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D etermination of E lemental S elenium P roduction by a F acultative A naerobe G rown U nder S equential A naerobic/ A erobic C onditions Suminda Hapuarachchi, Jerry Swearingen, Jr, and Thomas G. Chasteen Department of Chemistry Sam Houston State University.

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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. ChasteenDepartment of ChemistrySam Houston State University



What happens to toxic metalloids bioprocessed by metalloid resistant bacteria1
What happens to toxic metalloids bioprocessed by metalloid-resistant bacteria?

Soluble forms remain in solution.


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria2
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 bacteria3
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
Phototropic Bacteria metalloid-resistant bacteria?


Se 0 and te 0 from strict anaerobes
Se metalloid-resistant bacteria?0 and Te0 from Strict Anaerobes


Headspace yield from 6 phototrophs
Headspace yield from 6 phototrophs metalloid-resistant bacteria?

  • 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
Dimethyl telluride the headspace above production by Pseudomonas fluorescens K27

Amended with TeO32-


Ch 3 3 sb production by k27 amended with an inorganic sb salt
(CH the headspace above 3)3Sb production by K27amended with an inorganic-Sb salt

Trimethyl stibine

Dimethyl disulfide

Dimethyl trisulfide

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?

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 among solid, liquid, and gas phases in bacterial cultures?

  • Temperature controlled


3 L bioreactor among solid, liquid, and gas phases in bacterial cultures?

  • Temperature controlled

additions

  • pH control

acid

base


3 L bioreactor among solid, liquid, and gas phases in bacterial cultures?

  • Temperature controlled

  • pH control

  • Dissolved Oxygen

gas

purge

N2/O2

D.O.probe


3 L bioreactor among solid, liquid, and gas phases in bacterial cultures?

  • Temperature controlled

  • pH control

  • Dissolved Oxygen

  • Nutrient addition


3 L bioreactor among solid, liquid, and gas phases in bacterial cultures?

  • Temperature controlled

  • pH control

  • Dissolved Oxygen

  • Nutrient addition

  • Gas harvest

bubbler(s)


3 L bioreactor among solid, liquid, and gas phases in bacterial cultures?

  • Temperature controlled

  • pH control

  • Dissolved Oxygen

  • Nutrient addition

  • Gas harvest

  • Liquid harvest


Bacterial among solid, liquid, and gas phases in bacterial cultures?

Culture Conditions


Bacterial among solid, liquid, and gas phases in bacterial cultures?

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 among solid, liquid, and gas phases in bacterial cultures?

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 among solid, liquid, and gas phases in bacterial cultures?

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 among solid, liquid, and gas phases in bacterial cultures?

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


S among solid, liquid, and gas phases in bacterial cultures?e

Determination

Liquid phase selenium

Inductively coupled plasma spectrometry (ICP)


S among solid, liquid, and gas phases in bacterial cultures?e

Determination

Liquid phase selenium

Inductively coupled plasma spectrometry (ICP)

Solid phase selenium (Se0 and cells)

ICP following centrifugation and dissolution with HNO3


S among solid, liquid, and gas phases in bacterial cultures?e

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


ICP among solid, liquid, and gas phases in bacterial cultures?

Analysis

Simultaneous ICP


T among solid, liquid, and gas phases in bacterial cultures?e

Determination

Liquid phase tellurium

Hydride generation atomic absorption spectrometry (HGAAS)


T among solid, liquid, and gas phases in bacterial cultures?e

Determination

Liquid phase tellurium

Hydride generation atomic absorption spectrometry (HGAAS)

Solid phase tellurium (Te0 and cells)

HGAAS following centrifugation and dissolution with HNO3


T among solid, liquid, and gas phases in bacterial cultures?e

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
Hydride Generation AAS among solid, liquid, and gas phases in bacterial cultures?Movie not available


Te amendments
Te Amendments among solid, liquid, and gas phases in bacterial cultures?


Distribution of Te among supernatant among solid, liquid, and gas phases in bacterial cultures?and collected solids in four duplicate bioreactor runs

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
Se Amendments among solid, liquid, and gas phases in bacterial cultures?


Gas trapping efficiencies
Gas trapping efficiencies among solid, liquid, and gas phases in bacterial cultures?

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 n 2 purged 72 hour batch experiments with p fluorescens

Mass Balance of anaerobic, Se-amended bioreactors among solid, liquid, and gas phases in bacterial cultures?

Strictly anaerobic (but N2 purged) 72 hour batch experiments with P. fluorescens


Does shifting between aerobic anaerobic growth effect se 0 production for k27
Does shifting between aerobic/anaerobic growth effect Se among solid, liquid, and gas phases in bacterial cultures?0 production for K27?


Does shifting between aerobic anaerobic growth effect se 0 production for k271
Does shifting between aerobic/anaerobic growth effect Se among solid, liquid, and gas phases in bacterial cultures?0 production for K27?

Alternate between anaerobic and aerobic growth.


Does shifting between aerobic anaerobic growth effect se 0 production for k272
Does shifting between aerobic/anaerobic growth effect Se among solid, liquid, and gas phases in bacterial cultures?0 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 se 0 production for k273
Does shifting between aerobic/anaerobic growth effect Se among solid, liquid, and gas phases in bacterial cultures?0 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
Alternating anaerobic/aerobic purge cycles experiments with among solid, liquid, and gas phases in bacterial cultures?P. fluorescens



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
72-hour Anaerobic Experiment selenite-amended culture of

1 mM selenite amendmentPseudomonas fluorescens K27tryptic soy broth (with 3% nitrate), 30°C

QuickTime Time Lapse Movie

Movie not available


1 hour selenite-amended culture of


72 hours selenite-amended culture of


1 hour selenite-amended culture of

72 hours


Acknowledgements
Acknowledgements selenite-amended culture of

  • 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.


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