U N C L A S S I F I E D
U N C L A S S I F I E D
Bacterial Biotransformations for the
In situ Stabilization of Plutonium
Mary Neu, Hakim Boukhalfa, Gary Icopini, Larry Hersman, Joe Lack,
John Priester, Scott Olson, Patricia Holden
Chemistry & Biology Divisions, Los Alamos National Laboratory
Bren School of Environmental Science and Management, UCSB
NABIR P.I. Meeting, April, 2005
Plutonium contamination in the environment is generally low-leveland may be present and transported in a range of forms (IV, V, VI).
Current remediation strategies are costly, financially and in termsof increased exposure risk to people and the environment.In situ bacterial biostabilization is a promising alternative.
Aqueous Speciation Related to Environmental Conditions
• Plutonium(VI) vs Uranium(VI) Hydrolysis• Plutonium(IV/III) EDTA Speciation and Stability• Siderophore Stabilization of Plutonium(IV)• New Reduction Potentials
Bacterial Biotransformations
• Siderophore-mediated Accumulation by Aerobic Bacteria• EPS and Cell Adsorption by Aerobic Bacteria• Reduction by DMRB
Overview
Pu (VI) Hydrolysis9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
p[H
]
3.02.01.00.0-1.0Equivalents of NaOH
[An(VI)] = 1 mM0.1 M NaNO3
Pu
H
O
Pu
O
H
OH2
O
O
O
O
H2O
H2O
H2O OH2
OH2
U
H
O
U
O
OH2
O
O
O
O
H2O
H2O OH2
U
HO OH
O
O
OH2H2O
Why biostabilization methods being developed for U, e.g. reduction by DMRB,may or may not work for Pu
~600 papers on U(VI) hydrolysis 5 papers on Pu(VI) hydrolysis 2 papers on Pu(V) hydrolysis
100
80
60
40
20
0
% o
f [U
(VI)
] Tot
al
765432pH
UO22+
UO2(OH)+
(UO2)2(OH)22+
(UO2)3(OH)42+
(UO2)3(OH)5+
(UO2)4(OH)7+
[U(VI)]tot = 0.1 mM100
80
60
40
20
0
% o
f [P
u(V
I)] T
otal
765432pH
PuO22+
PuO2(OH)+
PuO2(OH)2
(PuO2)2(OH)22+
(PuO2)2(OH)4
[Pu(VI)]tot = 0.1 mM
Reilly, Neu, Inorg. Chem., submitted.
E mV (Ag/AgCl)
-800-600-400-2000200400600800
i, µ
A
-150
-100
-50
0
50
100
1501:1 (Pu:EDTA)
[H+] = 0.6 M
1:1 (Pu:EDTA)pH = 2.4
1:1: (Pu:EDTA:citrate)pH = 6.2
1:2 (Pu:EDTA)pH = 6.67
Cyclic Voltammetry
Pu(IV)EDTA Aqueous SpeciationNew Species Distributions (---)
Boukhalfa, Reilly, Neu, Inorg. Chem.,2004, 43(19), 5816.
Pu:EDTA = 1:1
Pu:EDTA = 1:2
New species are identified includingPu(EDTA)2, log = 35.43
Stability of Pu(IV) is enhanced atenvironmentally relevant pH by theformation Pu(IV)-EDTA-hydroxo andPu(IV)-EDTA-L mixed complexes.
Unusual speciation due to highcharge and large coordination sphere
Pu Speciation Under Environmental Conditions
PuO22+ logKsp = 5.5
PuO2+ logKsp = 5.0
Pu4+ logKsp = -2.0Pu3+ logKsp = 15.8
e.g., Pu4+/PuO2(hyd) [H+]4
Ered (mV) vs NHE, pH = 7
Solubility of primary (hydr)oxide phases
0
0.1
0.2
0.3
0.4
0.5
0 5 10 15 20 25
Time (hours)
0
0.1
0.2
0.3
0.4
0.5
0 5 10 15 20 25
Time (hours)
Pu(VI) as a Terminal Electron Acceptor for DMRBP
u C
oncentr
ation (
mM
)
No Cells
No Donor added
Heat Killed
Shewanella oneidensis MR1
10 mM Lactate
5 x 108 Cells/mL
Geobacter metallireducens GS15
10 mM Acetate
5 x 108 Cells/mL
-0.001
0.001
0.003
0.005
0.007
0.009
400 500 600 700 800 900
Ab
sorb
an
ce
Wavelength
(nm)
MR-1
No cell control DMRB reduce Pu(VI) and Pu(V)
Product appears to be PuO2 (hyd)
Characterization and dissolutionstudies in progress
DFEN
NH
O
NO
OH
O
NH
N NH
O
OH
OOH
O
+ Pu(IV)
+ Pu(IV)
+ Fe(III) ???
