Minerals Engineering xxx (2016) xxx–xxx

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Minerals Engineering
journal homepage: www.elsevier.com/locate/mineng

Fungal biomineralization of lead phosphates on the surface of lead metal

Cristina Povedano-Priego, Inés Martín-Sánchez, Fadwa Jroundi, Iván Sánchez-Castro,
Mohamed L. Merroun ⇑
Department of Microbiology, University of Granada, Campus Fuentenueva s/n 18071, Granada, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The present work describes lead phosphate biomineralization by a wood-decaying fungal isolate,
Received 28 July 2016 Penicillium chrysogenum A15, after incubation in solid medium with metallic lead (Pb shot pellets).
Revised 7 November 2016 This fungal isolate showed high tolerance to this element, being able to grow on solid medium containing
Accepted 9 November 2016
8 mM Pb(II). Environmental Scanning Electron Microscope (ESEM) observations of abiotic controls
Available online xxxx
showed the presence of lead oxide crystals on the lead shot surface after 8 weeks incubation.
However, in presence of P. chrysogenum, lead phosphate mineral phases were detected as aggregates
Keywords:
on the surface of lead shots after 2-weeks and 8-weeks incubation. The morphology of the biotically
Fungi
Tolerance
deposited secondary minerals showed significant differences in comparison to those produced under abi-
Lead phosphates otic conditions, appearing globular, prismatic and acicular crystals. High Resolution Transmission
Environmental Scanning Electron Electron Microscope (HRTEM) analysis indicated deposition of lead phosphates on the cell surface which
Microscopy could be considered as Pb resistance mechanism used by this isolate to cope with toxicity of lead. This
High Resolution Transmission Electron study extends the spectrum of fungal isolates with potential on the biomineralization of lead phosphates
Microscopy useful for the bioremediation of lead contaminated sites.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction (Merroun and Selenska-Pobell, 2008; Morcillo et al., 2014), intra-

Lead is a well-known and hazardous heavy metal that may
cause serious ecological and human health problems as was
reported by European Commission (2002). This element is widely
used in several industrial activities such as lead smelting, leaded
petrol production, and reactional shooting and hunting products
(Dermatas et al., 2006; Sullivan et al., 2012). Many occidental
countries like USA, Canada, England, etc. reported high annual
deposition rates of metallic Pb in the environment through anthro-
pogenic activities associated with angling, hunting and reactional
shooting (Sullivan et al., 2012). In soil, the speciation of metallic
lead used in shooting activity, is controlled by several processes
such as sorption, precipitation and complexation with various
organic and inorganic soil colloids (Zhang et al., 1997). However,
in highly heavy metal contaminated soils, the mobilization of
metallic lead may occur through processes such as dissolution,
hydration, carbonation or oxidation mediated by abiotic (exposure
to air, water, and varying pH conditions) or biotic factors (Cao et al.,
2003; Dermatas et al., 2004; Vilomet et al., 2003).
Regarding the biotic processes, it is well-known that soil micro-
bial populations play an important role in the biogeochemical cycle
of metals through different processes including biosorption
⇑ Corresponding author.
E-mail address: merroun@ugr.es (M.L. Merroun).
cellular accumulation (Brookshaw et al., 2012), biomineralization
(Merroun et al., 2011; Mondani et al., 2011) and chelation by
organic acid production, leading to the immobilization or mobiliza-
tion of heavy metal (Gadd et al., 2012). Among the soil microorgan-
isms, indigenous fungi are able to solubilize metal compounds
releasing phosphates and other ligands, which in turn lead to the
liberation of metal cations (e.g. Pb). Conversely, fungi interact with
lead through immobilization processes such as biomineralization
of lead oxalates, phosphates and carbonates (Gadd et al., 2012).
Lead phosphate biomineralization by fungi and bacteria is con-
sidered an attractive and efficient bioremediation strategy, due to
the long-term stability of this mineral phase. Pyromorphite (Pb5[-
PO4]3X, where X = F, Cl, Br, OH) is known as the most stable lead
mineral occurring in terrestrial environments (Casas and Sordo,
2006; Debela et al., 2010; Miretzky and Fernandez-Cirelli, 2008),
with a very low solubility product (Ksp) of 10 84.4, (Miretzky and
Fernandez-Cirelli, 2008; Nriagu, 1974). Therefore, pyromorphite
formation has previously been proposed as a remediation treat-
ment for immobilization of contaminant lead (Cao et al., 2003;
Hettiarachchi et al., 2001; Ma et al., 1993; Miretzky and
Fernandez-Cirelli, 2008). In this respect, Rhee et al. (2014)
described pyromorphite within a fungal biofilm community
growing on Pb sheeting in natural environment, thus providing
novel evidences on the fungal involvement in lead
biogeochemistry.

