The biogeochemistry of Pu mobilization and retention

B.D. Honeyman (PI)1, A.J. Francis2, C. J. Dodge2, J.B. Gillow2 and P.H. Santschi3

1Environmental Science and Engineering Division, Colorado School of Mines; 2Environmental Sciences Department, Brookhaven National Laboratory; 3Texas A&M University at Galveston

2

Historically, expectations of minimal aqueous Pu transport of have been confounded by the apparent contradiction: • an understanding of low Pu solubility (based on its inorganic speciation) and • the observed transport of Pu sometimes at substantial distances from its presumed source.

3

Of the relatively limited number of papers on Pu environmental speciation, a majority have implicated ‘organic matter’ as a transport agent*.

This project focuses on the role of bacteria in the production of EPS: as Pu mobilization and immobilization agents.

*(e.g., Nelson et al., 1990; Orlandini et al., 1990; Loyland et al., 2001; Honeyman, 1998; Santschi et al., 2002)

4

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

-400 -200 0 200 400 600 800EH (Volts)

Pu(OH)4o

PuO2(s) PuO

2(s)

PuO2CO

3−

β −UO2(OH)2(s)

(UO2)

2CO

3(OH)

3−

UO2(s)

U(OH)4o(aq)

Comparison of U and Pu solubility

Uncertainty overcontrolling phase

pH 7.1

A

239Pu≈2.2 dpm

5

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

-20 -18 -16 -14 -12 -10

log [Pu (M)]

Examples of environmental measurements

Rocky FlatsDischarge Std.(0.15 pCi/L)

Drinking WaterStandard

Rocky FlatsMaximum obs.SW conc.

Kersting et al. (1999):NTS

Dai and Beusseler (2004): Hanford

Honeyman et al.(1999): Rocky Flats

EH = 800 mV-150 mV

6

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

-20 -18 -16 -14 -12 -10

log [Pu (M)]

Examples of environmental measurements

Drinking WaterStandard

Rocky FlatsMaximum obs.SW conc.

Kersting et al. (1999):NTS

Dai and Beusseler (2004): Hanford

Honeyman et al.(1999): Rocky Flats

EH = 800 mV-150 mV

0

1

2

3

4

5

6

Total 0.4m - 10K Da <10kDa

7

Pu release from vegetated soils as a function of quality and quantity of DOC

Santschi et al. (2002) Environ. Sci. Technol., 36, 3711-3719.

8

PAGE (Gel electrophoresis) of 239,240Pu from Soil Resuspension Experiment

Santschi et al. (2002) Environ. Sci. Technol., 36, 3711-3719.

9

Pourbaix Diagram (Eh/pH ) diagram for the system Pu-O2-CO2-H2O at 25° 1 C and bar total pressure forΣ ( )=10Pu aq -8 M and

(total carbonate CT) = 10-2.0 M

A: Aerobic bacteriaB: DenitrifersC: Mn-reducersD. Fe reducersE: FermentersF: Sulfate reducers

After Langmuir (1997)

10

Pus

Puaq

adcb

Fe(II)

PusPuaq

EPS

Pus

‘solubilization’

‘trapping’

Pu

Puaq

PusPu

Pu mobility / immobility as a transformational process

Key: A. Pu carrier-phase dissolution; B. Trapping of colloidal Pu by EPS; C. Biodegradation of Pu-EPS; D. Enhanced Transport of Pu by EPS.

A

C DPu-EPS

EPS

B

Fe(II)

PuaqBiofilm on ironoxide solid phasecontaining adsorbedPu

Exopolymer trappingcolloidal Pu andadsorbed Pu

Fe-oxidecarrier phase

Extracellularpolymeric substance (EPS)

Pu-EPS

EPS

Puaq

1 m 1 m

SEM SEM

TEM of Clostridium sp.

1 m

12

Characterization of EPS

13

TEM of <0.45 m EPS from Shewanella putrefaciens

Bar = 200 nm

TEM image of Clostridium sp.

TEM image of Clostridium sp. after 48 hours growth showing polysaccharide surrounding cell (bar = 1000nm)

J.B.Gillow (unpublished)

14

Soil Bacterial culturesCentrifugation (3600 g, 30 min)

SupernatantEtOH precipitation (overnight, filtration)

Pellet

Removing Protein with proteinase or pronase for 12 hr at 37 C, then dialyzed, lyophilized

Dissolved in D.W. with 3% NaCl, stirring 1 hr, add EtOH

Crude exopolymerPurification of EPS for three times with EtOH,

Mixture

PelletDissolved in D.W. with 3% NaCl for 1 hr

Capsular exopolymer

Pure EPS

(C )

(C )

(C )

(C )

(C )

(C )

15

Objectives

• Determination of amphiphilic character of EPS through evaluation of chemical composition of hydrophilic carbohydrates and uronic acids, and more hydrophobic proteins, through the use of Hydrophobic Interaction Chromatography (HIC).

