Environmental Microbiology (2007) 9(2), 308–321
X-ray absorption spectroscopy (XAS) of toxic metal
mineral transformations by fungi
Marina Fomina,1 John Charnock,2 Andrew D. Bowen1
and Geoffrey M. Gadd1*
1
Division of Environmental and Applied Biology,
Biological Sciences Institute, School of Life Sciences,
University of Dundee, Dundee, DD1 4HN, UK.
2
SRS Daresbury Laboratory, Daresbury, Warrington,
WA4 4AD, UK.
Summary
Fungi can be highly efficient biogeochemical agents
and accumulators of soluble and particulate forms of
metals. This work aims to understand some of the
physico-chemical mechanisms involved in toxic
metal transformations focusing on the speciation of
metals accumulated by fungi and mycorrhizal
associations. The amorphous state or poor crystallin-
ity of metal complexes within biomass and relatively
low metal concentrations make the determination of
metal speciation in biological systems a challenging
problem but this can be overcome by using
synchrotron-based element-specific X-ray absorption
spectroscopy (XAS) techniques. In this research, we
have exposed fungi and ectomycorrhizas to a variety
of copper-, zinc- and lead-containing minerals. X-ray
absorption spectroscopy studies revealed that
oxygen ligands (phosphate, carboxylate) played a
major role in toxic metal coordination within the
fungal and ectomycorrhizal biomass during the accu-
mulation of mobilized toxic metals. Coordination of
toxic metals within biomass depended on the fungal
species, initial mineral composition, the nitrogen
source, and the physiological state/age of the fungal
mycelium.
Introduction
The influence of microbiological processes on mineral
transformations and metal speciation is of economic and
environmental significance. Certain processes solubilize
metals from minerals and bound locations thereby
increasing metal bioavailability, whereas others immobi-
lize them and reduce bioavailability (Gadd, 1993; Burford
Received 21 March, 2006; accepted 8 August, 2006. *For
correspondence. E-mail g.m.gadd@dundee.ac.uk; Tel.
(+44) 1382 384765; Fax (+44) 1382 388216.
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd
doi:10.1111/j.1462-2920.2006.01139.
et al., 2003). In terms of metal pollution, both processes of
metal mobilization (e.g. in situ soil washing, extraction and
filtration techniques) and immobilization (e.g. in situ sta-
bilization techniques) may be applied to remediate con-
taminated matrices (Cotter-Howells and Caporn, 1996;
Knox et al., 2000; Prasad and De Oliveira Freitas, 2003;
Brown et al., 2004). Fungi are a major component of the
biota in soils and mineral substrates, and can be very
tolerant to toxic metals: under certain environmental con-
ditions (e.g. low pH, pronounced toxic metal pollution)
they can become the dominant microbial group (Fomina
et al., 2005a). The involvement of fungi in mutualistic
symbiotic associations with plants (mycorrhizas), algae
and cyanobacteria (lichens) has major consequences for
the biogeochemical cycling of elements (Gorbushina
et al., 1993; Smith and Read, 1997; Meharg and Cairney,
2000; Sterflinger, 2000; Gadd et al., 2005). Free-living
and mycorrhizal fungi can be efficient biogeochemical
agents and bioaccumulators of soluble and particulate
forms of metals (Gadd, 1993; 2004; Burford et al., 2003;
Fomina et al., 2004; 2005a,b). More than 95% of all land
plants depend on symbiotic mycorrhizal fungi (Smith and
Read, 1997) which make fungal biogeochemical activity
of particular importance in any type of remediation of toxic
metal polluted soils, whether physico-chemical or
biological. Mycorrhizal fungi solubilize phosphate and
essential metals for the host plant in the course of ‘het-
erotrophic leaching’ as a result of protonation (acidolysis),
chelation (complexolysis) and metal accumulation by the
biomass (Burgstaller and Schinner, 1993; Burford et al.,
2003). Fungal and plant cell walls can act as a cation
exchanger due to their negative charge originating from
functional groups, e.g. carboxylic, phosphate, amine or
sulfhydryl, in different wall components (hemicelluloses,
pectin, lignin, chitin, etc.) (Gadd, 1993; Sarret et al.,
1998a; 1999). Mechanisms for metal immobilization within
plant and fungal biomass also include intracellular uptake
with complexation to ligands such as S-containing pep-
tides (metallothioneins, phytochelatins), carboxylic acids
(citrate, malate, oxalate) and phenolic acids (Gadd, 1993;
Sarret et al., 1998b; 2002; 2003; Fomina et al., 2004;
2005c; Bellion et al., 2006). Because of the amorphous
state or poor crystallinity of metal complexes within
biomass and relatively low metal concentrations, the
determination of metal speciation in biological systems
remains a challenging problem with only a few studies on
 Toxic metal speciation in fungi 309

Fig. 1. Diagram illustrating X-ray absorption spectroscopy.

A. The Cu K-edge X-ray absorption spectrum of copper phosphate.
B. The Cu K-edge EXAFS spectrum of copper phosphate. The oscillations are displayed as a function of k, the wave-vector, and the
amplitude (normalized to the size of the edge-step) is multiplied by k3, in order to enhance the features at high k (high energy).
C. The Fourier transform of the EXAFS spectrum of copper phosphate. The major peak is due to the four nearest neighbour oxygen atoms
around the copper, at a distance of 1.95 Å. The smaller peak is due to a combination of two copper atoms at 2.98 Å and three phosphorus
atoms at 3.15 Å.

fungal biomass that mainly clarify the nature of adsorption
sites on cell walls (Fourest et al., 1996; Sarret et al.,
1998a,b; 1999). However, synchrotron-based X-ray
absorption spectroscopy (XAS) provides a means for
studying element complexation in environmental samples
varying from biological to mineralogical in nature (Sarret
et al., 1998a,b; 1999; 2002; 2003; Kemner et al., 2005).
X-ray absorption spectroscopy is a technique increasingly
used to study biological systems. The theory is well-
documented (Hasnain, 1988; Charnock, 1995) and analy-
sis of the data gives information about the oxidation state
of the target element and its coordination environment,
including the number and identity of neighbouring atoms
(Gardea-Torresdey et al., 2004). It allows studies involv-
ing fast data collection, small samples, low concentra-
tions, crystalline and amorphous solids, and solutions.
X-ray absorption spectroscopy is also a non-destructive,
non-invasive method that could probe metal transforma-
tions at the mineral–microbe interface, and directly study
samples in their natural hydrated states. The basic
process is absorption of an X-ray photon by the atom of
interest (the absorber atom) in the sample. The spectrum,
plotting absorbance of the sample as a function of photon
energy (Fig. 1), shows a sudden increase, the absorption
edge, corresponding to the ionization of a 1s (Cu or Zn,
K-edge) or 2p [Pb, L(III)-edge] electron, the energy of
which is sensitive to the oxidation state of the absorber
(Fig. 1A). When the X-ray is absorbed the photoelectron
is emitted in the form of a simple spherical wave. If the
absorber atom is isolated the electron wave propagates
freely outwards, but if the atom is incorporated in a
molecule or dense matter the outgoing spherical wave
is elastically scattered by the surrounding atoms
(scatterers). Interactions between the outgoing and scat-
tered electron waves give rise to the oscillations in the
spectrum above the edge, which are the EXAFS
(extended X-ray absorption fine structure) (Fig. 1A). In
certain situations, such as a highly symmetrical octahe-
dral coordination site, the electron wave can interact with
more than one scatterer, giving rise to multiple scattering
effects.
The EXAFS data are isolated from the absorption spec-
trum by subtracting the ionization energy, E0, removal of
a smooth background, and normalization of the oscilla-
tions to the edge step (the difference in absorbance just
before and just after the edge) (Fig. 1B). The data are
displayed as a function of the wave-vector k (proportional
to the square root of the difference between the X-ray
energy and E0). In k-space each scatterer contributes a
damped sine wave to the total EXAFS, with a frequency
which depends on the absorber–scatterer distance and
an intensity which depends on the atomic number of the
scatterer. The damping is affected by thermal oscillations
along the absorber–scatterer axis. Thus, the observed
EXAFS spectrum is an interference pattern of the contri-
butions from all the surrounding scatterers (Fig. 1B).
Taking a Fourier transform of the EXAFS spectrum gives
peaks corresponding to the different frequencies, yielding
an approximate radial distribution function showing
‘shells’ of scatterers (i.e. groups of scatterers at a similar
distance) around the absorber atom (Fig. 1C). This can be
used to build up a model of distribution of scatterers
surrounding the absorber. The EXAFS spectrum is analy-
sed by calculating a spectrum based on the model of the
absorber coordination site using various possible scatter-
ers and refining parameters in the model (distances,

