Determination of Elemental
Selenium Production by a Facultative
Anaerobe Grown Under Sequential
Anaerobic/Aerobic Conditions
Suminda Hapuarachchi,
Jerry Swearingen, Jr, and
Thomas G. Chasteen
Department of Chemistry
Sam Houston State University
What happens to toxic metalloids
bioprocessed by metalloid-resistant bacteria?
What happens to toxic metalloids
bioprocessed by metalloid-resistant bacteria?
Soluble forms remain in solution.
What happens to toxic metalloids
bioprocessed by metalloid-resistant bacteria?
Soluble forms remain in solution.
Bioreduction also produces methylated, volatile forms.
What happens to toxic metalloids
bioprocessed by metalloid-resistant bacteria?
Soluble forms remain in solution.
Bioreduction also produces methylated, volatile forms.
Metalloids are converted to elemental (solid) form.
Phototropic Bacteria
0
Se
and
0
Te
from Strict Anaerobes
Headspace yield
from 6 phototrophs
•dimethyl sulfide
•dimethyl selenide
•dimethyl diselenide
(also dimethyl selenenyl sulfide)
The fluorine-induced
chemiluminescence GC
chromatogram of the
headspace above
Se-resistant bacteria.
Amended with SeO32-
Dimethyl telluride
production by
Pseudomonas
fluorescens K27
Amended with TeO32-
DMTe
Trimethyl stibine
Dimethyl disulfide
Dimethyl trisulfide
(CH3)3Sb production
by K27
amended with an
inorganic-Sb salt
Amended with KSb(OH)6
Methanethiol
Dimethyl sulfide
Can a mass balance be determined for
metalloids distributed among solid, liquid,
and gas phases in bacterial cultures?
Use 3 L batch cultures amended with Se oxyanions.
Incubate culture far into the stationary phases.
Determine metalloid content in each phase.
3 L bioreactor
• Temperature controlled
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3 L bioreactor
• Temperature controlled
• pH control
additions
acid
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base
3 L bioreactor
• Temperature controlled
• pH control
• Dissolved Oxygen
gas
N2/O2 purge
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D.O.probe
3 L bioreactor
• Temperature controlled
• pH control
• Dissolved Oxygen
• Nutrient addition
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3 L bioreactor
• Temperature controlled
• pH control
• Dissolved Oxygen
• Nutrient addition
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• Gas harvest
bubbler(s)
3 L bioreactor
• Temperature controlled
• pH control
• Dissolved Oxygen
• Nutrient addition
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• Gas harvest
• Liquid harvest
Bacterial
Culture Conditions
Bacterial
Culture Conditions
Pseudomonas fluorescens K27
Isolated by Ray Fall at CU Boulder
Facultative anaerobe (grows with or without oxygen)
Grown on tryptic soy broth with 3% nitrate added
Bacterial
Culture Conditions
Pseudomonas fluorescens K27
Isolated by Ray Fall at CU Boulder
Facultative anaerobe (grows with or without oxygen)
Grown on tryptic soy broth with 3% nitrate added
Selenium Amendments
1–10 mM SeO42- or SeO32- along with 10%/vol. inoculum
Bacterial
Culture Conditions
Pseudomonas fluorescens K27
Isolated by Ray Fall at CU Boulder
Facultative anaerobe (grows with or without oxygen)
Grown on tryptic soy broth with 3% nitrate added
Selenium Amendments
1–10 mM SeO42- or SeO32- along with 10%/vol. inoculum
Tellurium Amendments
0.01 to 1 mM TeO42- or TeO32- along with 10%/vol. inoculum
Bacterial
Culture Conditions
Pseudomonas fluorescens K27
Isolated by Ray Fall at CU Boulder
Facultative anaerobe (grows with or without oxygen)
Grown on tryptic soy broth with 3% nitrate added
Selenium Amendments
1–10 mM SeO42- or SeO32- along with 10%/vol. inoculum
