Marina Fomina, John M. Charnock, Stephen Hillier, Rebeca Alvarez, Francis Livens&. Geoffrey M. ... ericae (Read) Korf & Kernan (provided by Prof. A. Meharg) ...
Supplemental Data: Role of fungi in the biogeochemical fate of depleted uranium
Marina Fomina, John M. Charnock, Stephen Hillier, Rebeca Alvarez, Francis Livens & Geoffrey M. Gadd
Supplemental Results
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
Organisms 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. White).
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.
SEM/EDX 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 biomass.
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 PerkinElmer 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
atom); the peaks of the Fourier transform can be related to “shells” of surrounding back scattering atoms characterised by atom type, number of atoms in the shell, the absorberscatterer distance, and the Debye-Waller factor 2σ2. The data were fitted for each sample by defining a theoretical model and comparing the calculated EXAFS spectra and the associated Fourier Transforms with experimental data (see [S2] for extensive discussion of XAS spectroscopy of metal transformations by fungi).
X-ray Powder Diffraction (XRPD) Analyses Samples of fungal biomass and DU corrosion products were mounted onto single crystal silicon substrates and examined using a Panalytical X-pert Pro diffractometer equipped with a position sensitive ‘X-celerator’ detector. Diffraction patterns were identified by reference to patterns in the International Centre for Diffraction Data (ICDD) Powder Diffraction File (PDF) set 51 (2001), using Bruker AXS Diffrac Plus Eva software.
Supplementary References S1.
Fomina, M., Hillier, S., Charnock, J.M., Melville, K., Alexander, I.J., and Gadd, G.M. (2005). Role of oxalic acid over-excretion in toxic metal mineral transformations by Beauveria caledonica. Appl. Environ. Microbiol. 71, 371-381.
S2.
Fomina, M., Charnock, J., Bowen, A.D. and Gadd, G.M. (2007). X-ray absorption spectroscopy (XAS) of toxic metal mineral transformations by fungi. Environ. Microbiol. 9, 308-321.
