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

Natural

Non-carcinogenic

Insoluble (pH 7)

Trace element

Chromate, CrO42-

Contaminant

Carcinogenic

Soluble (pH 7)

Cr(III) Cr(VI)Bioremediation

Primarily industrial process

ReactiveMost stable

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

1From literature

Couple to anaerobic respiration 1 – Possible, but only one report

Riboflavin vitamin B2

FMN: flavin mononucleotide

FAD: flavin adeninedinucleotide

FMN and FAD are well known enzyme cofactors

FMNH2 and FADH2

FMN and FAD

NAD+

NADH + H+

O2

H2O2

Fre

Flavin Reductase (Fre) is Common in Cell

reduce metals, quinones

Cr(VI) Reduction rates by E. coli Fre

96.5 + 6.4Riboflavin

71.3 + 1.1FMN

76.7 + 0.6FAD

Anaerobic Cr(VI) Reduction

(mol mg-1 min-1)

Flavin

Formation of Soluble Complexes after Cr(VI) Reduction by Fre

Control 10 mM 25 mM

CrPO4 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.

Organo-Cr(III)

(End product)

Organo-Cr(III)

Organo-Cr(III)

Organo-Cr(III)

Organo-Cr(III)

N/A

Organo-Cr(III)

Control

5 mM Cr(VI)

10 mM dithionite

50 mM KPi (pH 7)

Cr(III) precipitates

Hypothesis: Organo-Cr(III) is readily formed during Cr(VI) reduction in the presence of organics

Geoff Puzon

Experiments:

With selected metabolites

5 mM Cr(VI)

10 mM dithionite

50 mM KPi (pH 7)

Organo-Cr(III)

Control No organic GSH-Cr(III)Serine-Cr(III)

Cysteine-Cr(III)Oxaloacetate-Cr(III)

Malate-Cr(III)Lactate-Cr(III)

Pyruvate-Cr(III)

Soluble Organo-Cr(III) end products

Complex solubility Organic ligand Soluble Cr(III) (mM) Percent soluble Cr(III)

Highly soluble organo-Cr(III) end products

Histidine 5.01 + 0.06 100%

Glutathione 4.76 + 0.15 95%

-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 organo-Cr(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%

KPi-Cr(III) Control 100 mM KPi pH 7.0 < 0.01 0%

0

0.05

0.1

0.15

0.2

0.25

0.3

410 460 510 560 610 660 710

Cysteine-Cr(III)

Malate-Cr(III) GSH-Cr(III)

Serine-Cr(III)

Oxaloacetate-Cr(III)

Cr(NO3)3

Wavelength (nm)

Ab

sorb

ance

Absorbance SpectraPeak AbsorbanceCr(NO3)3= 579nmCys-Cr(III)= 584nmMal-Cr(III)= 595nmSer-Cr(III)= 600nm GSH-Cr(III)= 604nmOx-Cr(III)= 607nm

Proposed Cr(III)-DNA adducts. Arakawa et al. 2005. Carcinogenesis 27:639-645.

Cr(III)-DNA Adducts are Formed from Cr(VI) Reduction

The adducts block DNA polymerase.

Zhicheng Zhang

Inorganic Cr(III) Cr(VI)Bioremediation

Primarily industrial process

Organo-Cr(III)

Microbialactivities

Mass balance of Cr after reduction by E. coli

Cr(VI)

Total Cr (In Supernatant)

Cr

(M

)

Days

0

50

100

150

200

250

0 1 2 3 4 5 6 7 8

Geoff Puzon

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

Formation of both soluble and insoluble Cr(III) from Cr(VI) reduction

Ranjeet Tokala

Cr(III) Cr(VI)Bioremediation

Primarily industrial process

Organo-Cr(III)

Recalcitrant

Microbialactivities

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 20 40 60 80 100 120

Time (h)

OD

600n

m

Malate-Cr(III)

Malate

Malate + Malate-Cr(III)

Malate-Cr(III) is recalcitrant but not toxic to R. eutropha JMP134

Geoff Puzon

Substrate: 2 mM

Cr(III) Cr(VI)Bioremediation

Primarily industrial process

Organo-Cr(III)

Recalcitrant Negatively charged

Mobile in soil

Microbialactivities

Malate-Cr(III) moves through a soil column

Tracer Vs Malate-Cr(III) complex

0

0.2

0.4

0.6

0.8

1

1.2

2 4 6 8 10 12 14 16 18 20 22 24Time (h)

C/C

0 Br tracer

GWM ctrl

Malate-Cr(III) complex

Br -tracer

Malate-Cr(III)

Cr(NO3)3

- NaBr: 10 ppmMalate-Cr(III): 10 ppmCr(NO3)3: 10 ppm

Mobile phase: simulated groundwater pH 7

Ranjeet Tokala

Immobile phase: Hanford soil

Fate of NAD+-Cr(III)?

- Bacterial utilization – slow process

PTX1

PTX2

Leifsonia sp. Rhodococcus sp.

- Bacteria enriched with NAD+-Cr(III)

Geoff Puzon

- Soluble Cr(III) decreased

Cr(III) Cr(VI)Bioremediation

Primarily industrial process

Organo-Cr(III)

Recalcitrant Negatively charged

Mobile in soil

Updated Biogeochemical Cycle of Cr

Microbialreduction

Microbialmineralization

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 supportsDepartment of EnergyERSD (NABIR)

NADH

Fre

Flavinox

Flavinred

H2O2

O2

Cr(VI)

Cr(III)

NAD+

Chromate Reduction by Flavin reductase (Fre)