Radhika Burra, Gonzalo A. Pradenas, Claudio C. Vásquez and Thomas G. Chasteen.
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Transcript of Radhika Burra, Gonzalo A. Pradenas, Claudio C. Vásquez and Thomas G. Chasteen.
![Page 1: Radhika Burra, Gonzalo A. Pradenas, Claudio C. Vásquez and Thomas G. Chasteen.](https://reader038.fdocuments.net/reader038/viewer/2022110322/56649d135503460f949e7e2e/html5/thumbnails/1.jpg)
Radhika Burra, Gonzalo A. Pradenas,Claudio C. Vásquez and Thomas G. Chasteen
![Page 2: Radhika Burra, Gonzalo A. Pradenas, Claudio C. Vásquez and Thomas G. Chasteen.](https://reader038.fdocuments.net/reader038/viewer/2022110322/56649d135503460f949e7e2e/html5/thumbnails/2.jpg)
Selenium identified as an element in 1917, named from the Greek word, ‘selene’
exists in different forms: metallic, water soluble and gaseous.
considered as an essential trace nutrient
used in the treatment of serious deficiency diseases
used as an anti-oxidant, in glass manufacturing industry, semi- conductor materials and in electronic applications
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Tellurium discovered in 1782, named after Latin word ‘tellus’
extremely rare element
chemically related to selenium and sulfur
mildly toxic, teratogenic
used in semiconductor and electronic industry
used in the treatment of syphilis and leprosy
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exposure is fatal to living beings.
considered as a severe environmental problem.
environmental problems include,
water contamination• Kesterson Reservoir of California• Power River Basin, Wyoming
soil contamination • selenium contamination affecting plants and animals
Why we are concerned???
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Environmental clean-up method includes:
Biological treatment-bioprocessing. Filtration after pH adjustment Evaporation and soil removal
Bioprocessing: also called bioremediation/bioreduction:
• use of microorganisms or their enzymes for detoxification.
• different microbial pathways for the metabolism of toxic
compounds.
• detoxify soluble toxic ions to insoluble and other less toxic forms.
What is Bioprocessing????
} Chemical detoxification methods
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Bacteria currently being used LHVE - species of interest.
characteristics include:
gram positive, rod shaped bacteria forms spores. gelatinase activity.
classified as a Bacillus spp.
isolated from Huerquehue National park, Chile.
selenium (Se) resistant.
reduce Se in solution to elemental Se.
can be seen as a blood-red precipitate.
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Chemical species of interest
Anions of selenium:
selenite (SeO32-)
selenate (SeO42-)
selenocyanate (SeCN-)
Oxyanions of tellurium:
tellurite (TeO32-)
tellurate (TeO42-)
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Gas chromatography with fluorine induced sulfur chemiluminescence detector (GC-SCD)
analyze and separate volatile compounds specific for Se, Te, and Sb compounds detection limits are in picogram range
Gas chromatography- mass spectrometry (GC-MS)
identification of structure of the unknown compounds
Instrumentation
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Sample preparation
Luria-Bertani (LB) medium: tryptone, sodium chloride, yeast extract, water.
pH adjusted to 7.
autoclave at 1200C.
preparation of preculture.
incubation at 370C for approximately 24 hrs.
growth curve and headspace samples preparation.
amendment with different metalloid concentrations.
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Growth curve analysis
performed using liquid culture absorbance at 526 nm
readings are taken at regular intervals of time
log phases of growth are estimated as the linear portion of the log of absorbance versus time plot
the specific growth rate gave a clear idea about the relative toxicity of each of the amended metalloid
lower specific growth rates suggest higher toxicity
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• Lag phase ( where the bacteria gets used to the new environment) • Log phase (growth phase of bacteria) • Stationary phase (no growth)• Death phase
www.bioc.rice.edu/.../NDL Bioreactor%20Page.htm
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Growth Curve Results
Figure 1: Growth Curve for LHVE with 5 mM metalloid amendment.
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Figure 2: Growth Curve for LHVE with 10 mM metalloid amendment.
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Zone of Inhibition
second method of estimating the relative toxicity
it is the clear region around the paper disc saturated with metalloid solution on the agar surface
this is an indication of the absence, or the effective inhibition, of microbial growth by the metalloid
zone of inhibition of 52 mm was observed for tellurite amended plate
tellurite was proved to be more highly toxic than all selenium anions
these set of experiments further confirmed the growth curve results
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control tellurite selenite
selenate selenocyanate
Zone of Inhibition of LHVE at 25 mM tellurite & 100 mM selenium anions
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part of the bioreduction process involves methylating Se
the headspace of the bacteria is sampled using solid-phase microextraction fiber (SPME)
fiber thickness is 75 µm (larger the surface area, the greater the adsorption)
fiber exposure time is about 20-45 minutes.
splitless injection of sample in 2750C injector.
temperature Program: 300C for 2 minutes, ramped 150/min and held at 2750C for 5 minutes.
Headspace Analysis
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What do you mean by headspace?