http://dx.doi.org/10.1016/j.mineng.2016.11.007
0892-6875/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Povedano-Priego, C., et al. Fungal biomineralization of lead phosphates on the surface of lead metal. Miner. Eng. (2016),
http://dx.doi.org/10.1016/j.mineng.2016.11.007
2 C. Povedano-Priego et al. / Minerals Engineering xxx (2016) xxx–xxx
The present study describes the ability of a fungal strain iso-
lated from decayed woods to transform metallic lead into lead
phosphates. The resulting lead mineral phase was characterized
by applying a multidisciplinary approach combining Environmen-
tal Scanning Electron Microscopy (ESEM) and High Resolution
Transmission Electron Microscopy (HRTEM).
2. Material and methods
2.1. Fungal strains and culture conditions
Fungal strains used in the present work, Penicillium chryso-
genum A15, Aspergillus niger A43 and Trichoderma viride B30, were
previously isolated from decayed wood collected from historic edi-
fications (Castro-Rodriguez, 2013). These fungal strains were main-
tained on potato dextrose agar (PDA: 4 g/L potato extract, 20 g/L
dextrose, 15 g/L agar) and grown in malt extract agar (MEA: 5 g/L
peptones, 15 g/L malt extract, 15 g/L agar) during 14 days at 28 °C.
2.2. Metal stock solution preparation
A stock solution of Pb(NO3)2 at 0.1 M was prepared by dissolv-
ing the appropriate quantity of metal salt in 0.1 M NaClO4. The
stock solution was sterilized by filtration through 0.22 lm nitro-
cellulose filters and stored at 4 °C until use. Working solutions
were prepared by dilutions of the stock solution.
2.3. Determination of lead minimal inhibitory concentration (MIC)
Tolerance of the tested fungi to Pb(II) was determined as the
Minimal Inhibitory Concentration (MIC). The MIC is defined as
the lowest metal concentration where no visible fungal growth
occurs. Discs of fungal mycelium (5 mm diameter) were placed
on MEA, solid medium containing increasing concentrations of
Pb(II) nitrate (1, 2, 4 and 8 mM). After inoculation, all plates were
incubated in the dark at 25 °C for 7 days.
2.4. Interaction of metal lead with Penicillium chrysogenum
For Pb-fungal interaction study, individual oven-sterilized
(105 °C for 3 days) lead shots (4.5 mm in diameter) were placed
aseptically at regular intervals on the surface of MEA plates leaving
free the center of the plate to place the fungal disc, as described by
Rhee et al. (2012). Afterwards, the plates were inoculated with
5 mm diameter discs of two weeks-old fungal mycelium. The
plates were incubated for 2, 4 and 8 weeks in the dark at 25 °C. Abi-
otic control consisted of lead shots placed on the surface of MEA
culture medium and incubated at the same conditions as described
above. Experimental and control plates were prepared in triplicate.
After incubation, the lead shots were aseptically recollected and
prepared for the different microscopic analyses.
2.4.1. Environmental scanning electron microscopy and
energy-dispersive X-ray analyses
Lead shots and their surrounding fungal hypha collected after 2,
4 and 8 weeks incubation were fixed in 3% glutaraldehyde
prepared in cacodylate buffer at 4 °C. Then, they were washed
three times for 15 min with cold-cacodylate buffer. Afterwards,
the samples were post-fixed in 1% OsO4 for one hour in the dark,
at room temperature. Next to washing was performed with
distilled water (3  5 min), samples were dehydrated through
gradients of ethanol (50, 70, 90 and 100%) for 15 min each wash,