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Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
310 M. Fomina, J. Charnock, A. D. Bowen and G. M. Gadd
damping factors) to minimize a least squares residual,
giving the best fit to the experimental data.
This XAS study of toxic metal speciation within fungal
and ectomycorrhizal biomass not only aims to further
understand the physico-chemical mechanisms involved in
toxic metal transformations and accumulation by fungi
and mycorrhizal associations, but also to demonstrate
that XAS is an ideal approach for investigating metal
transformations at the mineral–microbe interface in bio-
geochemical systems (Gardea-Torresdey et al., 2004;
Kemner et al., 2005).
Results and discussion
Copper
Copper makes a significant contribution to global pollu-
tion (Nriagu, 1996). Although only a small fraction of
copper content in sludge-amended soils is soluble, this is
found almost exclusively in low molecular weight com-
plexes with amino acids, small peptides or polycarboxylic
acids. This fraction is a major contributor to copper
mobility and bioavailability threatening lower trophic
levels of the food chain (Vulkan et al., 2002). In situ
EXAFS and X-ray absorption near edge structure
(XANES) spectroscopy of copper speciation in electroki-
netically remediated metal contaminated soil revealed
that 50% of the copper was chelated by humic sub-
stances, 28% formed CuCO3 and 22% formed Cu2O and
CuO (Liu and Wang, 2004). Fungi can play a significant
role in copper transformations (Fomina et al., 2005a,b,c)
and many soil fungi were able to withstand copper tox-
icity in heavily contaminated soils (500–11 500 mg Cu
per kg soil) (Fomina et al., 2005a). Nearly 60–70% of
tested ericoid and ectomycorrhizal fungal cultures were
able to grow in the presence of copper phosphate and
cuprite with more than half of them being able to solubi-
lize copper phosphate and over 40% of them solubilizing
cuprite (Fomina et al., 2005b).
In the present study, all tested samples of fungal and
ectomycorrhizal biomass (Beauveria caledonica, Rhizo-
pogon rubescens and R. rubescens/Scots pine) grown in
the presence of copper phosphate, and Aspergillus niger
grown on CuO, showed Cu coordination with oxygen
ligands, fitting carboxylate and/or phosphate coordination
(Table 1). B. caledonica, grown on copper phosphate-
containing medium, accumulated approximately
100 mg Cu g-1 dry weight (Fomina et al., 2005c). The
EXAFS spectrum and parameters, and the Fourier trans-
form were very similar to those of the copper oxalate
standard (Table 1) and the spectrum and the three clear
peaks in the Fourier transform could be fitted effectively
with the same parameters. In the standard, the outer
shells were slightly better defined which may be an effect
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
of grain size in the sample or a slight degree of non-
crystallinity. Copper precipitation on fungal hyphae is not
homogeneous, and related studies have shown that well-
structured moolooite (copper oxalate) crystals (10–
40 mm) on the mycelium comprised approximately 35% of
the total copper accumulated (Fomina et al., 2005c).
Various electron microscopic techniques (including wet
mode Environmental SEM) coupled with elemental analy-
sis EDX have shown that the majority of copper accumu-
lated by B. caledonica was deposited extracellularly,
being biomineralized within a well-hydrated mucilaginous
sheath of extracellular polymeric substances (EPS) sur-
rounding hyphae and hyphal cords (Fig. 2A).
The effect of nitrogen source on the coordination of
accumulated copper was examined for strains of the ecto-
mycorrhizal fungus R. rubescens in both axenic culture
and in mycorrhizal association with Scots pine (Table 1,
Fig. 3G and H). The nitrogen source is a factor that can
affect organic acid production by fungi with nitrate consid-
erably increasing oxalate excretion (Lapeyrie et al., 1991;
Gharieb and Gadd, 1999; Whitelaw et al., 1999). Analysis
of the EXAFS spectrum from biomass of R. rubescens
grown on nitrate and copper phosphate gave the best fit to
the inner shell with a coordination shell of four oxygens
bound to the copper at a distance of 1.96 Å and two
oxygens bound at 2.31 Å. This is a typical distortion from
perfect octahedral symmetry for a six-coordinate copper
(II) centre, and is known as a Jahn-Teller distortion
(Greenwood and Earnshaw, 1984). The EXAFS contrib-
uting to two further peaks in the Fourier transform were
best fitted with four carbons at 2.72 Å and four coppers at
3.82 Å, showing carboxylate coordination close to oxalate
(Table 1, Figs 3H and 4A). If ammonium was used as a
nitrogen source, the best fit to the inner shell was with a
Jahn-Teller distorted coordination shell of four oxygens at
1.94 Å and two oxygens at 2.43 Å. There was a small
outer shell which was best fitted with two coppers at
3.79 Å. However, attempts to fit phosphorus atoms at
3.1 Å (phosphate) or carbon atoms at 2.7 Å (oxalate/
carboxylic acid) did not improve the fit (Table 1, Fig. 3G).
For other biomass from organisms grown on ammonium-
containing medium, the fit with carbons was slightly better
and more consistent with oxalate coordination than with
phosphate, but was not conclusive probably indicating
mixed carboxylate/phosphate coordination of copper.
Phosphate coordination of copper was observed for
A. niger grown on CuO (Table 1). The best fit to the inner
coordination sphere was with four oxygens at 1.94 Å and
two oxygens at 2.48 Å, typical of six-coordinate octahe-
dral Cu(II) with a Jahn-Teller distortion which was
improved by the addition of 2 phosphorus scatterers at
3.00 Å.
In the microcosm experiments with A. niger,
B. caledonica and Penicillium simplicissimum grown on
© 2006 The Authors
 Toxic metal speciation in fungi 311
Table 1. Cu K-edge EXAFS parameters.

