concentra tions in ppm Se (SD)
SeO32SeO42SeCN0
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
Conclusions
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Each of the selenium-amended solutions had a pronounced effect on the
specific growth rate of 1VH. Of those oxyanions, selenite had the most
toxic effect. The relative toxicity of the selenocyanate anion was similar
to that of the selenate anion, only slightly more toxic.
The tellurite-amended solution had the most toxic effect of all the
amended solutions, reducing the growth rate by almost 86% compared to
the control culture.
Headspace analysis showed a diverse production of sulfur- and seleniumcontaining volatiles. Of these, the culture amended with KSeCN
produced the widest array of compounds in the largest quantities.
Selenite-amended cultures of P. fluorescens produced detectable amounts
of selenocyanate, SeCN1-. Therefore the presence of this anion in
selenium-contaminated industrial streams may be from biological sources.
Acknowledgements
This work was supported by
SHSU Faculty Enhancement Research Fund
Robert A. Welch Foundation