• Pu(IV,V) partitioning to EPS from Pseudomonas with(w/) and without(w/o) proteins.

16

Pus

Puaq

EPS

Pus

‘trapping’

Key: A. Pu carrier-phase dissolution; B. Trapping of colloidal Pu by EPS; C. Biodegradation of Pu-EPS; D. Enhanced Transport of Pu by EPS.

A

C DPu-EPS

EPS

B

Fe(II)

PuaqBiofilm on ironoxide solid phasecontaining adsorbedPu

Exopolymer trappingcolloidal Pu andadsorbed Pu

Fe-oxidecarrier phase

Extracellularpolymeric substance (EPS)

Pu-EPS

EPS

Puaq

1 m 1 m

SEM SEM

TEM of Clostridium sp.

1 m

18

GC-MS Spectra of EPS (C.-C. Hung): Clostridium sp. Exudates (> 6 kDa)

RT: 12.17 - 65.49

15 20 25 30 35 40 45 50 55 60 65

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

R

e

l

a

t

i

v

e

A

b

u

n

d

a

n

c

e

9

8

7

6

5

4

1

2

3

NL:

2.29E6

TIC MS

08140202

1, 2, 6, 8, 9: unknown3: glucose4: galactose5: galacturonic acid7: mannuronic acid

D-GalacturonateC6H10O7

MW- 194.14 g/mol

D-MannuronateC6H10O7

MW- 194.14 g/mol

L-GuluronateC6H10O7

MW- 194.14 g/mol

Alginic acid

19

0

10

20

30

40

50

RhamnoseFucoseRiboseArabinoseXyloseMannoseGalactoseGlucose

(%)

Particulate EPS

0

10

20

30

40

50

RhamnoseFucoseRiboseArabinoseXyloseMannoseGalactoseGlucose

(%)

Dissolved EPS

20

Hydrophobicity and MW determinations:

• Waters HPLC• Amersham HiTrap, 2 butyl 1 ml columns in series

• Waters HPLC• Tosoh Biosciences

G4000 PWxl, 7.8 mm x 30 cm, particle size 10 µm, with guard column 6 mm x 4 cm

21

ResultsHCA, Protein, Charged Groups

Shewanella, particulate

Shewanella, dissolved

Pseudomonas, with protein

Pseudomonas, no protein

C/SO4 (1000 * mole ratio)

C/PO4 (1000 * mole ratio)

URA/OC%

Protein-C/OC%

HCA Hydrophobic Contact Area( Å2 molecule-1)

22

ResultsMolecular Weight (MW) by SEC

23

ResultsMolecular Weight (MW) by SEC

24

Summary of characterization work:

• The neutral monosaccharides in this EPS consist of rhamnose, fucose, ribose, arabinose, xylose, mannose, galactose and glucose.

• The acidic groups in this EPS are mainly composed of carboxylic acid and minor polyanionic groups, e.g., sulphate and phosphate.

• 45 - 70 % of total carbohydrates are uronic acids, and total carbohydrates made up 26-31% of organic carbon.

• Besides the neutral and acidic sugars in the EPS, EPS also contained 9 % of proteins (% of carbon), which makes the EPS amphiphatic (amphiphilic).

25

Pus

Puaq

EPS

Pus

‘trapping’

26

Pu / EPS complexes

• Evaluation of Pu binding sites (‘empirical adequacy’): affinity distribution

• Determination of conditional Pu /EPS complexes: ligand competition method

27

-0.00012

-0.0001

-0.00008

-0.00006

-0.00004

-0.00002

0

0.00002

0.00004

4 5 6 7 8 9 10pH

[COM] = 143 mg/LI = 0.1 M

4 6 8 10

4 6 8 10

Affinity distributions

‘SmoothnessHypothesis’

‘Discrete’ peaks

Linear combination ofindependent ionizable sites?