Sample Scatterers r (Å) 2s2 (Å2) Residual

Cu(acetate) standard 4¥O 1.95 0.012 25.2

1 ¥ Cu 2.60 0.007
4¥C 2.83 0.017
Cu(gluconate) standard 4¥O 1.93 0.009 25.1
2¥O 2.30 0.024
4¥C 2.73 0.037
4 ¥ Cu 3.78 0.027
Cu(malate) standard 4¥O 1.92 0.011 34.8
2¥O 2.38 0.020
4¥C 2.75 0.017
Cu2O standard 2¥O 1.85 0.012 29.3
12 ¥ Cu 3.03 0.024
6 ¥ Cu 4.27 0.029
24 ¥ Cu 5.27 0.035
12 ¥ Cu 6.06 0.025
Cu(oxalate) standard 4¥O 1.96 0.011 27.8
2¥O 2.35 0.024
4¥C 2.66 0.010
4 ¥ Cu 3.73 0.019
4 ¥ Cu 4.48 0.026
4 ¥ Cu 5.52 0.014
Cu3(PO4)2 standard 4¥O 1.95 0.011 25.3
2 ¥ Cu 2.98 0.020
3¥P 3.15 0.041
B. caledonica on copper phosphate-containing MMN agar 4¥O 1.99 0.021 41.0
2¥O 2.39 0.028
4¥C 2.70 0.013
4 ¥ Cu 3.74 0.025
4 ¥ Cu 4.00 0.039
B. caledonica on azurite-containing malt extract agar 3¥S 2.20 0.027 36.4
2 ¥ Cu 2.60 0.030
B. caledonica on malachite-containing malt extract agar 3¥S 2.16 0.025 37.6
2 ¥ Cu 2.58 0.031
A. niger on azurite-containing malt extract agar (margin) 3¥S 2.23 0.033 33.6
2 ¥ Cu 2.60 0.040
A. niger on azurite-containing malt extract agar (centre) 4¥O 1.95 0.022 34.4
2¥O 2.53 0.022
2¥P 2.89 0.018
2 ¥ Cu 3.70 0.018
A. niger on malachite-containing malt extract agar (margin) 3¥S 2.26 0.017 37.5
2 ¥ Cu 2.68 0.028
A. niger on malachite-containing malt extract agar (centre) 4¥O 1.94 0.032 31.9
2¥O 2.48 0.022
2¥P 3.00 0.046
A. niger on CuO-containing AP1 agar 4¥O 1.93 0.028 30.6
2¥O 2.48 0.035
2¥P 2.97 0.039
2 ¥ Cu 3.75 0.039
P. simplicissimum on azurite-containing malt extract agar 3¥S 2.14 0.022 45.4
1 ¥ Cu 2.56 0.018
P. simplicissimum on malachite-containing malt extract agar 3¥S 2.13 0.033 35.2
1 ¥ Cu 2.58 0.040
R. rubescens axenic culture grown on copper phosphate 4¥O 1.96 0.012 29.8
and nitrate MMN agar medium 2¥O 2.31 0.022
4¥C 2.72 0.019
2 ¥ Cu 3.82 0.039
R. rubescens axenic culture grown on copper phosphate 4¥O 1.94 0.013 27.1
and ammonium MMN agar medium 2¥O 2.37 0.037
R. rubescens/P. sylvestris ectomycorrhizas grown in a 4¥O 1.99 0.021 27.2
square Petri dish mesocosm with copper phosphate 2¥O 2.39 0.028
and nitrate Ingestad agar medium 4¥C 2.78 0.006
2 ¥ Cu 3.79 0.028
R. rubescens/P. sylvestris ectomycorrhizas grown in a 4¥O 1.94 0.012 33.7
square Petri dish mesocosm with copper phosphate 2¥O 2.42 0.054
and Ingestad agar medium 2 ¥ Cu 3.79 0.034

r is the copper–scatterer distance in Angstroms, ⫾0.02 Å inner shells, ⫾0.05 Å outer shells; 2s2 is the Debye–Waller type factor, ⫾15% inner shells, ⫾30%
outer shells (a measure of both the thermal motion between the absorber and scatterer and of the static disorder or range of absorber–scatterer distances);
the residual is a least squares residual from fitting the spectrum of the model to the experimental data.

© 2006 The Authors

Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
312 M. Fomina, J. Charnock, A. D. Bowen and G. M. Gadd

Fig. 2. Electron microscopic observations of toxic metal localization in fungal biomass. (A) High-vacuum mode ESEM image of air-dried
Au/Pd-coated sample of extracellular moolooite (white arrows) precipitation by B. caledonica hyphae and cords when grown on copper
phosphate agar medium. (B) Low-vacuum (auxiliary gas) mode ESEM (uncoated samples) and (C) TEM images of zinc-containing crystals
(white arrows) found within cells of the extraradical mycelium of the R. rubescens/P. sylvestris ectomycorrhizal association grown in a
mesocosm on Ingestad agar with zinc phosphate and nitrate. Scale bars are (A) 20 mm, (B) 10 mm, (C) 1 mm.

agar medium containing azurite or malachite, sulfur coor-
dination of copper accumulated by biomass was found in
most samples (Table 1). A typical example of the best fit to
the inner coordination sphere was with three sulfurs at
2.26 Å which was significantly improved by the addition of
two copper scatterers at 2.68 Å clearly indicating the for-
mation of a copper sulfide phase.
The age and therefore the physiological and reproduc-
tive state of the fungal mycelium was found to play a
crucial role in metal coordination within the biomass. On
both azurite- and malachite-containing media, samples
from the central part of A. niger colonies with mature
ageing mycelium and abundant conidiophores with dark-
coloured conidia demonstrated phosphate coordination of
copper (Table 1, Figs 3J and 4B, D, E). In contrast,
samples from the marginal zone of colonies with young
vegetative mycelium showed sulfide coordination of
copper (Table 1, Figs 3I and 4C and D).
The variation in copper coordination in A. niger could
demonstrate the relative significance and spatial distribu-
tion of copper resistance mechanisms within the colony.
We suggest that the sulfur coordination of copper in the
immature outer mycelia originates from the binding of
copper with metallothionein. These are small cysteine-rich
peptides primarily produced in response to copper, and
play an important role in Cu homeostasis and sequestra-
tion (Jaeckel et al., 2005; Kumar et al., 2005). Thus, it is
likely that the majority of copper sequestration in young
mycelia occurs intracellularly by this mechanism. Proteins
are not ideal for long-term sequestration of copper and we
propose that there is a shift to a more permanent mode of
storage reflected in the phosphate coordination in the
mature parts of the colony. The possible activity of acid
phosphatases, which have been linked to toxic metal
resistance in fungi, increases with increased copper con-
centration and varies with colony age (Tsekova et al.,
2002; Tsekova and Galabova, 2003). These enzymes
could mediate the transfer of polypeptide-bound copper to
a more stable form such as polyphosphates. Polyphos-
phates have long been invoked in fungal metal
sequestration/tolerance mechanisms (Gadd, 1993) and,
for example, are used by ectomycorrhizal fungi to immo-
bilize metals within their vacuoles (Bucking and Heyser,
1999) which would provide longer-term storage and pro-
tection from toxic effects.
Zinc
In some metal-contaminated soils, a proportion of phos-
phorus may exist as toxic metal phosphate minerals with
very low solubility, e.g. hopeite [Zn3(PO4)2·4H2O] with a
Ksp = 10-35.5. Analysis of some highly contaminated