G = the gas phase (headspace)The gas phase referred to as the headspace and lies above the condensed sample phase
S = the sample phaseThe sample phase contains the compound(s) of interest which are volatile in nature that diffuse into the gas phase until equilibrium is attained
Ref:duiblog.arizonaduicenter.com/tags/defense/
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Solid Phase MicroExtraction
Ref: www.chem.sc.edu/.../lab/images/RGFig1.JPG
rapid, simple, sensitive, solvent-free extraction technique
works on adsorption and desorption principle
concentrate the headspace gases
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Headspace Results
Time (min)
Chemilumines
cenceIntensi
ty
0 5 10 15 2020
MeSH, 2.55
DMeDS, 8.78
DMeTS, 12.6
Figure 3: Chromatogram of LHVE control after 48 h.
MeSH- methanethiol
DMeDS- dimethyl disulfide
DMeTS- dimethyl trisulfide
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Time (min)
Chemilumines
cenceIntensi
ty
0 5 10 15 20
MeSH, 2.63
DMeSe, 5.58
DMeDSeS, 15.64
DMeDSe, 11.29
DMeTS, 12.67
DMeSeDS, 13.68
DMeSeS, 10.09
DMeDS, 8.78
DMeTSe, 17.34
DMeTSe, 17.34
MeSH- methanethiol, 2.63
DMeSe- dimethyl selenide, 5.58
DMeDS- dimethyl disufide, 8.78
DMeSeS- dimethyl selenenyl sulfide, 10.09
DMeDSe- dimethyl diselenide, 11.29
DMeTS- dimethyl trisulfide, 12.67
DMeSeDS- dimethyl selenenyl disulfide, 13.68
DMeDSeS- dimethyl diselenenyl disulfide, 15.64
DMeTSe- dimethyl triselenide, 17.34
Figure 4: Chromatogram of LHVE amended with 1.0 mM selenite, after 48 h.
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Figure 5: Chromatogram of LHVE amended with 1 mM tellurite, after 48 h.
Chemilumines
cenceIntensi
ty
Time (min)
0 5 10 15 20
MeSH, 2.60
DMeDS, 8.76
DMeTS, 12.66
MeSH- methanethiol, 2.60
DMeDS- dimethyl disufide, 8.76
DMeTS- dimethyl trisulfide, 12.66
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Compound Formula Boiling Point (0C)
Retention Time (min)
Methanethiol CH3SH 6 2.63
Dimethyl selenide CH3SeCH3 58 5.58
Dimethyl disulfide CH3SSCH3 110 8.78
Dimethyl selenenyl sulfide
CH3SeSCH3 131 10.09
Dimethyl diselenide CH3SeSeCH3 153 11.29
Dimethyl trisulfide CH3SSSCH3 170 12.67
Dimethyl selenenyl disulfide
CH3SeSSCH3 190 13.68
Dimethyl diselenenyl sulfide
CH3SeSeSCH3 217* 15.64
Dimethyl triselenide CH3SeSeSeCH
3
236* 17.34
Table of Retention Times of Headspace compounds in GC-SCD
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GC-MS Results
Figure 6: Total ion chromatogram of an empty SPME fiber.
Time (min)
Abundan
ce
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Time (min)
Butamine
To beconfirmed, may be fromSPME fiber
Abundan
ce
1- Butanamine
Figure 7: Total ion chromatogram of LHVE control after 72 h.
From the SPME fiber
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Figure 8: Total ion chromatogram of LHVE amended with selenite after 72 h.
DMeSeS- dimethyl selenenyl sulfide, 6.3
DMeDSe- dimethyl diselenide, 7.32
DMeSeDS- dimethyl selenenyl disulfide, 9.47
*DMeDSeS- dimethyl diselenenyl disulfide, 10.38
*DMeTSe- dimethyl triselenide, 11.17
Abundan
ce
Time (min)
DMeSeS, 6.3DMeDSe, 7.32
DMeSeDS, 9.41
*DMeDSeS, 10.38
*DMeTSe, 11.17
DMeSeDS, 9.47
* TWO NEW COMPOUNDS
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Figure 9: Mass spectrum of dimethyl diselenenyl sulfide at 10.38 min.
Abundan
ce
m/z
222
80
93
110
127
160216207190175
184142
m/z Fragment80 Se
93 CH3-Se-
110 CH3-Se-CH3
127 CH3-Se-S-
142 CH3-Se-S-CH3
160 -Se-Se-
175 CH3-Se-Se-
190 CH3-Se-Se-CH3
207 CH3-Se-Se-S-
216 CH3-Se-Se-S-CH3
222 CH3-Se-Se-S-CH3
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Abundan
ce
m/z
80
95
160
175
255190
270
Figure 14: Mass spectrum of dimethyl triselenide at 11.17 min.
m/z Fragment80 Se
95 CH3-Se-
160 -Se-Se-
175 CH3-Se-Se-
190 CH3-Se-Se-CH3
255 CH3-Se-Se-Se-
270 CH3-Se-Se-Se-CH3
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Conclusions
amendments had pronounced effect on the specific growth rate (SGR) of LHVE
TeO3
2- > > SeO32- > SeO4
2- = SeCN-
zone of inhibition experiments, further confirmed the SGR results
headspace analysis showed a diverse production of organo-sulfur and -selenium containing volatiles, but no organo-tellurium
identification of two new compounds: DMDSeS, DMTSe
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Acknowledgements
• Department of Chemistry, Sam Houston State University
• Ms. Rachelle Smith, Analytical Laboratory Manager, TRIES Lab
• Funding from Robert A. Welch Foundation
• Rekha Raghavendra, for guiding in toxicity experiments
• Dr. Stacey Edmonson, UWGRE
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Thank You…
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Questions????