followed by three additional times in 100% ethanol for 15 min
each. The dehydrated samples were then critical point dried
(Anderson, 1951) using a Leica EM CPD300 Critical Point Dryer.
Please cite this article in press as: Povedano-Priego, C., et al. Fungal biomineralization of lead phosphates on the surface of lead metal. Miner. Eng. (2016),
http://dx.doi.org/10.1016/j.mineng.2016.11.007
Samples were immediately mounted on aluminum stubs using car-
bon adhesive tapes and coated with carbon by EMITECH K975X
coater. The coated samples were observed using a Quanta 400 FEI
ESEM operating at an accelerating voltage of 15 kV and 20 kV for
the observation of biocorrosion products formed on the surface
and around the lead shots.
The elemental composition of the secondary minerals formed
on the surface and the periphery of lead shots was determined
by energy-dispersive X-ray (EDX) analysis system. Spectra were
acquired using EDX analysis system embedded within an environ-
mental scanning electron microscope (Quanta 400 FEI ESEM) oper-
ating at an accelerating voltage of 20 kV. EDX provides elemental
information via analysis of the X-ray emission caused by a high
electron energy beam.
2.4.2. High resolution transmission electron microscopy and energy-
dispersive X-ray analyses
Lead-treated fungal biomass was fixed in 2% glutaraldehyde (in
0.1 M cacodylate buffer, pH 7.4) for 2 h at 4 °C and then washed
with the same cacodylate buffer three times for 10 min. Samples
were dehydrated with ethanol gradients from 30% to 100%, fol-
lowed by propylene oxide treatment for 10 min, then embedded
in a mixture of propylene oxide-resin (EPON) (1:1) and kept over-
night in pure resin, to be afterwards polymerized in heater at 60 °C
for 14 h. The samples were thin-sectioned using a Reichert Ultracut
S ultramicrotome, and the sections were supported on copper grids
and coated with carbon. Samples were examined using a Philips CM
200 high-resolution transmission electron microscope at an accel-
eration voltage of 200 kV and MegaViewIII camera under standard
operating conditions with the liquid nitrogen anticontaminator in
place. EDX analysis was also performed at 200 kV using a spot size
of 7 nm and a live counting time of 20 s.
3. Results
3.1. Tolerance of fungal strains to Pb(II): minimal inhibitory
concentration (MIC)
The MIC of Pb(II) for the growth of the three tested fungal iso-
lates is presented in Table 1. The results indicated that the strains
A. niger A43 and P. chrysogenum A15 were able to grow at 8 mM
lead-amended MEA medium. The MIC of Pb for the growth of T. vir-
ide B30 was 8 mM.
The contact of the mycelium with incremented Pb concentra-
tions resulted in phenotypic changes including sporulation inhibi-
tion (more relevant in P. chrysogenum), colored-mycelium changes
and variations in the morphology and the size of the fungal colo-
nies (T. viridae) (Figs. 1 and 2).
3.2. Interaction of Penicillium chrysogenum A15 with metallic lead
The interaction of fungal hyphae with the lead shot was macro-
scopically monitored for a total of two months (Fig. 3). After 9 days
incubation, the mycelium covering lead shots presented a greenish
color in comparison to the free-lead shot mycelium, which
Table 1
Minimal inhibitory concentration (mM) of lead for the growth of the three tested
fungal species.
Fungal strain MIC (mM Pb)
Aspergillus niger A43 >8
Penicillium chrysogenum A15 >8
Trichoderma viride B30 8
 C. Povedano-Priego et al. / Minerals Engineering xxx (2016) xxx–xxx 3

