Chromate Bioremediation: Formation and
Fate of Organo-Cr(III) Complexes
Luying Xun1, Brent Peyton2, Sue Clark1 , Dave
Younge1
Washington State University1
Montana State University2
Common Valence States of Chromium
Primarily industrial process
Cr(VI)
Cr(III)
Natural
Bioremediation
Contaminant
Non-carcinogenic
Carcinogenic
Insoluble (pH 7)
Chromate, CrO42-
Trace element
Soluble (pH 7)
Most stable
Reactive
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.
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
FMN and FAD are well known enzyme cofactors
Riboflavin
vitamin B2
FMN: flavin
mononucleotide
FAD: flavin adenine
dinucleotide
Flavin Reductase (Fre) is Common in Cell
FMN and FAD
NADH + H+
H 2O 2
Fre
NAD+
O2
FMNH2 and FADH2
reduce metals, quinones
Cr(VI) Reduction rates by E. coli Fre
Flavin
Anaerobic Cr(VI)
Reduction
(mmol mg-1 min-1)
FAD
76.7 + 0.6
FMN
71.3 + 1.1
Riboflavin
96.5 + 6.4
Formation of Soluble Complexes after Cr(VI)
Reduction by Fre
Control
CrPO4
10 mM
25 mM
Organo-Cr(III)
Geoff Puzon
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
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
1In
the presence of organic ligands.
(End product)
Organo-Cr(III)
Organo-Cr(III)
Organo-Cr(III)
Organo-Cr(III)
Organo-Cr(III)
Organo-Cr(III)
N/A
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
Soluble Organo-Cr(III) end products
Control
No organic
GSH-Cr(III)
Serine-Cr(III)
Lactate-Cr(III)
Oxaloacetate-Cr(III)
Malate-Cr(III)
Cysteine-Cr(III)
Pyruvate-Cr(III)
Complex solubility
Organic ligand
Highly soluble organoCr(III) end products
Histidine
5.01 + 0.06
100%
Glutathione
4.76 + 0.15
95%
a-ketoglutarate
4.65 + 0.05
93%
Citrate
4.30 + 0.10
86%
Malate
3.88 + 0.04
78%
Serine
3.62 + 0.14
72%
Cysteine
3.43 + 0.10
69%
Pyruvate
3.25 + 0.17
65%
Oxaloacetate
2.86 + 0.05
57%
Slightly soluble organoCr(III) end products
Leucine
0.71 + 0.04
14%
Glycine
0.68 + 0.01
13%
Insoluble organo-Cr(III)
end products
Succinate
0.02 + 0.01
0.4%
Fumarate
< 0.01
0%
Lactate
< 0.01
0%
Tyrosine
< 0.01
0%
Acetate
< 0.01
0%
Ethanol
< 0.01
0%
100 mM KPi pH 7.0
< 0.01
0%
KPi-Cr(III) Control
Soluble Cr(III) (mM)
Percent soluble Cr(III)
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
0.3
Cysteine-Cr(III)
Absorbance
0.25
0.2
GSH-Cr(III)
Malate-Cr(III)
Serine-Cr(III)
0.15
Oxaloacetate-Cr(III)
0.1
Cr(NO3)3
0.05
0
410
460
510
560
610
Wavelength (nm)
660
710
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
Primarily industrial process
Cr(VI)
Inorganic Cr(III)
Bioremediation
Organo-Cr(III)
Microbial
activities
Mass balance of Cr after reduction by E. coli
250
Total Cr (In Supernatant)
Cr (mM)
200
150
Cr(VI)
100
50
0
0
1
2
3
4
5
6
7
8
Days
Geoff Puzon
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
Primarily industrial process
Cr(VI)
Cr(III)
Bioremediation
Organo-Cr(III)
Recalcitrant
Microbial
activities
Malate-Cr(III) is recalcitrant but not toxic
to R. eutropha JMP134
0.35
Malate + Malate-Cr(III)
0.3
Substrate:
2 mM
OD600nm
0.25
Malate
0.2
0.15
0.1
Malate-Cr(III)
0.05
0
0
20
40
60
80
100
120
Time (h)
Geoff Puzon
Primarily industrial process
Cr(VI)
Cr(III)
Bioremediation
Organo-Cr(III)
Recalcitrant
Microbial
activities
Negatively charged
Mobile in soil
Malate-Cr(III) moves through a soil column
Tracer Vs Malate-Cr(III) complex
NaBr: 10 ppm
Malate-Cr(III): 10 ppm
Cr(NO3)3: 10 ppm
Br- -tracer
1.2
1
0.8
C/C0
Malate-Cr(III)
Mobile phase: simulated groundwater
pH 7
Br tracer
0.6
Immobile phase: HanfordGWM
soilctrl
Malate-Cr(III) complex
0.4
0.2
Cr(NO3)3
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (h)
Ranjeet Tokala
Fate of NAD+-Cr(III)?
- Bacteria enriched with NAD+-Cr(III)
- Bacterial utilization – slow process
- Soluble Cr(III) decreased
PTX2
PTX1
Leifsonia sp.
Rhodococcus sp.
Geoff Puzon
Updated Biogeochemical Cycle of Cr
Primarily industrial process
Cr(VI)
Cr(III)
Bioremediation
Microbial
mineralization
Organo-Cr(III)
Recalcitrant
Microbial
reduction
Negatively charged
Mobile in soil
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)
Chromate Reduction by Flavin reductase (Fre)
Flavinred
NADH
O2
Cr(VI)
Fre
Cr(III)
+
NAD
Flavinox
H2O2