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

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

Se 0 and Te 0 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 SeO 3 2 -

Dimethyl telluride production by Pseudomonas fluorescens K27 Amended with TeO 3 2-

(CH 3 ) 3 Sb 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.

3 L bioreactor Temperature controlled

3 L bioreactor Temperature controlled pH control acidbase additions

3 L bioreactor Temperature controlled pH control Dissolved Oxygen D.O.probe gas purge N 2 /O 2

3 L bioreactor Temperature controlled pH control Dissolved Oxygen Nutrient addition

3 L bioreactor Temperature controlled pH control Dissolved Oxygen Nutrient addition Gas harvest bubbler(s)

3 L bioreactor Temperature controlled pH control Dissolved Oxygen Nutrient addition Liquid harvest Gas 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 SeO 4 2- or SeO 3 2- 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 SeO 4 2- or SeO 3 2- along with 10%/vol. inoculum Tellurium Amendments 0.01 to 1 mM TeO 4 2- or TeO 3 2- 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 SeO 4 2- or SeO 3 2- along with 10%/vol. inoculum Tellurium Amendments 0.01 to 1 mM TeO 4 2- or TeO 3 2- along with 10%/vol. inoculum Batch cultures at 30˚C 15 hr to 72 hr bacterial cultures; ~ 3 L liquid volume

S e Determination Liquid phase selenium Inductively coupled plasma spectrometry (ICP)

S e Determination Liquid phase selenium Inductively coupled plasma spectrometry (ICP) Solid phase selenium (Se 0 and cells) ICP following centrifugation and dissolution with HNO 3

S e Determination Liquid phase selenium Inductively coupled plasma spectrometry (ICP) Solid phase selenium (Se 0 and cells) ICP following centrifugation and dissolution with HNO 3 Gas phase selenium (volatile organo-Se compounds) Species identified via GC/fluorine-induced chemiluminescence Trapping in serial HNO 3 bubblers Analysis via ICP

Simultaneous ICP ICP Analysis

T e Determination Liquid phase tellurium Hydride generation atomic absorption spectrometry (HGAAS)

T e Determination Liquid phase tellurium Hydride generation atomic absorption spectrometry (HGAAS) Solid phase tellurium (Te 0 and cells) HGAAS following centrifugation and dissolution with HNO 3

T e Determination Liquid phase tellurium Hydride generation atomic absorption spectrometry (HGAAS) Solid phase tellurium (Te 0 and cells) HGAAS following centrifugation and dissolution with HNO 3 Gas phase tellurium Capillary gas chromatography/F 2 -induced chemiluminescence

Hydride Generation AAS Movie not available

Te Amendments

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

Gas trapping efficiencies Se % recovery observed for 50% HNO 3 trapping solution, followed by ICP analysis. Se added as dimethyl diselenide to Trap-1 then purged continuously for 24 h with N 2, 50 mL/min.

Strictly anaerobic (but N 2 purged) 72 hour batch experiments with P. fluorescens Mass Balance of anaerobic, Se-amended bioreactors

Does shifting between aerobic/anaerobic growth effect Se 0 production for K27?

Alternate between anaerobic and aerobic growth.

Does shifting between aerobic/anaerobic growth effect Se 0 production for K27? Alternate between anaerobic and aerobic growth. Alternate N 2 with air purging over relatively long times.

Does shifting between aerobic/anaerobic growth effect Se 0 production for K27? Compare Se 0 yield between anaerobic and aerobic runs. Alternate between anaerobic and aerobic growth. Alternate N 2 with air purging over relatively long times.

Alternating anaerobic/aerobic purge cycles experiments with P. fluorescens

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 N 2 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 N 2 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 QuickTime Time Lapse Movie Movie not available

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.