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Common Valence States of Chromium

Chromate Bioremediation: Formation and Fate of Organo-Cr(III) Complexes Luying Xun 1 , Brent Peyton 2 , Sue Clark 1 , Dave Younge 1 Washington State University 1 Montana State University 2. Cr(VI). Cr(III). Bioremediation. Primarily industrial process. Common Valence States of Chromium.

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Common Valence States of Chromium

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  1. Chromate Bioremediation: Formation and Fate of Organo-Cr(III) ComplexesLuying Xun1, Brent Peyton2, Sue Clark1 ,Dave Younge1Washington State University1Montana State University2

  2. Cr(VI) Cr(III) Bioremediation Primarily industrial process Common Valence States of Chromium Natural Contaminant Non-carcinogenic Carcinogenic Insoluble (pH 7) Chromate, CrO42- Trace element Soluble (pH 7) Reactive Most stable

  3. Many microorganisms can reduce Cr(VI) Examples: Shewanella spp. Geobacter spp. Desulfovibrio spp. Deinococcus radiodurans Cellulomonas spp. Enterobacter spp. Pseudomonas spp. Escherichia coli Streptomyces spp. Fungi and more.

  4. Mechanisms of Chromate Reduction Fortuitous reduction by: • Glutathione 1 • Ascorbate (Vit. C) 1 • H2S or Fe(II) 1 • Flavin reductase • Quinone reductase 1 • Cytochrome C 1 • Hydrogenase 1 Couple to anaerobic respiration 1 • Possible, but only one report 1From literature

  5. FMN and FAD are well known enzyme cofactors Riboflavin vitamin B2 FMN: flavin mononucleotide FAD: flavin adenine dinucleotide

  6. FMN and FAD FMNH2 and FADH2 reduce metals, quinones Flavin Reductase (Fre) is Common in Cell NADH + H+ H2O2 Fre O2 NAD+

  7. Cr(VI) Reduction rates by E. coli Fre Anaerobic Cr(VI) Reduction (mmol mg-1 min-1) Flavin 76.7 + 0.6 FAD 71.3 + 1.1 FMN Riboflavin 96.5 + 6.4

  8. Control 10 mM 25 mM Formation of Soluble Complexes after Cr(VI) Reduction by Fre Organo-Cr(III) CrPO4 Geoff Puzon

  9. The Product is NAD+-Cr(III) Complex • - NAD+:Cr(III) ratio is 2:1 • Identified as a polymer by using • Dialysis • Size Exclusion Chromatography • Electron Paramagnetic Resonance Geoff Puzon

  10. Organo-Cr(III) production is common (End product) Fortuitous reduction by: • Glutathione • Ascorbate (Vit. C) • H2S or Fe(II)1 • Quinone reductase • Flavin reductase • Cytochrome C • Hydrogenase Organo-Cr(III) Organo-Cr(III) Organo-Cr(III) Organo-Cr(III) Organo-Cr(III) Organo-Cr(III) N/A 1In the presence of organic ligands.

  11. Hypothesis: Organo-Cr(III) is readily formed during Cr(VI) reduction in the presence of organics Experiments: Control 5 mM Cr(VI) 10 mM dithionite 50 mM KPi (pH 7) Cr(III) precipitates With selected metabolites 5 mM Cr(VI) 10 mM dithionite 50 mM KPi (pH 7) Organo-Cr(III) Geoff Puzon

  12. Soluble Organo-Cr(III) end products Control No organic GSH-Cr(III) Serine-Cr(III) Lactate-Cr(III) Malate-Cr(III) Cysteine-Cr(III) Oxaloacetate-Cr(III) Pyruvate-Cr(III)

  13. Absorbance Spectra Peak Absorbance Cr(NO3)3= 579nm Cys-Cr(III)= 584nm Mal-Cr(III)= 595nm Ser-Cr(III)= 600nm GSH-Cr(III)= 604nm Ox-Cr(III)= 607nm Cysteine-Cr(III) GSH-Cr(III) Malate-Cr(III) Serine-Cr(III) Absorbance Oxaloacetate-Cr(III) Cr(NO3)3 Wavelength (nm)

  14. Cr(III)-DNA Adducts are Formed from Cr(VI) Reduction The adducts block DNA polymerase. Proposed Cr(III)-DNA adducts. Arakawa et al. 2005. Carcinogenesis 27:639-645. Zhicheng Zhang

  15. Microbial activities Bioremediation Primarily industrial process Cr(VI) Inorganic Cr(III) Organo-Cr(III)

  16. Mass balance of Cr after reduction by E. coli Total Cr (In Supernatant) Cr (mM) Cr(VI) Days Geoff Puzon

  17. Formation of both soluble and insoluble Cr(III) from Cr(VI) reduction Initial Cr(VI) concentration is 4 ppm Ranjeet Tokala

  18. Microbial activities Bioremediation Primarily industrial process Cr(VI) Cr(III) Organo-Cr(III) Recalcitrant

  19. Malate + Malate-Cr(III) Malate Malate-Cr(III) Malate-Cr(III) is recalcitrant but not toxic to R. eutropha JMP134 Substrate: 2 mM Geoff Puzon

  20. Microbial activities Bioremediation Primarily industrial process Negatively charged Mobile in soil Cr(VI) Cr(III) Organo-Cr(III) Recalcitrant

  21. - Br -tracer Malate-Cr(III) Cr(NO3)3 Malate-Cr(III) moves through a soil column NaBr: 10 ppm Malate-Cr(III): 10 ppm Cr(NO3)3: 10 ppm Mobile phase: simulated groundwater pH 7 Immobile phase: Hanford soil Ranjeet Tokala

  22. PTX2 PTX1 Fate of NAD+-Cr(III)? - Bacteria enriched with NAD+-Cr(III) - Bacterial utilization – slow process - Soluble Cr(III) decreased Leifsonia sp. Rhodococcus sp. Geoff Puzon

  23. Microbial Microbial mineralization reduction Bioremediation Primarily industrial process Updated Biogeochemical Cycle of Cr Cr(VI) Cr(III) Organo-Cr(III) Recalcitrant Negatively charged Mobile in soil

  24. ACKNOWLEDGMENTS Dr. Geoff Puzon – organo-Cr(III)/enzyme, recalcitrance, and mineralization Dr. Ranjeet Tokala – organo-Cr(III)/cell and soil columns Zhicheng Zhang – organo-Cr(III) characterization Financial supports Department of Energy ERSD (NABIR)

  25. Flavin NADH O red 2 Cr(VI) Fre Cr(III) + Flavin H O NAD ox 2 2 Chromate Reduction by Flavin reductase (Fre)

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