© 2006 The Authors

Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321

Fig. 3. Copper coordination within fungal biomass: Fourier
transforms of Cu K-edge XANES, EXAFS (solid lines) and fits
(broken lines) for standard compounds (A) Cu(acetate), (B)
Cu(gluconate), (C) Cu(malate), (D) Cu(oxalate), (E) Cu(phosphate)
and (F) Cu(I) oxide. Fungal biomass samples are the
ectomycorrhizal fungus R. rubescens grown in the presence of
copper phosphate on MMN agar medium containing (G) ammonium
and (H) nitrate, and A. niger grown on malt agar amended with
azurite; (I) analysis of marginal part of A. niger colony with new
vegetative mycelium, and (J) central part of colony with older
conidiating mycelium.
organic soils at moderate pH showed that most of the zinc
was organically bound (~45%) and Zn-sorbed phosphate
comprised only ~10% of the total (Sarret et al., 2004).
However, as the formation of metal phosphates in metal-
contaminated soils [phosphate-induced metal stabiliza-
tion (PIMS)] has been proposed as an in situ remediation
technology, this may lead to an increased proportion in
soils of reaction products such as hopeite and mixed-
metal hopeites (Conca, 1997; Chen et al., 1997; Brown
et al., 2004). It is well known that many fungi, including
symbiotic fungi, can solubilize insoluble zinc-bearing min-
erals, e.g. zinc phosphate and zinc oxide (Sayer et al.,
1995; Sayer and Gadd, 1997; Martino et al., 2003;
Fomina et al., 2004; 2005b,c; 2006a). It has been also
shown that Paxillus involutus/pine ectomycorrhizas
exposed to zinc phosphate under phosphorus-deficient
conditions demonstrated a greater zinc mobilization from
zinc phosphate compared with abiotic controls with con-
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
Toxic metal speciation in fungi 313
sequent re-immobilization of zinc within the plant and
fungal biomass (Fomina et al., 2006b).
The results of bulk XAS-analysis of zinc coordination
in biomass (A. niger and P. simplicissimum grown on
ZnO; ectomycorrhizal R. rubescens grown on zinc phos-
phate supplied by either nitrate or ammonium as nitrogen
source; R. rubescens-, P. involutus- and Thelephora
terrestris-Scots pine ectomycorrhizal roots exposed to
zinc phosphate) were typically fitted with six oxygens at
distances between 1.99 and 2.07 Å with no clear evi-
dence for outer shells in any of the samples, although a
peak at c. 4 Å could be fitted well with multiple scattering,
assuming octahedral geometry (Table 2) (Fomina et al.,
2006a,b). This implies either that the zinc is not present in
a coordination environment with a well-defined long range
order, or that there are several zinc species present in
different coordination sites, most likely with carboxylate
ligands, although some phosphate coordination cannot be
ruled out. These data of mixed carboxylate/phosphate
coordination of zinc within the fungi are consistent with
synchrotron-based X-ray microfluorescence and
microEXAFS studies on zinc speciation in the zinc-
hyperaccumulating plant Arabidopsis halleri which
showed that zinc was distributed in Zn-malate, Zn-citrate
and Zn-phosphate (Sarret et al., 2002). Fungal and myc-
orrhizal biomass exposed to toxic metal minerals may
accumulate metals coordinated by different ligands (e.g.
carboxylate and phosphate). One drawback to the tech-
nique used is that it is a bulk measurement, with a sample
area of c. 2 mm ¥ 2 mm, collecting a signal from all the
absorber atoms present, so that if the metal is in several
different coordination sites only an average is seen. This
may be overcome by using micro-XAS techniques if their
sensitivity and resolution is high enough (at least few
microns) to distinguish different metal speciation within
biosystem.
The phosphate groups in fungal biomass can be of
organic and inorganic origin (e.g. nucleic acids, adenosine
phosphates, polyphosphates) and the precipitation of sec-
ondary metal phosphates originating from the initial metal
phosphates can also occur. Carboxyl groups can be
derived from, e.g. low molecular weight organic acids,
proteins, polysaccharides, (poly)phenols/quinones, and
melanin, and these groups have been previously reported
to account for up to 55% and 70% of zinc binding by cell
walls of Penicillium chrysogenum and Trichoderma reesei
respectively (Sarret et al., 1998a). However, in other
EXAFS studies of Zn binding sites in P. chrysogenum cell
walls, zinc was predominantly complexed to four PO4
groups in a tetrahedral configuration (Fourest et al., 1996).
For R. rubescens/Pinus sylvestris ectomycorrhizas
grown in a square Petri dish mesocosms with zinc phos-
phate and nitrate Ingestad agar medium, we have
observed micropatches of zinc precipitation in extramatri-
314 M. Fomina, J. Charnock, A. D. Bowen and G. M. Gadd
cum mycelium. Electron microscopy techniques coupled
with elemental analysis by EDX revealed that zinc was
deposited intracellularly (Fig. 2B and C). Though full
XRPD identification of precipitates was not possible (due
to either very small amount or poor crystallinity), the
absence of a phosphorus peak in the EDX spectra com-
bined with our EXAFS results (Table 2) indicated carboxy-
late coordination of zinc within intracellular deposits.
Some precipitates reached a size more than 1 mm across,
filling more than one-third of the inner space of fungal
cells, probably making them not functional and viable.
This could mean that mycelial system sacrificed some
hyphae to immobilize and detoxify the metal by locking it
within cell walls, enabling it to survive and advance across
the ameliorated environment.
Lead
Lead is the most common toxic metal which becomes
concentrated through the food chain, posing a toxic risk to
many species, including humans (Nriagu, 1996). Pyro-
morphite, or lead chlorophosphate [Pb5(PO4)3Cl], is the
most geochemically stable lead mineral which forms in
urban and industrially contaminated soils and is also one
of the reaction products of PIMS remediation of lead-
contaminated soils (Cotter-Howells and Caporn, 1996;
Conca, 1997; Zhang et al., 1997; Brown et al., 2004).
However, pyromorphite can be solubilized and trans-
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
Fig. 4. Models of copper coordination within
fungal biomass.
A. Model of Cu oxalate coordination within
biomass of R. rubescens in axenic culture and
ectomycorrhizal association with Scots pine
grown in the presence of copper phosphate
on media containing nitrate as a nitrogen
source.
The effect of age/physiological state of
mycelium on copper coordination within
A. niger biomass grown in the presence of
azurite or malachite.
B. Model of phosphate coordination of Cu in
central part of colony.
C. Model of sulfide coordination of Cu in
marginal zone of colony.
D. Colony of A. niger on malt extract agar
medium with white marginal zone of young
vegetative mycelium and black central zone
with profuse conidia production. Scale bar is
1 cm.
E. Abundant conidiophores carrying conidia in
black central zone of A. niger colony. Scale
bar is 200 mm.
formed by some saprotrophic and mycorrhizal fungi
(Sayer et al., 1999; Fomina et al., 2004; 2005b,c). This
ability of fungi to transform pyromorphite should be taken
into account in assessments of the long-term environmen-
tal consequences of in situ chemical remediation
techniques. Some fungi excrete strong chelators (oxalic
acid) that shift the mechanism of pyromorphite dissolution
from acidification to much more efficient ligand-promoted
lead mobilization (Fomina et al., 2004). Fungi are also
able to immobilize lead released from pyromorphite by
extracellular precipitation as lead oxalate (Fomina et al.,
2005c) and by Pb accumulation within the biomass
(Fomina et al., 2004; 2005b). Axenic cultures of ericoid
and ectomycorrhizal fungi grown with pyromorphite accu-
mulated from 2 to 5 mg Pb per g dry wt biomass: isolates
of the ectomycorrhizal fungus Suillus bovinus were able to
accumulate up to 12 mg Pb per g dry wt biomass (Fomina
et al., 2005b). Here, in general, biomass of the ectomyc-
orrhizal fungi (P. involutus, R. rubescens, S. bovinus and
Suillus collimitus), the ericoid mycorrhizal fungus
Hymenoscyphus ericae, and B. caledonica exposed to
pyromorphite demonstrated phosphate coordination of
accumulated Pb with the inner coordination sphere best
fitted with a split shell of one oxygen at 2.14 Å, four
oxygens at 2.40 Å and two oxygens at 3.04 Å and a
significant improvement in the fit on addition of a shell of
two phosphorus scatterers at 3.45 Å (Table 3, Figs 5G
and H and 6A).
© 2006 The Authors
 Toxic metal speciation in fungi 315
Table 2. Zn K-edge EXAFS parameters.

