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

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?

Metalloids are converted to elemental (solid) form.

Soluble forms remain in solution.

Bioreduction also produces methylated, volatile forms.

Phototropic Bacteria

Se0 and Te0 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 tellurideproduction by Pseudomonas

fluorescens K27

a

DMTe

Amended with TeO32-

(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

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

BacterialCulture Conditions

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

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

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

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

SeDetermination

Liquid phase selenium

Inductively coupled plasma spectrometry (ICP)

SeDetermination

Liquid phase selenium

Inductively coupled plasma spectrometry (ICP)

Solid phase selenium (Se0 and cells)

ICP following centrifugation and dissolution with HNO3

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

Simultaneous ICP ICPAnalysis

TeDetermination

Liquid phase tellurium

Hydride generation atomic absorption spectrometry (HGAAS)

TeDetermination

Liquid phase tellurium

Hydride generation atomic absorption spectrometry (HGAAS)

Solid phase tellurium (Te0 and cells)

HGAAS following centrifugation and dissolution with HNO3

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

Hydride Generation AAS Movie not available

Te Amendments

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.

Se Amendments

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.

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

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?

Compare Se0 yield between anaerobic and aerobic runs.

Alternate between anaerobic and aerobic growth.

Alternate N2 with air purging over relatively long times.

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)

Comparison of strictly anaerobic to mixed

anaerobic/aerobic conditions

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.

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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

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AirAirAirAir

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.

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0 10 20 30 40 50 60 70 80

1 mM selenite amended K27 culture

Dissolved Oxygen (% Saturation)

Time (hours)

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AirAirAirAir

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.