1. Chromate Bioremediation: Formation and Fate of Organo-Cr(III) Complexes Luying Xun1, Brent Peyton2, Sue Clark1 , Dave Younge1 Washington State University1 Montana State University2

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

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

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

7. Cr(VI) Reduction rates by E. coli Fre
Anaerobic Cr(VI) Reduction
(mmol mg-1 min-1)
Flavin
FAD
FMN
Riboflavin

8. Formation of Soluble Complexes after Cr(VI) Reduction by Fre
Control
10 mM
25 mM
CrPO4
Organo-Cr(III)
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
Fortuitous reduction by:
Glutathione
Ascorbate (Vit. C)
H2S or Fe(II)1
Quinone reductase
Flavin reductase
Cytochrome C
Hydrogenase
(End product)
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
Serine-Cr(III)
GSH-Cr(III)
Lactate-Cr(III)
Malate-Cr(III)
Cysteine-Cr(III)
Oxaloacetate-Cr(III)
Pyruvate-Cr(III)

13. Complex solubility
Organic ligand
Soluble Cr(III) (mM)
Percent soluble Cr(III)
Highly soluble organo-Cr(III) end products
Histidine
100%
Glutathione
95%
a-ketoglutarate
93%
Citrate
86%
Malate
78%
Serine
72%
Cysteine
69%
Pyruvate
65%
Oxaloacetate
57%
Slightly soluble organo-Cr(III) end products
Leucine
14%
Glycine
13%
Insoluble organo-Cr(III) end products
Succinate
0.4%
Fumarate
< 0.01 0%
Lactate
Tyrosine
Acetate
Ethanol
KPi-Cr(III) Control
100 mM KPi pH 7.0

14. Absorbance Spectra Absorbance Wavelength (nm) 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)
Malate-Cr(III)
GSH-Cr(III)
Serine-Cr(III)
Absorbance
Oxaloacetate-Cr(III)
Cr(NO3)3
Wavelength (nm)

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

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

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

18. Formation of both soluble and insoluble Cr(III) from Cr(VI) reduction
Bacteria
Soluble Cr(III)(ppm)
Insoluble Cr(III)(ppm)
Cellulomonas sp. ES6
4.12  0.02
0.49  0.01
S. oneidensis MR1
3.44  0.06
2.22  0.13
Ps. putida MK1
3.01  0.30
1.61  0.30
Ps. aeruginosa PAO1
3.17  0.01
1.71  0.01
D. vulgaris Hildenborough
1.25  0.30
2.60  0.44
D. desulfurreducens G20
3.18  0.30
1.84  0.20
Leafsonia sp.
2.02  0.06
2.55  0.04
Rhodococcus sp.
2.70  0.09
1.84  0.02
Initial Cr(VI) concentration is 4 ppm
Ranjeet Tokala

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

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

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

22. Malate-Cr(III) moves through a soil column
Br -tracer
Malate-Cr(III)
Cr(NO3)3 -
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

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

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

25. Financial supports 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)

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