Sample Scatterers r (Å) 2s2 (Å2) Residual

Zn(acetate) standard 4¥O 1.93 0.019 29.7

4¥C 2.86 0.015
4¥O 3.08 0.016
2 ¥ Zn 3.34 0.025
2 ¥ Zn 3.95 0.026
Zn(citrate) standard 6¥O 2.03 0.017 19.6
4¥C 2.78 0.031
Zn3(PO4)2 standard 4¥O 1.96 0.014 23.6
2¥P 3.13 0.032
2 ¥ Zn 3.33 0.016
Zn(gluconate) standard 6¥O 2.06 0.014 17.4
6¥C 2.96 0.047
2 ¥ Zn 3.96 0.026
Zn(malate) standard 6¥O 2.01 0.022 33.6
6¥C 2.81 0.052
2 ¥ Zn 3.88 0.026
Zn(oxalate) standard 6¥O 2.09 0.012 23.2
4¥C 2.81 0.028
12 ¥ O 3.81 0.043
ZnS standard 4¥S 2.35 0.010 31.1
12 ¥ Zn 3.84 0.017
12 ¥ S 4.49 0.022
6 ¥ Zn 5.42 0.016
A. niger on ZnO-containing AP1 agar 6¥O 2.06 0.018 32.0
P. simplicissimum on ZnO-containing AP1 agar 6¥O 2.03 0.024 37.7 (40.9)
R. rubescens axenic culture grown on copper phosphate and ammonium 6¥O 2.07 0.024 27.2
MMN agar medium
R. rubescens/P. sylvestris ectomycorrhizas grown in a square 6¥O 2.02 0.031 22.7
medium Petri dish mesocosm with zinc phosphate and nitrate Ingestad agar
R. rubescens/P. sylvestris ectomycorrhizas grown in a square Petri 6¥O 2.07 0.016 28.8 (22.6)
dish mesocosm with zinc phosphate and Ingestad agar medium
P. involutus/P. sylvestris ectomycorrhizas grown in a square Petri 6¥O 2.06 0.017 33.8 (27.9)
dish mesocosm with zinc phosphate and Ingestad agar medium
T. terrestris/P. sylvestris ectomycorrhizas grown in a square Petri 6¥O 2.05 0.023 33.1 (25.4)
dish mesocosm with zinc phosphate and Ingestad agar medium

r is the zinc–scatterer distance in Angstroms, ⫾0.02 Å inner shells, ⫾0.05 Å outer shells; 2s2 is the Debye–Waller type factor, ⫾15% inner shells,
⫾30% outer shells (a measure of both the thermal motion between the absorber and scatterer and of the static disorder or range of absorber–
scatterer distances); the residual is a least squares residual from fitting the spectrum of the model to the experimental data. For some samples
inclusion of multiple scattering, assuming an octahedral coordination site, improved the fit but didn’t change the fitting parameters. In these cases
the residuals in brackets in the final column are the residuals without multiple scattering.

Fungal ability to solubilize other lead-bearing com-
pounds (lead phosphate, tetraoxide, carbonate and
sulfide) has also been shown previously (Sayer and
Gadd, 1997; Fomina et al., 2005c). For most of the
EXAFS spectra recorded for A. niger, B. caledonica and
P. simplicissimum grown on nitrate- or ammonium-
containing media with lead oxides (PbO or Pb3O4), only
the inner coordination sphere could be fitted, in all cases
with oxygen scatterers (Table 3). The nitrogen source did
not seem to alter lead coordination. For most samples, the
spectra were noisy with useful data up to a k-range of
7–8 Å-1 and the best fit with a single shell of oxygen
scatterers at 2.39 Å. For B. caledonica grown on PbO-
and ammonium-containing medium a fit of six oxygens at
2.44 Å could be improved by splitting into two, at 2.14 Å
and 2.40 Å (Table 3). However, in two cases when
A. niger was grown on PbO- and nitrate-containing
medium and B. caledonica was grown on Pb3O4- and
ammonium-containing medium, where outer shells could
be fitted, the indication was that carboxylic groups were
the main ligands rather than phosphates (Table 3, Fig. 5I
and J). For example, EXAFS data for A. niger were col-
lected to a k-range of 11 Å-1. The main peak could be
fitted with a shell of six to eight oxygens at 2.55 Å, but the
fit was improved by splitting this into two shells, at 2.45 Å
and 2.56 Å. The coordination numbers should not be
regarded as definitive, because with the large Pb ion there
is usually significant disorder in the coordinated Pb-O
distances and consequently large errors in coordination
numbers. Outer shells were best fitted with carbons at
3.31 Å, implying carboxylic acid coordination, and a
heavier element, such as iron but not lead, at 3.81 Å
(Fig. 6B).
It has been reported that coordination of lead within
fungal biomass can be affected by lead concentration
(Sarret et al., 1999). Previous EXAFS studies of lead

© 2006 The Authors

Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
316 M. Fomina, J. Charnock, A. D. Bowen and G. M. Gadd
Table 3. Pb L(III)-edge EXAFS parameters.