28

Pseudomonas Clostridium Shewanella0

1

2

3

4

5

6

7

Bacterial EPS type

4

6

8

Total

Comparison of EPS ‘ligand’ specific concentrations

pKa

29K1,1 K1,2

Pu4+ + HL1 =K1,1

PuL13+ + H+

Pu4+ + 2HL 2 =K1,2

Pu(L 2 )22+ + 2H+

14

13

12

11

10

Log Ki,j

Pseud.

Clos.

Shew.

Comparison of Pu / EPS binding

Citrate

Galacturonic acid

30

Microbially-enhanced Pu mobilization

• Soil analysis• Batch studies• ‘Static’ columns

31

Isotope Half-Life

(years)

Specific Activity (Ci/g)

Decay Mode

Alpha(MeV)

Beta(MeV)

Gamma(MeV)

Pu-238 88 17 5.5 0.011 0.0018

Pu-239 24,000 0.063 5.1 0.0067 <

Pu-240 6,500 0.23 5.2 0.011 0.0017

Pu-241 14 100 β < 0.0052 <

Pu-242 380,000 0.0040 4.9 0.0087 0.0014

[US DOE, Plutonium Fact Sheet, ANL, 2001]

Radioactive Properties of Key Plutonium Isotopes

239,240Pu: in situ (‘aged)241Pu: tracer242Pu: tracer

32

-XRF of Rocky Flats Soil

1x100

1x101

1x102

1x103

1x104

1x105

1x106

5 10 15 20Energy (KeV)

Fe + Mn

Zr

Y

Sr

Rb

PbReAs

Fe

Mn+ Zresc.

InelasticCompton

[1 mm2 map of Fe (red) and Sr (blue)of ‘as-rec’d’ RF soil; incident beam energy 17.5 KeV]

[Microbeam (10 x 20 m spot) X-rayfluorescence of RF soil elements characteristic of loam/sandy soil]

[Analyses performed at NSLS Envirosuite beamline X27A; note that ‘as-rec’d’ the soil contained 1.6 ng 239,240Pu g-1 dry wt., 5 mg Fe g-1 dry wt. (0.5 wt. %), 1.97% TOC]

Key: A. Pu carrier-phase dissolution; B. Trapping of colloidal Pu by EPS; C. Biodegradation of Pu-EPS; D. Enhanced Transport of Pu by EPS.

A

C DPu-EPS

EPS

B

Fe(II)

PuaqBiofilm on ironoxide solid phasecontaining adsorbedPu

Exopolymer trappingcolloidal Pu andadsorbed Pu

Fe-oxidecarrier phase

Extracellularpolymeric substance (EPS)

Pu-EPS

EPS

Puaq

1 m 1 m

SEM SEM

TEM of Clostridium sp.

1 m

34

Pus

Puaq

Fe(II)

Biotransformation of Pu in Soil from Rocky Flats

4 .05 .06 .07 .08 .09 .001 02 03 04 05 06 07 08 0D a y sU n a m e n d e d (D I w a te r)L a c ta te A m e n d e dG lu c o s e A m e n d e d

4.0

5.0

6.0

7.0

8.0

9.0

0 10 20 30 40 50 60 70 80

Days

Unamended (DI water)

Lactate Amended

Glucose Amended

0.0

1.0

2.0

3.0

4.0

5.0

0 10 20 30 40 50 60 70 80

Days

Glucose Amended

Lactate AmendedUnamended (DI water)

A

B

Biostimulation of RF soil:

A. The pH dropped to 7 due to metabolism of lactate; and to 5 due to glucose fermentation.25 days, EH glucose = -180 mV, lact. = -123 mV.

B. Glucose fermentation released 50 wt. % of thetotal reducible iron oxide from the soil; lactatemetabolism released 3.5 wt. %.

Incubation time = 10 days

36

Formation of Pu Colloids in Soil due to Microbial Action

1.0x10-9

1.0x10-8

1.0x10-7

1.0x10-6

0 10 20 30 40 50 60 70 80

Days

Glucose Amended

Lactate Amended

[242Pu nitrate (1.5 x 10-6M) added to soil resulted in 1) inorganic colloid formation, 2) sorption to soil and 3) remobilization as a colloid (<0.45 m) upon incubation with glucose and lactate. <0.1% of total 242Pu was in this fraction of unamended samples.]

Bacteria (blue) and inorganicmicroparticles (unfiltered supernatant liquid); glucosesample.

