# Presentation: 146409

## 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

## 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.

## 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)

## Comparison of U and Pu solubility

- _Comparison of U and Pu solubility_

- Uncertainty over
- controlling phase

- pH 7.1

## Examples of environmental measurements

- _Examples of environmental measurements_

- Rocky Flats
- Discharge Std.
- (0.15 pCi/L)

- Drinking Water
- Standard

- Rocky Flats
- Maximum obs.
- SW conc.

- Kersting et al. (1999):
- NTS

- Dai and Beusseler
- (2004): Hanford

- Honeyman *et al.*
- (1999): Rocky Flats

- EH = 800 mV

- -150 mV

## Slide 6

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

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

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

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

## A:	Aerobic bacteria

- A:	Aerobic bacteria
- B:	Denitrifers
- C:	Mn-reducers
- D.	Fe reducers
- E:	Fermenters
- F:	Sulfate reducers

- After Langmuir (1997)

## Pu mobility / immobility as a transformational process

- _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.

## Characterization of EPS

## TEM of <0.45 m EPS from Shewanella putrefaciens

_*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)

## (C )

**(C )**

**(C )**

**(C )**

**(C )**

**(C )**

**(C )**

## 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.

## Slide 16

## 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.

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

## Slide 19

## 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

## ResultsHCA, 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)

**Notes:**
Charge density as inferred by the presence of charged groups (SO4, PO4, and COOH in URA) contribute to solubility and hydrophilicity in the EPS. Protein and the HCA relate to the hydrophobic nature of the amphiphilic EPS. Using these characteristics along with MW size fraction from SEC and the protein distribution relative to MW from the 2D-PAGE, these data suggest that:
when  the MW distribution in a species, i.e., Pseudomonas f., is very similar but the protein content differs, the EPS with the higher protein content is more hydrophobic even though it has charged functional groups.
Shewanella disssolved with its high protein (14%) content in the 14 to 20 kDa range (Ag stain) has about the same HCA (10 Ǻ2molecule-1) as Shewanella particulate. This is perhaps due the spread in its MW distribution (74% at <8kDa,10% at 14 kDa, 16% at 40 kDa) enabling the “shielding” of the protein by long fibrils along with its lower charge density and solubility through a lower content of functional groups. 

Future research should provide more information on charge distribution and flocculation capabilities. The HCA of these EPS will be used as an indicator of enhanced scavenging tendency in marine environments.

## ResultsMolecular Weight (MW) by SEC

**Notes:**
SEC chromatograms for dissolved Pseudomonas with protein and dissolved Pseudomonas, no protein, show about the same MW size distribution. Chromatograms are shown with polystyrene sulfonate standards and calibration curve.

## ResultsMolecular Weight (MW) by SEC

**Notes:**
Here is another comparison of the MW size distribution of the dissolved Pseudomonas with protein and dissolved Pseudomonas, no protein, along with Shewanella dissolved and particulate. Again, the MW size fractions of the 2 Pseudomonas are very similar while the dissolved Shewanella fractions are predominately smaller than the particulate.

## 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).

## Slide 25

## Pu / EPS complexes

- Evaluation of Pu binding sites (‘empirical adequacy’): affinity distribution
- Determination of conditional Pu /EPS complexes: ligand competition method

## Affinity distributions

- ‘Smoothness
- Hypothesis’

- ‘Discrete’ peaks

- Linear combination of
- independent ionizable
- sites?

## Comparison of EPS ‘ligand’ specific concentrations

_*Comparison of EPS ‘ligand’ specific concentrations*_

- pKa

## K1,1

- K1,1

- K1,2

- 14

- 13

- 12

- 11

- 10

- Log Ki,j

- Pseud.

- Clos.

- Shew.

- _Comparison of Pu / EPS binding_

- Citrate

- Galacturonic acid

## Microbially-enhanced Pu mobilization

- Soil analysis
- Batch studies
- ‘Static’ columns

## Radioactive Properties of Key Plutonium Isotopes

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

- _Radioactive Properties of Key Plutonium Isotopes_

- 239,240Pu: *in situ* (‘aged)
- 241Pu: tracer
- 242Pu: tracer

## -XRF of Rocky Flats Soil

- [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-ray
- fluorescence 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.

## Slide 34

## Biotransformation of Pu in Soil from Rocky Flats

- 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 the
- total reducible iron oxide from the soil; lactate
- metabolism released 3.5 wt. %.

- Incubation time = 10 days

## Formation of Pu Colloids in Soil due to Microbial Action

- [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 inorganic
- microparticles (unfiltered supernatant liquid); glucosesample.

- 1) inorganic colloid

- 2) sorption to soil

- 3) remobilization

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

- [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%

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

- [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

## 242Pu and 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

## 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 <0.45m, >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.

## ‘Static column’ experiments

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

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

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

## Pu transport enhancement by EPS

- _Pu transport enhancement by EPS_

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

- _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

**Notes:**
SUMMARY
Sorbed Pu-241 to RF soil for 24hrs then introduced Pseudomonas EPS/L to the soil
22 mg/L EPS ~ 10 mg/L OC.  Fairly high but enough to provide an excess of binding sites for experimental Pu concentration (2x10-11 M).

Key points:
EPS increased the mobilization of Pu-241 significantly at both time periods.  Presence of EPS can impact fate of Pu contamination.
Still need to conduct a similar experiment with the aged Pu-239,240.

## 239,240Pu vs. 241Pu Tracer

**Notes:**
Conceptual figure of aged versus tracer Pu in RF soil.
Demonstrates hypothetical difference in Pu distribution.
Assumed Pu-241 mostly in exchangeable after only 24 hrs.
Saw that incubation resulted in a difference for Pu-241 and 239,240Pu, where Pu-239,240 mobilization was enhanced.

May be due to the fact that the Pu-241 tracer distributes itself mainly in the exchangeable fraction.  
Labile phase of Pu241 is different than Pu239,240 under these experimental conditions.  This is important considering that tracers are widely used to assess the mobility of an aged radionuclide.  The amount of time Pu remains in a system will determine the fraction that is labile under different biogeochemistries.

IMPORTANT: Know solution chemistry of Pu-241 addition.  239,240Pu source term (mainly as PuO2(s) – TALK TO BRUCE ABOUT THIS)

## Summary:Pu mobility as a transformational process

- _Summary:__Pu mobility as a transformational process_

## Acknowledgements

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