Sample Scatterers r (Å) 2s2 (Å2) Residual

Pb5Cl(PO4)3 standard 0.5 ¥ O 2.31 0.009 59.2

5¥O 2.56 0.066
1 ¥ Cl 2.87 0.075
2¥P 3.24 0.035
1 ¥ Pb 3.32 0.018
Pb3O4 standard 4.67 ¥ O 2.19 0.021 37.5
0.67 ¥ Pb 3.30 0.012
2.67 ¥ Pb 3.61 0.015
3.33 ¥ Pb 3.78 0.020
Pb(oxalate) standard 3¥O 2.39 0.026 45.7
4¥O 2.59 0.066
6¥C 3.22 0.026
4 ¥ Pb 3.99 0.040
PbS standard 6¥S 2.95 0.022 37.7
12 ¥ Pb 4.19 0.021
8¥S 5.04 0.047
6 ¥ Pb 5.99 0.019
Pb3(PO4)2 standard 4¥O 2.38 0.044 56.4
4¥O 2.61 0.039
4¥P 3.29 0.056
PbO standard 4¥O 2.26 0.016 55.1
4 ¥ Pb 3.59 0.027
4 ¥ Pb 3.72 0.011
4 ¥ Pb 3.93 0.017
Pb(II) (acetate) standard 4¥O 2.49 0.046 71.2
Pb(IV) (acetate) standard 8¥O 2.24 0.020 42.8
4¥C 2.68 0.007
4¥C 4.15 0.012
Pb(malate) standard 4¥O 2.46 0.070 65.4
B. caledonica grown on pyromorphite-containing MMN agar 4¥O 2.62 0.010 51.0
4¥O 2.79 0.007
4¥P 3.25 0.044
H. ericae DGC3 grown on pyromorphite-containing MMN agar 2¥O 2.02 0.036 45.4
4¥O 2.31 0.027
2¥P 3.49 0.061
H. ericae C2 grown on pyromorphite-containing MMN agar 1¥O 1.89 0.013 54.7
4¥O 2.20 0.024
2¥O 2.42 0.015
2¥P 3.52 0.033
P. involutus 23 grown on pyromorphite-containing MMN agar 1¥O 2.11 0.015 50.6
4¥O 2.38 0.030
2¥O 3.13 0.090
2¥P 3.68 0.050
R. rubescens B20 grown on pyromorphite-containing MMN agar 1¥O 2.13 0.021 49.4
4¥O 2.39 0.031
2¥O 3.02 0.030
2¥P 3.55 0.054
S. bovinus Lst8 grown on pyromorphite-containing MMN agar 1¥O 2.14 0.010 51.5
4¥O 2.40 0.025
2¥O 3.04 0.012
2¥P 3.45 0.021
S. collimitus 22 grown on pyromorphite-containing MMN agar 1¥O 2.18 0.015 40.7
4¥O 2.42 0.017
2¥O 2.62 0.015
2¥P 3.61 0.029
A. niger grown on PbO and nitrate-containing AP1 agar 4¥O 2.45 0.027 31.4
4¥O 2.56 0.010
4¥C 3.31 0.014
4 ¥ Fe 3.81 0.026
A. niger grown on Pb3O4 and ammonium-containing AP1 agar 6¥O 2.55 0.034 81.8
A. niger grown on Pb3O4 and nitrate-containing AP1 agar 6¥O 2.49 0.042 53.2
B. caledonica grown on PbO and nitrate-containing AP1 agar 6¥O 2.42 0.051 59.4
B. caledonica grown on Pb3O4 and nitrate-containing AP1 agar 6¥O 2.39 0.059 48.0
B. caledonica grown on PbO and ammonium-containing AP1 agar 2¥O 2.14 0.043 46.8
4¥O 2.40 0.023
B. caledonica grown on Pb3O4 and ammonium-containing AP1 agar 2¥O 2.17 0.056 29.0
4¥O 2.41 0.032
4¥C 3.30 0.042
P. simplicissimum grown on Pb3O4 and nitrate-containing AP1 agar 2¥O 2.22 0.059 56.4
4¥O 2.48 0.014
P. simplicissimum grown on PbO and ammonium-containing AP1 agar 6¥O 2.40 0.060 49.8
P. simplicissimum grown on Pb3O4 and ammonium-containing AP1 agar 6¥O 2.40 0.072 50.1

r is the lead–scatterer distance in Angstroms, ⫾0.02 Å inner shells, ⫾0.05 Å outer shells; 2s2 is the Debye–Waller type factor, ⫾15% inner shells, ⫾30% outer shells
(a measure of both the thermal motion between the absorber and scatterer and of the static disorder or range of absorber–scatterer distances); the residual is a least
squares residual from fitting the spectrum of the model to the experimental data.