Tellurium Amendments
0.01 to 1 mM TeO42- or TeO32- along with 10%/vol. inoculum
Batch cultures at 30˚C
15 hr to 72 hr bacterial cultures; ~ 3 L liquid volume
Se
Determination
Liquid phase selenium
Inductively coupled plasma spectrometry (ICP)
Se
Determination
Liquid phase selenium
Inductively coupled plasma spectrometry (ICP)
Solid phase selenium (Se0 and cells)
ICP following centrifugation and dissolution with HNO3
Se
Determination
Liquid phase selenium
Inductively coupled plasma spectrometry (ICP)
Solid phase selenium (Se0 and cells)
ICP following centrifugation and dissolution with HNO3
Gas phase selenium (volatile organo-Se compounds)
Species identified via GC/fluorine-induced chemiluminescence
Trapping in serial HNO3 bubblers
Analysis via ICP
Simultaneous ICP
ICP
Analysis
Te
Determination
Liquid phase tellurium
Hydride generation atomic absorption spectrometry (HGAAS)
Te
Determination
Liquid phase tellurium
Hydride generation atomic absorption spectrometry (HGAAS)
Solid phase tellurium (Te0 and cells)
HGAAS following centrifugation and dissolution with HNO3
Te
Determination
Liquid phase tellurium
Hydride generation atomic absorption spectrometry (HGAAS)
Solid phase tellurium (Te0 and cells)
HGAAS following centrifugation and dissolution with HNO3
Gas phase tellurium
Capillary gas chromatography/F2-induced chemiluminescence
Hydride Generation AAS
Movie not available
Te Amendments
Distribution of Te among supernatant
and collected solids in four duplicate bioreactor runs
Run
Distribution between solid/liq uid
Solid phase Te Solution phase Te
S.D.
(n=4 aliquots
from each r un)
1
2
3
4
Averag e
(4 runs)
42
18
33
43
34%
58
82
67
57
66%
% Recovery
of added Te
6.5
1.1
18.1
5.4
7.8%
107
84
111
87
97%
Anaerobic cultures of Pseudomonas fluorescens K27 were amended with 0.1 mM
sodium tellurite, maintained at 30°C for 92 h, and then 1) spun-down cells and solids
and 2) liquid medium were analyzed for tellurium by HGAAS.
Four samples harvested at the same time from each run were analyzed.
Se Amendments
Gas trapping efficiencies
Run
1
2
3
Averag e
Trap-1
Se µg
276.21
271.32
291.04
279.52
Trap-2
Se µg
25.58
23.84
24.74
24.72
Trap-3
Se µg
22.28
22.56
19.95
21.60
Total
Se µg
324.07
317.72
335.73
325.84
% Recovery
102.6
100.6
106.3
103.17
Se % recovery observed for 50% HNO3 trapping solution,
followed by ICP analysis. Se added as dimethyl diselenide to
Trap-1 then purged continuously for 24 h with N2, 50 mL/min.
Mass Balance of anaerobic, Se-amended bioreactors
1 mM of SeO32- (n=5 runs )
Phase
% Recovery (± SD)
Liqu id
66.68 (±18.29)
Soli d
32.44 (±19.81)
Gas
0.04 (±0.07)
Total Recove ry 99.16% (±0.62)
Phase
Liqu id
Soli d
Gas
10 mM of SeO32- (n=3)
% Recovery
92.17 (±8.13)
6.90 (±1.32)
0.004 (±0.002)
Total Recove ry 99.071 (±8.07)
Phase
Liqu id
Soli d
Gas
10 mM of SeO42- (n=3)
% Recovery
95.07 (±6.98)
0.73 (±0.06)
0.001 (±0.001)
Total Recove ry 95.80 ( ±6.93)
Strictly anaerobic (but N2 purged) 72 hour batch experiments with P. fluorescens
Does shifting between aerobic/anaerobic
growth effect Se0 production for K27?
Does shifting between aerobic/anaerobic
growth effect Se0 production for K27?
Alternate between anaerobic and aerobic growth.
Does shifting between aerobic/anaerobic
growth effect Se0 production for K27?
Alternate between anaerobic and aerobic growth.
Alternate N2 with air purging over relatively long times.
Does shifting between aerobic/anaerobic
growth effect Se0 production for K27?
Alternate between anaerobic and aerobic growth.
Alternate N2 with air purging over relatively long times.
Compare Se0 yield between anaerobic and aerobic runs.
