radhika burra, gonzalo a. pradenas, claudio c. vásquez and thomas g. chasteen

Radhika Burra, Gonzalo A. Pradenas,Claudio C. Vásquez and Thomas G. Chasteen

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

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

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???

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

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.

Chemical species of interest

Anions of selenium:

selenite (SeO32-)

selenate (SeO42-)

selenocyanate (SeCN-)

Oxyanions of tellurium:

tellurite (TeO32-)

tellurate (TeO42-)

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

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.

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

• 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

Growth Curve Results

Figure 1: Growth Curve for LHVE with 5 mM metalloid amendment.

Figure 2: Growth Curve for LHVE with 10 mM metalloid amendment.

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

control tellurite selenite

selenate selenocyanate

Zone of Inhibition of LHVE at 25 mM tellurite & 100 mM selenium anions

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

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/ 

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

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

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.

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

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

GC-MS Results

Figure 6: Total ion chromatogram of an empty SPME fiber.

Time (min)

Abundan

ce

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

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

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

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

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

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

Thank You…

Questions????