© 2006 The Authors

Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321

Fig. 5. Lead coordination within fungal biomass: Fourier transforms
of Pb L(III)-edge XANES, EXAFS (solid lines) and fits (broken
lines) for standard compounds: (A) Pb(malate), (B) Pb(oxalate), (C)
Pb(II) (acetate), (D) Pb3(PO4)2, (E) PbO, (F) Pb3O4. Fungal biomass
samples are (G) S. bovinus Lst8 and (H) B. caledonica grown in
the presence of pyromorphite, and (I) A. niger grown on AP1 agar
medium containing nitrate and PbO, and (J) B. caledonica grown
on AP1 agar medium containing ammonium and Pb3O4.
bound to cell walls of P. chrysogenum showed that car-
boxyl groups have greater affinity for lead ions and pref-
erentially bound Pb at low metal concentrations whereas
at higher concentrations phosphoryl groups accounted for
up to 95% of Pb binding, with carboxyl accounting for the
remaining 5% (Sarret et al., 1999). Our data have shown
that the Pb-O distances were all consistent with Pb(II)
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
Toxic metal speciation in fungi 317
species (Table 3). This means that the fungi which grew
on and solubilized red lead oxide (Pb3O4) with mixed lead
valencies [Pb(II) + Pb(IV)] selectively accumulated Pb(II)
in the biomass. However, it is known that the Pb(II) oxi-
dation state is more stable than Pb(IV), and there is a
strong tendency for lead(IV) compounds to react to give
lead(II) compounds, especially under acidic conditions.
Conclusions
Our goal was to determine the speciation of toxic metals
accumulated by fungi and mycorrhizal associations
exposed to toxic metal minerals. XAS techniques gave us
a means of revealing the speciation of toxic metals that
underwent a cycle of chemical transformations caused by
fungi.
Our data have shown that:
(i) Oxygen ligands (phosphate and carboxylate) played
a major role in metal coordination within fungal and
ectomycorrhizal biomass during the accumulation of
toxic metals mobilized by fungi from metal phos-
phates and oxides.
(ii) A copper sulfide phase was found in fungi grown on
azurite and malachite.
(iii) Metal precipitation by fungal and mycorrhizal
biomass was not homogeneous in many cases dem-
onstrating mixed metal coordination and indicating
some limitations of bulk XAS analysis of biomass.
(iv) Coordination of toxic metals within biomass
depended on the fungal species, the supplied
mineral, nitrogen source, and the physiological state/
age of the fungal mycelium.
Metal-contaminated terrestrial environments are
dynamic systems with metal contaminants changing their
speciation with time depending on physico-chemical con-
ditions and biogeochemical activities of the biota. The ‘in
situ’ situation in metal-polluted soils has a much more
complex geochemical environment than that used in our
experiments. However, our experiments with ectomycor-
rhizal associations grown in the zinc phosphate spiked
Fig. 6. Models of lead coordination within
fungal biomass.
A. Model of phosphate coordination of lead
accumulated within biomass of a variety of
mycorrhizal and saprotrophic fungi (e.g.
H. ericae, P. involutus, S. bovinus,
S. collimitus) grown in the presence of
pyromorphite.
B. A model for carboxylate coordination of
lead accumulated by A. niger grown on PbO-
and nitrate-containing AP1 agar medium with
outer shells also fitting a metal element (Met)
such as iron or nickel.
318 M. Fomina, J. Charnock, A. D. Bowen and G. M. Gadd
mesocosms, where vermiculite/peat matrix represented
both inorganic and organic soil components, simulated
metal-polluted ‘field’ conditions. It has been observed that
ectomycorrhizas immobilized half of total zinc mobilized
from zinc phosphate whereas only one-tenth of mobilized
zinc was immobilized by the vermiculite/peat matrix (see
Fomina et al., 2006b). Thus, in phytoremediation and
reforestation/revegetation sites with abundant vegetation,
phyto- and fungal biomass may play a crucial role in toxic
metal transformations and immobilizations.
The conditions of growth both in vitro and ex vitro may
change the nature of ligands provided by fungal and myc-
orrhizal biomass. For example, the change of nitrogen
source from ammonium to nitrate may increase the pro-
portion of oxalate excretion and subsequently shift
towards oxalate coordination of toxic metal (copper)
within biomass (R. rubescens). However, it appears that
for certain toxic metal minerals fungal/mycorrhizal
biomass provides the same selection of functional groups
(e.g. phosphate, carboxylate, sulfhydryl) in both agar and
vermiculite/peat and in both axenic culture and ectomyc-
orrhizal association, with an obvious predominance of
oxygen-containing ligands. It is likely that ‘in situ’ fungal/
mycorrhizal biomass, comprising the largest biomass in
soils, will also provide those functional groups for metal
immobilization.
The roles of free-living and symbiotic fungi have previ-
ously been a much underestimated component of metal
biogeochemistry but these organisms are involved in pro-
cesses of both metal mobilization and immobilization.
Understanding the molecular nature of toxic metal immo-
bilization by fungi is of relevance to the development and
maintenance of efficient remediation techniques as well
as a fuller understanding of the chemical and biological
interactions of organisms to environmental pollutants.
Experimental procedures
Fungi and mycorrhizas
Fungal cultures (saprotrophic Aspergillus niger, Penicillium
simplicissimum, entomopathogenic Beauveria caledonica,
ericoid mycorrhizal Hymenoscyphus ericae and Oidioden-
dron maius and ectomycorrhizal Lactarius turpis, Paxillus
involutus, Rhizopogon rubescens, Suillus bovinus, Suillus
collimitus) and ectomycorrhizal roots of associations of Scots
pine (Pinus sylvestris) with P. involutus, R. rubescens and
T. terrestris were used in this study (see Fomina et al.,
2005b). We exposed fungi and ectomycorrhizas to a variety
of copper-, zinc- and lead-containing minerals at a final metal
concentration equivalent to 15 mM (copper and zinc phos-
phates and oxides [Cu3(PO4)2.2H2O (Fluka), Zn3(PO4)2.2H2O
(Alfa) (naturally occurring as hopeite), CuO (Aldrich), Cu2O
(BDH) (naturally occurring as cuprite), ZnO], azurite
[Cu3(CO3)2(OH)2], malachite [Cu2(CO3)(OH)2], pyromorphite
[Pb5(PO4)3Cl] (Fomina et al., 2004), lead oxide and tetraoxide
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
[PbO and Pb3O4 (Aldrich) (red lead)]). Axenic cultures of fungi
were grown at 23–25°C in sterile Petri-dish microcosms on
top of cellophane membranes on a variety of media: malt
extract agar, AP1 medium [(NH4)2SO4 (5 g l–l), KH2PO4
(0.5 g l–l), MgSO4.7H2O (0.2 g l–l), CaCl2.6H2O (50 mg l–l),
NaCl (100 mg l–l), FeCl3.6H2O (2.5 mg l–l), ZnSO4.7H2O,
MnSO4.4H2O, and CuSO4.5H2O (each 4 mg l–l), D-glucose
(20 g l-1), agar No. 1 (Laboratory M, Bury UK) (15 g l–l)], and
modified Melin-Norkrans medium for mycorrhizal fungi
(MMN) [(NH4)2HPO4 (0.5 g l–l), KH2PO4 (0.3 g l–l), MgSO4.
7H2O (0.14 g l–l), CaCl2.6H2O (50 mg l–l), NaCl (25 mg l–l),
D-glucose (10 g l-1), glutamic acid (1 mg l–l), thiamine
(100 mg l–l) and agar No. 1 (Laboratory M, Bury UK) (14 g l–l)]
(Fomina et al., 2005b). Previously it was shown that all tested
fungi were able to solubilize zinc- and copper-containing min-
erals and pyromorphite with oxalic and citric acid overproduc-
ing fungi A. niger and B. caledonica being very efficient
mineral transformers (Sayer et al., 1999; Fomina et al., 2004;
2005b,c). Biomass of fast-growing saprotrophic fungi A. niger
and P. simplicissimum was harvested after 7 day growth,
other slow growing fungi were harvested after 2 months.
Synthesis of ectomycorrhizas was carried out under sterile
conditions using a test tube technique (Peterson and Chakra-
varty, 1991) in autoclaved (120°C, 60 min) glass tubes
(culture tubes 25 mm ¥ 150 mm, Sigma-Aldrich Cat C 5916,
Dorset, UK) filled with 30 ml vermiculite (Sinclair, Lincoln, UK)
and peat (Irish Moss Peat, B and Q plc, Chandlers Ford, UK)
mixture 5:1 and 10 ml of Ingestad medium comprising: K2SO4
(12.25 mg l–l); KH2PO4 (8.58 mg l–l); K2HPO4 (10.11 mg l–l);
KNO3 (9.71 mg l–l); NH4NO3 (58.56 mg l–l); Ca(NO3)2
(8.61 mg l–l); Mg(NO3)2 15.84 mg l–l; HNO3 (13.7 mg l–l);
H3BO3 (0.57 mg l–l); Mn(NO3)2 (0.913 mg l–l); Zn(NO3)2
(0.07 mg l–l); CuCl2 (0.04 mg l–l); Na2MoO4 (0.009 mg l–l);
FeNaEDTA (1.149 mg l–l) (Ingestad, 1979). D-glucose
(0.5 g l–l) was also added to the medium to initiate fungal
growth in the matrix. Seeds of Pinus sylvestris L. (Chiltern
seeds, Cat no. 1014, Ulverston, Cumbria, UK) were surface-
sterilized in 30% hydrogen peroxide with one drop of Tween
60 for 30 min, then thoroughly washed with sterile water and
transferred into Petri dishes containing MMN agar medium to
germinate for at least 14 days. Germinating pine seeds were
planted into vermiculite/peat matrix together with four discs of
7 mm diameter discs of mycelium cut from the leading edge
of colonies which had been maintained on MMN at 25°C for
at least 14 days. Seedlings were cultivated for 3–4 months in
the growth chamber (Fisons Fi-totron 600 Growth Cabinet,
Suffolk, UK) with 200 mmol m-2 s-1 PAR, at least 60% relative
air humidity and at day/night regime of 18/6 h and a tempera-
ture of 23/15°C. Mycorrhizal colonization of the roots was
nearly 85–90%. After successful colonization, uniform pine
seedlings were carefully taken out from tubes, washed with
sterile water and transferred into sterile square Petri dishes
mesocosms. Square Petri dishes were filled with Ingestad
agar amended with zinc or copper phosphate to a final metal
concentration equivalent to 15 mM as for axenic cultures of
fungi. The roots of mycorrhizal seedlings were placed over
sterile cellophane membrane on top of the agar medium. The
upper cellophane membrane and an autoclaved piece of
foam, soaked in sterile water, were placed on top of the roots
to prevent them from desiccation and to fix the position of the
root system. The shoot was kept outside of the Petri dish by
© 2006 The Authors

