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Determination of Elemental Selenium Production by a Facultative Anaerobe Grown Under Sequential Anaerobic/Aerobic ConditionsSuminda Hapuarachchi,Jerry Swearingen, Jr, andThomas G. Chasteen
Department of ChemistrySam Houston State University
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What happens to toxic metalloids bioprocessed by metalloid-resistant bacteria?
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What happens to toxic metalloids bioprocessed by metalloid-resistant bacteria?
Soluble forms remain in solution.
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What happens to toxic metalloids bioprocessed by metalloid-resistant bacteria?
Soluble forms remain in solution.
Bioreduction also produces methylated, volatile forms.
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What happens to toxic metalloids bioprocessed by metalloid-resistant bacteria?
Metalloids are converted to elemental (solid) form.
Soluble forms remain in solution.
Bioreduction also produces methylated, volatile forms.
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Phototropic Bacteria
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Se0 and Te0 from Strict Anaerobes
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Headspace yield from 6 phototrophs
•dimethyl sulfide•dimethyl selenide•dimethyl diselenide(also dimethyl selenenyl sulfide)
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The fluorine-induced chemiluminescence GC chromatogram of the headspace above Se-resistant bacteria.
Amended with SeO32-
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Dimethyl tellurideproduction by Pseudomonas
fluorescens K27
a
DMTe
Amended with TeO32-
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(CH3)3Sb production by K27
amended with an inorganic-Sb salt
Dimethyl disulfide
Dimethyl trisulfide
Trimethyl stibine
Methanethiol
Dimethyl sulfide
Amended with KSb(OH)6
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Can a mass balance be determined for metalloids distributed among solid, liquid, and gas phases in bacterial cultures?
Determine metalloid content in each phase.
Use 3 L batch cultures amended with Se oxyanions.
Incubate culture far into the stationary phases.
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3 L bioreactor• Temperature controlled
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3 L bioreactor• Temperature controlled
• pH controlacid base
additions
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3 L bioreactor• Temperature controlled
• pH control
• Dissolved Oxygen
D.O.probe
gas purgeN2/O2
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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
• Gas harvest
bubbler(s)
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3 L bioreactor• Temperature controlled
• pH control
• Dissolved Oxygen
• Nutrient addition
• Liquid harvest
• Gas harvest
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BacterialCulture Conditions
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BacterialCulture Conditions
Pseudomonas fluorescens K27Isolated by Ray Fall at CU BoulderFacultative anaerobe (grows with or without oxygen)Grown on tryptic soy broth with 3% nitrate added
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BacterialCulture Conditions
Pseudomonas fluorescens K27Isolated by Ray Fall at CU BoulderFacultative anaerobe (grows with or without oxygen)Grown on tryptic soy broth with 3% nitrate added
Selenium Amendments1–10 mM SeO4
2- or SeO32- along with 10%/vol. inoculum
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BacterialCulture Conditions
Pseudomonas fluorescens K27Isolated by Ray Fall at CU BoulderFacultative anaerobe (grows with or without oxygen)Grown on tryptic soy broth with 3% nitrate added
Selenium Amendments1–10 mM SeO4
2- or SeO32- along with 10%/vol. inoculum
Tellurium Amendments0.01 to 1 mM TeO4
2- or TeO32- along with 10%/vol. inoculum
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BacterialCulture Conditions
Pseudomonas fluorescens K27Isolated by Ray Fall at CU BoulderFacultative anaerobe (grows with or without oxygen)Grown on tryptic soy broth with 3% nitrate added
Selenium Amendments1–10 mM SeO4
2- or SeO32- along with 10%/vol. inoculum
Tellurium Amendments0.01 to 1 mM TeO4
2- or TeO32- along with 10%/vol. inoculum
Batch cultures at 30˚C15 hr to 72 hr bacterial cultures; ~ 3 L liquid volume
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SeDetermination
Liquid phase selenium
Inductively coupled plasma spectrometry (ICP)
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SeDetermination
Liquid phase selenium
Inductively coupled plasma spectrometry (ICP)
Solid phase selenium (Se0 and cells)
ICP following centrifugation and dissolution with HNO3
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SeDetermination
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
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Simultaneous ICP ICPAnalysis
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TeDetermination
Liquid phase tellurium
Hydride generation atomic absorption spectrometry (HGAAS)
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TeDetermination
Liquid phase tellurium
Hydride generation atomic absorption spectrometry (HGAAS)
Solid phase tellurium (Te0 and cells)
HGAAS following centrifugation and dissolution with HNO3
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TeDetermination
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
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Hydride Generation AAS Movie not available
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Te Amendments
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Distribution of Te among supernatant and collected solids in four duplicate bioreactor runs
Distribution between solid/liquidRun Solid phase Te Solution phase Te S.D.
