Role of fungi in the biogeochemical fate of depleted uranium
Marina Fomina, John M. Charnock, Stephen Hillier, Rebeca Alvarez, Francis Livens &
Geoffrey M. Gadd
Figure S1. DU-containing microcosms. (a) 90 mm-plate with Rhizopogon rubescens. (b)
The DU coupon, overgrown by R. rubescens, with black and yellow DU corrosion
products. (c) DU colonization by Beauveria caledonica. (d) Corroded DU coupon from a
B. caledonica microcosm. (e-h) SEM images of DU coupons taken from B. caledonica
microcosms showing (e) fungal colonization and (f-h) uranium biomineralization
associated with phosphorus formed on the exterior surfaces of (g) cord-like hyphal
aggregates and (h) individual hyphae. Scale bars are (b, d) 1 mm, (c) 500 µm, (e) 100
µm, (f) 5 µm, (g) 1 µm, (h) 10 µm.
Figure S2. X-ray absorption spectroscopy (XAS) and X-ray powder diffraction (XRPD)
data. (a) Fourier transforms of the U L(III)-edge EXAFS spectra (solid lines) and
associated fits (broken lines) for standards of (blue) uranyl phosphate and (lilac) UO3,
and typical samples of (green) DU corrosion products after exposure to fungi and (red)
DU-treated fungal biomass. The insert shows a schematic model of the coordination
geometry for a uranyl moiety coordinated by phosphate for uranium accumulated by
fungi. (b) XRPD patterns of (top) a mixture of black corrosion products with H. ericae
hyphae (Fig. 1a, b), (bottom) biomineralized mycelium from the colony marginal zone of
R. rubescens. Example reference patterns from the Powder Diffraction File (PDF) for
uraninite (red), U3O7 (blue) and chernikovite (green) are also shown.
Supplemental Experimental Procedures
The selected fungi have previously shown efficient mineral dissolution and toxic metal
transformations [S1]. These included an ectomycorrhizal fungus Rhizopogon rubescens
Tulasne (provided by Dr. H. Wallander); an ericoid mycorrhizal fungus Hymenoscyphus
ericae (Read) Korf & Kernan (provided by Prof. A. Meharg) a soil saprotroph and
entomopathogen Beauveria caledonica Bissett & Widden (provided by Dr. D. Genney);
and a wood-rotting fungus Serpula himantioides (Fries:Fries) Karst (provided by Dr. N.
DU samples and microcosm design
The samples of DU alloy (provided by Defence Science and Technology Laboratories
(Dstl), Porton Down and machined by AWE, UK) were triangular sectors weighing
approx. 6.5-8.5 g with approximate dimensions (mm): 15x15x11 and 5 mm in height. A
specific radioactivity of 12.5 kBq/g was assumed. Before use, they were sequentially
washed with dichloromethane and isopropyl alcohol, and sterilized with absolute ethanol.
The DU coupons were exposed to fungi in Petri dish microcosms. The microcosm design
in this study was intended to simulate a nutritionally and mineralogically heterogeneous
environment typical for soil filamentous fungi. Each DU coupon was inserted into a hole
of corresponding size that had been cut from the centre of the modified Melin-Norkrans
[S1] agar in the Petri dish, leaving a 2 mm gap between the DU coupon and agar to
prevent any DU-agar interaction. A sterile dialysis membrane with a hole of the same
size as the DU coupon was placed on the top of the agar to ease biomass harvesting.
Three 7mm-square blocks of fungal inoculum cut from the edge of fungal colonies were
placed on top of the membrane at each side of the DU coupon, also leaving a 2 mm gap.
Plates were incubated for three months at 21±1°C. Growth of the fungi was determined
by dry weight measurement: DU-tolerance was expressed as a tolerance index calculated
as the percentage of the DU-treated biomass weight of the non-treated control. The pH of
the agar surface under growing fungal colonies was measured using a surface
combination pH electrode (Orion, Model 720A, BDH, Poole, UK).
Organic (carboxylic) acid analyses
To determine carboxylic acid exudation by the fungi, agar samples (5 blocks with a total
volume ~2.5 cm3) were cut from the areas beneath fungal colonies and subjected to water
extraction in test tubes containing 7.5ml distilled deionised (dd)H2O at 80ºC for 20 min.
Analyses of the water extracts were carried out using a HPLC Waters system with
Aminex HPX-87H HPLC organic acid analysis ion-exclusion column [S1]. SigmaStat
(Release 3.1) was used for statistical analysis. At least three replicate determinations were
used in experiments.
Following light microscopic observations of DU transformations by fungi, a Philips
XL30 environmental scanning electron microscope (ESEM) field emission gun (FEG)
operating at an accelerating voltage of 15 or 25 kV coupled with energy dispersive X-ray
microanalysis (EDX) was used in high vacuum mode for air-dried and Au/Pd-coated DU
coupons, or in low temperature SEM mode for cryo-preserved samples of fungal
ICP-AES analyses of uranium in biomass
The harvested mycelia, following dry weight measurement, concentrated HNO3-digestion
and appropriate dilution with ddH2O, were analyzed for uranium content using a Perkin-
Elmer 5300 Optima dual view inductively-coupled plasma-atomic emission spectrometer.
Uranium L(III)-edge XAS Measurements
X-ray absorption spectra at the U L(III)-edge for samples of fungal biomass and DU
corrosion products were collected in the fluorescence mode on Station 16.5 at the
CCLRC Daresbury SRS operating at 2 GeV with an average current of 150 mA, using a
vertical focussing mirror and a sagitally bent focussing Si(220) double crystal
monochromator detuned to 70 % transmission to minimise harmonic contamination.
Background subtracted EXAFS spectra were analysed in EXCURV98 using full curved
wave theory. Multiple scattering effects from the linear O=U=O uranyl moiety were
included in the fits. Fourier transforms of the EXAFS spectra were used to obtain an
approximate radial distribution function around the central uranium atom (the absorber