Gram -P. putida
Gram +A. flavesens,
JG-9
N
OOHHN
O
C
HN
OCH
HN
O
CH
CH2
HO
CH
H3C
HO
CH
HN
O
OH
HN N
HN
O
CH
NH
O
N NHHO
HO NH
O
CH2
H2C
COR
CH2 OH
H
H2C
NH2
4
L-Lys
D-allo-Thr
L-Ser
L-c(OH)Orn
D-Ser
Pyoverdine
Can Pu be accumulatedby other bacteria
via other types of siderophores?
Siderophore-Mediated Pu AccumulationFe(III) and Pu(IV) siderophore structures
Time (min)
0 20 40 60 80 100 120 140 160 180 200
55
Fe a
ccum
ula
tio
0
50
100
150
200
no Fe
Fe-NTA
Fe-tiron
Fe-DFB
Fe-EDTA
Fe-pyoverdin
Fe uptake profile suggests that exogenous ligands release iron to the siderophoreeither in solution or at the membrane surface.
P. putida Metal Acquisition from Multiple Chelates
0
0.5
1
1.5
0 100 200 300 400 500
Pu-NTAPu-pyoverdinPu-pyoverdin #2
Pu
acc
um
ula
tio
n (
pm
ole
s)time (min.)
Uptake inversely proportional to Fe-L complexes stability.
Time in min
0 50 100 150 200
0.0
0.5
1.0
1.5
2.0
2.5
Cr
Al
Ga
Ga 2 uM
pu NTA uptake
Fe-NTA uptake
1:1 competmition
Time (min)
0 50 100 150 200 250
Pu accum
ulatio
0
50
100
150
200
Pu-pyoverdin
Pu-(pyoverdin)2
Pu-NTA
Requirements for Metal–Siderophore Uptake
Siderophore binding, membrane protein recognition, metal shuttle, intra-cellularrelease thought to require specific physico-chemical characteristics.
P. Putida cells pre-incubated with 2 µM of pyoverdincomplexes of Cr(III), Ga(III) and Al(III) unable to acquireFe from NTA, EDTA or pyoverdin complexes.
P. Putida cells take up NTA and pyoverdincomplexes, both in the presence and absence of Fe.
Characteristics required for complete translocation? specific radii, trivalent charge, specific molecular conformation, neutral molecular charge, metal reduction, ligand exchange (solution or membrane).…
Pu(IV) and Th(IV) uptake reveal combination of ligand exchange and reduction is key
Metal Binding of Microbial Extracellular Polymers
HN
NH
HN
NH
CO2H
O CO2H
O CO2H
O
PGA of B. licheniformis
0
0.1
0.2
0.3
0.4
0.5
0 0.5 1 1.5 2 2.5 3 3.5
Fe(III)
U(VI)
Pu(IV)µm
ol M
etal
Bou
nd
µmol Metal Added
~800 kDa, forms soluble metal complexes,generally >10:1 glu to M
0
0.1
0.2
0.3
0.4
0.5
0 0.1 0.2 0.3 0.4 0.5
Fe(III) U(VI)Pu(IV)
Fe(III)U(VI)Pu(IV)
µm
ol m
etal
bou
nd
µmol metal added
PGA OnlyWhole Cells
~0.12 mmol metal bound per mg PGA (alone)
Pu(IV) remains associated with PGA duringrepeated pH cycling 2-12
Whole cells (in culture media) take up more Pu per mass than does polyglutamate
Siderophores and EPS associate Pu with cells
EPS, but not cell growth, affected by U.
U(VI) adsorbed onto Fe(III) minerals increasedEPS produced by P. putida
Effect of U(VI) on P. putida Biofilms
EPS, but not cell growth, affected by U.
U(VI) adsorbed onto Fe(III) minerals increasedEPS produced by P. putida
P. Putida grown on membrane discs onU-containing agar withminimal nutrients
[U] = 10-4 Mor 50 µg/g72 hr growthFractionation by cent.
Distribution of U(VI) within P. putida Biofilms
Less U removed from substrate by P. putida in the presence of EDTA
U preferentially associated with cell fraction
Pu Biogeochemistry
Advances in Pu aqueous geochemistry
Solubilization, biosorption, bioaccumulation, mineralizationbiotransformation mechanisms all affect Pu
Stability of products, rates of combined processes,…?
cknowledgementcknowledgement
U.S. DOE, U.S. DOE, OScOSc, OBER, ERSD, NABIR, OBER, ERSD, NABIR
Dr. Hakim Dr. Hakim BoukhalfaBoukhalfaDr. GaryDr. Gary Icopini IcopiniMrMr. Sean Reilly. Sean Reilly
Dr. LarryDr. Larry Hersman HersmanProf. Patricia HoldenProf. Patricia HoldenDr. CherylDr. Cheryl Kuske Kuske
