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

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What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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 bacteria

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


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


Se 0 and te 0 from strict anaerobes

Se0 and Te0 from Strict Anaerobes


Headspace yield from 6 phototrophs

Headspace yield from 6 phototrophs

  • dimethyl sulfide

  • dimethyl selenide

  • dimethyl diselenide

  • (also dimethyl selenenyl sulfide)


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

The fluorine-induced chemiluminescence GC chromatogram of the headspace above Se-resistant bacteria.

Amended with SeO32-


Dimethyl telluride production by pseudomonas fluorescens k27

Dimethyl tellurideproduction by Pseudomonas fluorescens K27

Amended with TeO32-


Ch 3 3 sb production by k27 amended with an inorganic sb salt

(CH3)3Sb production by K27amended with an inorganic-Sb salt

Trimethyl stibine

Dimethyl disulfide

Dimethyl trisulfide

Methanethiol

Dimethyl sulfide

Amended with KSb(OH)6


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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.


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

3 L bioreactor

  • Temperature controlled


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

3 L bioreactor

  • Temperature controlled

additions

  • pH control

acid

base


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

3 L bioreactor

  • Temperature controlled

  • pH control

  • Dissolved Oxygen

gas

purge

N2/O2

D.O.probe


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

3 L bioreactor

  • Temperature controlled

  • pH control

  • Dissolved Oxygen

  • Nutrient addition


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

3 L bioreactor

  • Temperature controlled

  • pH control

  • Dissolved Oxygen

  • Nutrient addition

  • Gas harvest

bubbler(s)


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

3 L bioreactor

  • Temperature controlled

  • pH control

  • Dissolved Oxygen

  • Nutrient addition

  • Gas harvest

  • Liquid harvest


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

Bacterial

Culture Conditions


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

Se

Determination

Liquid phase selenium

Inductively coupled plasma spectrometry (ICP)


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

Se

Determination

Liquid phase selenium

Inductively coupled plasma spectrometry (ICP)

Solid phase selenium (Se0 and cells)

ICP following centrifugation and dissolution with HNO3


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

ICP

Analysis

Simultaneous ICP


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

Te

Determination

Liquid phase tellurium

Hydride generation atomic absorption spectrometry (HGAAS)


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

Te

Determination

Liquid phase tellurium

Hydride generation atomic absorption spectrometry (HGAAS)

Solid phase tellurium (Te0 and cells)

HGAAS following centrifugation and dissolution with HNO3


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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

Hydride Generation AASMovie not available


Te amendments

Te Amendments


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

Distribution of Te among supernatant 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


Gas trapping efficiencies

Gas trapping efficiencies

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

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 Se0 production for K27?


Does shifting between aerobic anaerobic growth effect se 0 production for k271

Does shifting between aerobic/anaerobic growth effect Se0 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 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 se 0 production for k273

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

Alternating anaerobic/aerobic purge cycles experiments with P. fluorescens


Comparison of strictly anaerobic to mixed anaerobic aerobic conditions

Comparison of strictly anaerobic to mixed anaerobic/aerobic conditions


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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.


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

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

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

QuickTime Time Lapse Movie

Movie not available


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

1 hour


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

72 hours


What happens to toxic metalloids bioprocessed by metalloid resistant bacteria

1 hour

72 hours


Acknowledgements

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.


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