Alternating anaerobic/aerobic purge cycles experiments with P. fluorescens
Phase
Liqu id
Soli d
Gas
1 mM of SeO32- (n=3 )
16 h N2 /8 h air@ 50 mL/mi n
% Recovery (± SD)
52.31 (±4.43)
37.58 (±7.99)
0.04 (±0.07)
Total Recove ry 89 .89% (±11.22)
Phase
Liqu id
Soli d
Gas
10 mM of SeO32- (n=3)
12 h N2/6 h air @50 mL/mi n
% Recovery
83.05 (± 3.04)
8.53 (±1.90)
0.002 (±0.001)
Total Recove ry 91 .58 (±4.43)
Phase
Liqu id
Soli d
Gas
1 mM of SeO32- (n= 3)
12 h N2/6 h air @ 50 mL/mi n
% Recovery
59.47(±19.65)
32.99(±18.71)
0.011(±0.014)
Total Recove ry 92 .50 (±0.99)
Phase
Liqu id
Soli d
Gas
1 mM of SeO32- (n= 3)
12 h N 2/6 h air @ 250 mL/min
% Recovery
45.759 (±10.80)
43.152 (±10.86)
NA due to air purge rate
Total Recove ry 88 .91 (±4.37)
Comparison of strictly
anaerobic to mixed
anaerobic/aerobic conditions
1 mM of SeO32- (n=5 runs )
Phase
% Recovery (± SD)
Liqu id
66.68 (±18.29)
Soli d
32.44 (±19.81)
Gas
0.04 (±0.07)
Total Recove ry 99.16% (±0.62)
Phase
Liqu id
Soli d
Gas
10 mM of SeO32- (n=3)
% Recovery
92.17 (±8.13)
6.90 (±1.32)
0.004 (±0.002)
Total Recove ry 99.071 (±8.07)
Phase
Liqu id
Soli d
Gas
10 mM of SeO42- (n=3)
% Recovery
95.07 (±6.98)
0.73 (±0.06)
0.001 (±0.001)
Total Recove ry 95.80 ( ±6.93)
1 mM of SeO32- (n=3 ) air 50 mL/mi n
Phase
% Recovery (± SD)
Liqu id
52.31 (±4.43)
Soli d
37.58 (±7.99)
Gas
0.04 (±0.07)
Total Recove ry 89 .89% (±11.22)
10 mM of SeO32- (n=3) air 50 mL/mi n
Phase
% Recovery
Liqu id
83.05 (± 3.04)
Soli d
8.53 (±1.90)
Gas
0.002 (±0.001)
Total Recove ry 91 .58 (±4.43)
1 mM of SeO32- (n= 3) air 50 mL/mi n
Phase
% Recovery
Liqu id
59.47(±19.65)
Soli d
32.99(±18.71)
Gas
0.011(±0.014)
Total Recove ry 92 .50 (±0.99)
1 mM of SeO32- (n= 3) air 250 mL/m in
Phase
% Recovery
Liqu id
45.759 (±10.80)
Soli d
43.152 (±10.86)
Gas
NA due to air purge rate
Total Recove ry 88 .91 (±4.37)
Alternating anaerobic/aerobic cycling in a 1 mM selenite amended culture of P.
fluorescens K27. The alternating cycles were 12 h N2 then 6 h air purging at 50 mL.
Alternating anaerobic/aerobic cycling in a 1 mM selenite-amended culture of P.
fluorescens K27. The alternating cycles were 12 h N2 then 6 h air purging at 250 mL.
72-hour Anaerobic Experiment
1 mM selenite amendment
Pseudomonas fluorescens K27
tryptic soy broth (with 3% nitrate), 30°C
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Acknowledgements
• Suminda Hapuarachchi and Jerry Swearingen Jr.
• Verena Van Fleet-Stalder
• Hakan Gürleyük, Rui Yu, Mehmet Akpolat
•
•
•
•
Robert A. Welch Foundation
SHSU Faculty Enhancement Grants
Ruth Hathaway/ACS Environmental Division
Richard Courtney “Cajun Support”
• Dr. John W. Birks above and beyond everyone else
Thank you John for 16 years of friendship, support, and love.