Chromate Bioremediation: Formation and Fate of OrganoCrIII Complexes Luying Xun1, Brent Peyton2, Sue

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Chromate Bioremediation Formation and Fate of
Organo-Cr(III) ComplexesLuying Xun1, Brent
Peyton2, Sue Clark1 , Dave Younge1 Washington
State University1 Montana State University2
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Common Valence States of Chromium
Natural
Contaminant
Non-carcinogenic
Carcinogenic
Insoluble (pH 7)
Chromate, CrO42-
Trace element
Soluble (pH 7)
Reactive
Most stable
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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.
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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
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FMN and FAD are well known enzyme cofactors
Riboflavin vitamin B2 FMN flavin
mononucleotide FAD flavin adenine dinucleotide
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Flavin Reductase (Fre) is Common in Cell
NADH H
H2O2
Fre
O2
NAD
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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
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Formation of Soluble Complexes after Cr(VI)
Reduction by Fre
Organo-Cr(III)
CrPO4
Geoff Puzon
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The Product is NAD-Cr(III) Complex

• - NADCr(III) ratio is 21
• Identified as a polymer by using
• Dialysis
• Size Exclusion Chromatography
• Electron Paramagnetic Resonance

Geoff Puzon
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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.
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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
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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)
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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)
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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 27639-645.
Zhicheng Zhang
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Cr(VI)
Inorganic Cr(III)
Organo-Cr(III)
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Mass balance of Cr after reduction by E. coli
Total Cr (In Supernatant)
Cr (mM)
Cr(VI)
Days
Geoff Puzon
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Formation of both soluble and insoluble Cr(III)
from Cr(VI) reduction
Initial Cr(VI) concentration is 4 ppm
Ranjeet Tokala
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Cr(VI)
Cr(III)
Organo-Cr(III)
Recalcitrant
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Malate-Cr(III) is recalcitrant but not toxic to
R. eutropha JMP134
Substrate 2 mM
Geoff Puzon
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Cr(VI)
Cr(III)
Organo-Cr(III)
Recalcitrant
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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
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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
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Updated Biogeochemical Cycle of Cr
Cr(VI)
Cr(III)
Organo-Cr(III)
Recalcitrant
Negatively charged Mobile in soil
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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)
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Chromate Reduction by Flavin reductase (Fre)