(n=4 aliquotsfrom each run)
% Recoveryof added Te
1 42 58 6.5 1072 18 82 1.1 843 33 67 18.1 1114 43 57 5.4 87Average(4 runs)
34% 66% 7.8% 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.
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Se Amendments
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Gas trapping efficiencies
Run Trap-1 Trap-2 Trap-3 Total % RecoverySe µg Se µg Se µg Se µg
1 276.21 25.58 22.28 324.07 102.62 271.32 23.84 22.56 317.72 100.63 291.04 24.74 19.95 335.73 106.3Average 279.52 24.72 21.60 325.84 103.17
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.
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Strictly anaerobic (but N2 purged) 72 hour batch experiments with P. fluorescens
1 mM of SeO32- (n=5 runs )
Phase % Recovery (± SD)Liquid 66.68 (±18.29)Solid 32.44 (±19.81)Gas 0.04 (±0.07)
Total Recovery 99.16% (±0.62)
10 mM of SeO32- (n=3)
Phase % RecoveryLiquid 92.17 (±8.13)Solid 6.90 (±1.32)Gas 0.004 (±0.002)
Total Recovery 99.071 (±8.07)
10 mM of SeO42- (n=3)
Phase % RecoveryLiquid 95.07 (±6.98)Solid 0.73 (±0.06)Gas 0.001 (±0.001)
Total Recovery 95.80 (±6.93)
Mass Balance of anaerobic, Se-amended bioreactors
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Does shifting between aerobic/anaerobic growth effect Se0 production for K27?
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Does shifting between aerobic/anaerobic growth effect Se0 production for K27?
Alternate between anaerobic and aerobic growth.
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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.
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Does shifting between aerobic/anaerobic growth effect Se0 production for K27?
Compare Se0 yield between anaerobic and aerobic runs.
Alternate between anaerobic and aerobic growth.
Alternate N2 with air purging over relatively long times.
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Alternating anaerobic/aerobic purge cycles experiments with P. fluorescens
1 mM of SeO32- (n=3 ) 16 h N2 /8 h air@ 50 mL/min
Phase % Recovery (± SD)Liquid 52.31 (±4.43)Solid 37.58 (±7.99)Gas 0.04 (±0.07)
Total Recovery 89.89% (±11.22)10 mM of SeO3
2- (n=3) 12 h N2/6 h air @50 mL/minPhase % RecoveryLiquid 83.05 (± 3.04)Solid 8.53 (±1.90)Gas 0.002 (±0.001)
Total Recovery 91.58 (±4.43)1 mM of SeO3
2- (n= 3) 12 h N2/6 h air @ 50 mL/minPhase % RecoveryLiquid 59.47(±19.65)Solid 32.99(±18.71)Gas 0.011(±0.014)
Total Recovery 92.50 (±0.99)1 mM of SeO3
2- (n= 3) 12 h N2/6 h air @ 250 mL/minPhase % RecoveryLiquid 45.759 (±10.80)Solid 43.152 (±10.86)Gas NA due to air purge rate
Total Recovery 88.91 (±4.37)
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Comparison of strictly anaerobic to mixed
anaerobic/aerobic conditions
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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.
-20
0
20
40
60
80
100
0 10 20 30 40 50 60 70 80
1 mM selenite amended K27 culture
Dissolved Oxygen (% Saturation)
Time (hours)
N
2
N
2
N
2
N
2
AirAirAirAir
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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.
-20
0
20
40
60
80
100
120
140
0 10 20 30 40 50 60 70 80
1 mM selenite amended K27 culture
Dissolved Oxygen (% Saturation)
Time (hours)
N
2
N
2
N
2
N
2
AirAirAirAir
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72-hour Anaerobic Experiment
1 mM selenite amendmentPseudomonas fluorescens K27tryptic 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 elseThank you John for 16 years of friendship, support, and love.