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

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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  1. 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

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

  3. What happens to toxic metalloids bioprocessed by metalloid-resistant bacteria? Soluble forms remain in solution.

  4. What happens to toxic metalloids bioprocessed by metalloid-resistant bacteria? Soluble forms remain in solution. Bioreduction also produces methylated, volatile forms.

  5. 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.

  6. Phototropic Bacteria

  7. Se0 and Te0 from Strict Anaerobes

  8. Headspace yield from 6 phototrophs • dimethyl sulfide • dimethyl selenide • dimethyl diselenide • (also dimethyl selenenyl sulfide)

  9. The fluorine-induced chemiluminescence GC chromatogram of the headspace above Se-resistant bacteria. Amended with SeO32-

  10. Dimethyl tellurideproduction by Pseudomonas fluorescens K27 Amended with TeO32-

  11. (CH3)3Sb production by K27amended with an inorganic-Sb salt Trimethyl stibine Dimethyl disulfide Dimethyl trisulfide Methanethiol Dimethyl sulfide Amended with KSb(OH)6

  12. 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.

  13. 3 L bioreactor • Temperature controlled

  14. 3 L bioreactor • Temperature controlled additions • pH control acid base

  15. 3 L bioreactor • Temperature controlled • pH control • Dissolved Oxygen gas purge N2/O2 D.O.probe

  16. 3 L bioreactor • Temperature controlled • pH control • Dissolved Oxygen • Nutrient addition

  17. 3 L bioreactor • Temperature controlled • pH control • Dissolved Oxygen • Nutrient addition • Gas harvest bubbler(s)

  18. 3 L bioreactor • Temperature controlled • pH control • Dissolved Oxygen • Nutrient addition • Gas harvest • Liquid harvest

  19. Bacterial Culture Conditions

  20. 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

  21. 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

  22. 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

  23. 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

  24. Se Determination Liquid phase selenium Inductively coupled plasma spectrometry (ICP)

  25. Se Determination Liquid phase selenium Inductively coupled plasma spectrometry (ICP) Solid phase selenium (Se0 and cells) ICP following centrifugation and dissolution with HNO3

  26. 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

  27. ICP Analysis Simultaneous ICP

  28. Te Determination Liquid phase tellurium Hydride generation atomic absorption spectrometry (HGAAS)

  29. Te Determination Liquid phase tellurium Hydride generation atomic absorption spectrometry (HGAAS) Solid phase tellurium (Te0 and cells) HGAAS following centrifugation and dissolution with HNO3

  30. 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

  31. Hydride Generation AASMovie not available

  32. Te Amendments

  33. 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.

  34. Se Amendments

  35. 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.

  36. Mass Balance of anaerobic, Se-amended bioreactors Strictly anaerobic (but N2 purged) 72 hour batch experiments with P. fluorescens

  37. Does shifting between aerobic/anaerobic growth effect Se0 production for K27?

  38. Does shifting between aerobic/anaerobic growth effect Se0 production for K27? Alternate between anaerobic and aerobic growth.

  39. 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.

  40. 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.

  41. Alternating anaerobic/aerobic purge cycles experiments with P. fluorescens

  42. Comparison of strictly anaerobic to mixed anaerobic/aerobic conditions

  43. 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.

  44. 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.

  45. 72-hour Anaerobic Experiment 1 mM selenite amendmentPseudomonas fluorescens K27tryptic soy broth (with 3% nitrate), 30°C QuickTime Time Lapse Movie Movie not available

  46. 1 hour

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