means of a hole in the side of the plastic dish and sealed
around the stem with sterile lanolin. Mesocosms were incu-
bated for at least 3 months under the same conditions as
described above. In some experiments with axenic fungal
cultures and the R. rubescens/P. sylvestris ectomycorrhizal
association we also used a nitrate-only version of MMN/AP1
and Ingestad media substituting KNO3 (0.8 g l–l and
17.34 mg l–l) for (NH4)2HPO4 and NH4NO3 respectively.
Biomass of fungi and ectomycorrhizas grown over cello-
phane membrane in Petri dishes was harvested by peeling
from the membrane. Ectomycorrhizal roots of seedlings
grown in vermiculite/peat mixture were removed from the
matrix, washed with distilled water, dried with paper towels,
and then cut with a razor blade. Freshly harvested biomass of
fungi and ectomycorrhizas, enclosed in cellotape and
quenched in liquid nitrogen, was used for X-ray absorption
spectrometry.
Electron microscopy
Accumulation of toxic metals by fungal mycelia was studied
using scanning and transmission electron microscopy. For
scanning electron microscopy we used air-dried samples,
uncoated or coated with 30 nm Au/Pd using a Cressington
208 HR sputter coater. Samples were examined using a
Philips XL30 environmental scanning electron microscope
(ESEM) field emission gun (FEG) operating at an accelerat-
ing voltage of 15 or 25 kV. For transmission electron micro-
scopy we used samples of mycelium fixed in 2.5%
glutaraldehyde in 5 mM 1,4-piperazinediethanesulfonic acid
(PIPES) buffer, pH 6.5, washed in PIPES buffer, then dehy-
drated with an ascending series of ethanol in distilled water
and embedded into L.R. White resin. Unstained ultra-thin
sections (cut on a Reichert OMU-3 microtome) mounted on
pioloform-copper grids were examined using a Jeol-1200 EX
transmission electron microscope.
X-ray absorption spectrometry
Cu and Zn. X-ray absorption spectra at the Cu and Zn
K-edges were collected on Station 7.1 at the CCLRC Dares-
bury SRS operating at 2 GeV with an average current of
140 mA, using a vertically collimating plane mirror and a
sagittally bent focusing Si(111) double crystal monochroma-
tor detuned to 80% transmission to minimize harmonic
contamination. Sample data were collected with the station
operating in fluorescence mode using a nine-element solid
state Ge diode detector with high count-rate XPRESS pro-
cessing electronics: spectra of model compounds were col-
lected in the transmission mode. The monochromator was
calibrated using a 5 mm Cu or Zn foil. Experiments were
performed using a liquid nitrogen cooled cryostat. Single
scans were collected for the model compounds, and three to
four scans were collected and summed for each sample.
Model compounds used were Cu-acetate, Cu-gluconate,
Cu-malate, Cu-oxalate, Cu3(PO4)2 and Cu2O for Cu and
Zn-acetate, Zn-citrate, Zn-gluconate, Zn-malate, Zn-oxalate,
Zn3(PO4)2 and ZnS for Zn.
Pb. X-ray absorption spectra at the Pb L(III)-edge were col-
lected on Station 16.5 at the CCLRC Daresbury SRS oper-
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 308–321
Toxic metal speciation in fungi 319
ating at 2 GeV with an average current of 150 mA, using a
vertically focusing mirror and a sagittally bent focusing
Si(220) double crystal monochromator detuned to 70% trans-
mission to minimize harmonic contamination. Data were col-
lected with the station operating in fluorescence mode using
an Ortec 30 element solid state Ge detector. The monochro-
mator calibration was checked using a Pb3(PO4)2 standard.
Experiments were performed in a liquid nitrogen cooled
cryostat. Spectra of model compounds were collected in
transmission mode. Single scans were collected for the
model compounds, and three to four scans were collected
and summed for each sample. Model compounds were
Pb-oxalate, Pb3(PO4)2, Pb5Cl(PO4)3, Pb3O4, PbO, Pb-malate,
Pb(II)-acetate, Pb(IV)-acetate and PbS.
EXAFS spectra analysis. Background-subtracted EXAFS
spectra were analysed in EXCURV98 using full curved wave
theory (Gurman et al., 1984; Binsted, 1998). Phaseshifts
were derived in the program from ab initio calculations using
Hedin–Lundqvist potentials and von Barth ground states
(Hedin and Lundqvist, 1969). Fourier transforms of the
EXAFS spectra were used to obtain an approximate radial
distribution function around the central metal (Cu, Zn or Pb)
(the absorber atom): the peaks of the Fourier transform can
be related to ‘shells’ of surrounding back scattering atoms
characterized by atom type, number of atoms in the shell, the
absorber–scatterer distance, and the Debye–Waller factor,
2s2 (a measure of both the thermal motion between the
absorber and scatterer and of the static disorder or range of
absorber–scatterer distances). The data were fitted for each
sample by defining a theoretical model and comparing the
calculated EXAFS spectrum with the experimental data.
Shells of back scatterers were added around the absorber
and by refining an energy correction Ef (the Fermi energy),
the absorber–scatterer distance, and Debye–Waller factor for
each shell, a least squares residual [the R-factor (Hedin and
Lundqvist, 1969; Binsted et al., 1992) was minimized]. Where
appropriate, multiple scattering effects were included in the
fits (Gurman et al., 1986).
Acknowledgements
G.M.G. gratefully acknowledges funding from the BBSRC/
BIRE programme (94/BRE13640) and CCLRC Daresbury
SRS (SRS user grants 40107 and 43079). The authors grate-
fully acknowledge the help of the 7.1 and 16.5 stations sci-
entist Drs Fred Mosselmans, Lorrie Murphy, Bob Bilsborrow
and Steve Fiddy (CCLRC Daresbury SRS) and Dundee Uni-
versity team members Mr Ewan Starke, Miss Karrie Melville,
Mr Martin Kierans, Miss Louise McGregor, Dr Euan Burford,
Dr Simon Hockin and Dr Ademola Adeyemi. A.D.B. gratefully
acknowledges receipt of a BBSRC postgraduate research
studentship. We thank Professor Andy Meharg and Dr David
Genney (University of Aberdeen, Scotland), Dr Elena Martino
(University of Torino, Italy), Dr Derek Mitchell (University
College Dublin, Ireland), Dr Jan Colpaert (Limburgs Univer-
sity Centre, Belgium), Dr Claude Plassard (INRA, France),
and Dr Håkan Wallander (Lund University, Sweden) for the
provision of fungal strains, and Mr Martin Kierans [Centre for
High Resolution Imaging and Processing (CHIPs), School of
Life Sciences, University of Dundee, Scotland] for assistance
with electron microscopy.
320 M. Fomina, J. Charnock, A. D. Bowen and G. M. Gadd
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