1) inorganic colloid

2) sorption to soil

3) remobilization

37

239,240Pu and 242Pu Fate at 77 Days Incubation

1.0x10-12

1.0x10-11

1.0x10-10

1.0x10-9

1.0x10-8

Treatment

Pu-239

Pu-242

[Indigenous 239,240Pu and spiked 242Pu were mobilized (<0.45 m) from RF soil with both glucose and lactate; the majority of the 239,240Pu in the ‘as-rec’d’ soil was associate with the organic and inert fraction.] [mol % of total added]

0.18%

0.01%

0.7% 0.2%

0.33%

0.15%

38

Re-distribution of 242Pu in Soil at 77 Days Incubation

Unamended Glucose Lactate0

20

40

60

80

100

120

Iron

Plutonium

[242Pu was recovered with the reducible iron oxide fraction (CBD extraction) in unamended samples, however, it was redistributed to another phase after biostimulation with glucose or lactate]

50 wt.%Fe dissolved

39

242Pu and Total Carbohydrates

1.0x10-10

1.0x10-9

1.0x10-8

1.0E-5

1.0E-4

1.0E-3

Treatment

Pu-242

Total Carbohydrates

[At 77 days, the colloidal 242Pu was correlated with an increase in suspended carbohydrates (<0.45 m); this indicates that microbial exudates may play a role in Pu mobilization in the incubation experiments]

microbial exudates

40

Summary of Soil Biotransformation Studies

• Pu was below detection for microprobe XRF, however discrete Fe phases were observed.

• Biostimulation with glucose released a significant amount of Fe while Fe(II) was readsorbed in lactate amended samples.

• A 242Pu spike rapidly sorbed to soil, however it was remobilized due to microbial action; under highly reducing, fermentative conditions 0.7% of the total was detected in the <0.45m, >30kDa fraction.

• The majority of Pu was released with the reducible iron oxide fraction of unamended samples, however this was not the case after biostimulation.

• The indigenous 239,240Pu was remobilized, to a lesser extent than the spike, but this may be due to different mineralogical association (majority resided with the organic and inert fraction).

• There was an increase in soluble carbohydrates in the biostimulated samples implicating microbial exudates in stabilizing Pu in the colloidal fraction.

Key: A. Pu carrier-phase dissolution; B. Trapping of colloidal Pu by EPS; C. Biodegradation of Pu-EPS; D. Enhanced Transport of Pu by EPS.

A

C DPu-EPS

EPS

B

Fe(II)

PuaqBiofilm on ironoxide solid phasecontaining adsorbedPu

Exopolymer trappingcolloidal Pu andadsorbed Pu

Fe-oxidecarrier phase

Extracellularpolymeric substance (EPS)

Pu-EPS

EPS

Puaq

1 m 1 m

SEM SEM

TEM of Clostridium sp.

1 m

42

‘Static column’ experiments

Assess Pu mobility under solid / solution ratios appropriate to in situ conditions

43

Static Column Incubation Experiment: 10 vs. 47 Day Incubation

0

2E-13

4E-13

6E-13

8E-13

1E-12

1.2E-12

1.4E-12

1.6E-12

1.8E-12

2E-12

239,240

Pu in Solution (M)

10 Days 10 Days47 Days 47 Days

Control

Incubated

0.0056% Solubilized 0.017%

Solubilized0.0040% Solubilized

0.018% Solubilized

Error Bars = 95%

Confidence Interval

239,240Pu = ~70 pCi / g; 0.5 w/v glucose; 0.015% w/v NH4Cl

44

Pus

Pu

Pu transport enhancement by EPS

EPS Facilitated Pu Mobilization

0

2

4

6

8

10

12

14

16

% Total

241

Pu Mobilized

n = 3

n = 3n = 3n = 3

Control ControlEPS EPS

Error bars = 95% confidence interval.

t = 0

t = 24 hrs

Sorbed Pu-241 to RF soil for 24hrs then 22 mg/L EPS (~ 10 mg/L OC) injected

239,240Pu mobilized: 0.018% v. 0.004% in the control

• 239,240Pu vs. 241Pu Tracer

Exchangeable

Carbonates

Oxides/hydroxides

Organics

Residual (“inert”)

241Pu 239,240Pu

Exchangeable (hypothetical)

47

Pus

Puaq

adcb

Fe(II)

PusPuaq

EPS

Pus

‘solubilization’

‘trapping’

Pu

Puaq

PusPu

Summary:Pu mobility as a transformational process

48

Acknowledgements

• Cetin Kantar (Ph.D. Student / post-doc)• Ruth Tinnacher (Ph.D. student)• Angelique Diaz (Ph.D. student)• NABIR Program