Fig. 1. Fungal colonies growing in presence of increasing Pb concentrations (4 mM and 8 mM): Aspergillus niger A43 (A), Penicillium chrysogenum A15 (B) and Trichoderma
viride B30 (C). Growth inhibition is observed in 8 mM lead-amended MEA medium (T. viride), along with morphological changes induced by the metal.

Fig. 2. Penicillium chrysogenum A15 colonies growing in Pb-containing MEA medium: control, 4 mM and 8 mM Pb. Darkening of mycelium is observed.

remained white (Fig. 3A). After 8 weeks incubation, the fungal bio-
mass exhibited more uniform color (Fig. 3D).
3.2.1. ESEM and EDX analyses of lead shots
The surface of the uninoculated lead shots (abiotic controls) and
those incubated with P. chrysogenum for 2 and 8 weeks exhibited
differing morphological changes. ESEM images showed that the
abundant mycelium covering the lead shots after 2 weeks of incu-
bation with P. chrysogenum disappeared over time (Fig. 4).
Corrosion products were observed on the surface of the lead
shots in all cases (abiotic and biotic conditions). Control lead shots
showed the formation of crystals with different sizes on the surface

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4 C. Povedano-Priego et al. / Minerals Engineering xxx (2016) xxx–xxx

Fig. 3. Penicillium chrysogenum A15 growth in MEA medium in contact with lead shot over time: 9 days (A), 2 weeks (B), 4 weeks (C) and 8 weeks (D).

Fig. 4. Environmental scanning electron micrographs of the lead shots. (A and B) Abiotic controls: lead shots after 2 and 8 weeks incubation, respectively; (C and D) lead shots
after 2 and 8 weeks incubation with Penicillium chrysogenum A15, respectively.

Please cite this article in press as: Povedano-Priego, C., et al. Fungal biomineralization of lead phosphates on the surface of lead metal. Miner. Eng. (2016),
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 C. Povedano-Priego et al. / Minerals Engineering xxx (2016) xxx–xxx 5

Fig. 5. Environmental scanning electron micrographs of lead shot surfaces in absence and in presence of fungal biomass. (A and B) Control lead shots after 2 and 8 weeks
incubation, respectively; (C and D) lead shots after 2 weeks incubation with Penicillium chrysogenum A15; (E and F) lead shots after 8 weeks incubation with Penicillium
chrysogenum A15.

(Fig. 5B) produced by abiotic corrosion (as lead oxides), though
after 2 weeks of incubation no mineral formation was observed
(Fig. 5).
Microscopic observation showed the presence of lead secondary
mineral precipitates of various shapes and sizes at the different
incubation times. After 2 weeks incubation, coexistence of lead
mineral crystals with fungal hyphae was observed, while the sam-
ples incubated during 8 weeks showed a significant increase in the
size and abundance of the lead secondary minerals, but no pres-
ence of fungal hyphae was detected. The most peculiar crystals
were small spheroids, and those of tabular and acicular morpholo-
gies. Botryoidal shape also appeared in the 8-weeks sample
(Fig. 5F). Morphological differences were observed between the
mineral precipitates in fungal treatments and those formed in
the abiotic controls, where the crystals appeared like thin layers.
EDX analysis revealed differences in the elemental composition
between the precipitated formed under biotic and abiotic condi-
tions. In control lead shots, at both incubation time (2 and
8 weeks), lead and oxygen were determined as main elements
(Figs. 6A and 3B), whereas in the presence of P. chrysogenum at dif-
ferent incubation periods, EDX spectra of the secondary minerals
showed a peak for phosphorus together with the peaks of lead
and oxygen in the EDX spectra (Fig. 6C and D).
3.3. Interaction of Penicillium chrysogenum with metallic lead at the
interphase metal-mycelium
3.3.1. ESEM and EDX analyses
Pb-fungal interaction study was performed at the metal-
mycelium interphase using ESEM coupled with EDX analysis. After

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6 C. Povedano-Priego et al. / Minerals Engineering xxx (2016) xxx–xxx
Fig. 6. Energy-dispersive X-ray spectra of secondary minerals deposited on the surface of the lead shots. (A) Control after 2 weeks, (B) control after 8 weeks. Lead shot
incubated with Penicillium chrysogenum A15 after 2 weeks (C) and after 8 weeks incubation (D).
2 weeks incubation, bright areas were observed around the spot
where lead shot were placed (Fig. 7A). These bright areas consisted
of secondary lead minerals precipitated on the surface and inside
the fungal hyphae (Fig. 7B and C).
EDX analysis revealed the elemental composition of the sec-
ondary minerals (Fig. 7). The minerals formed around the fungal
hyphae showed similar EDX spectra to those biominerals found
on the lead shot surface (presence of lead, phosphorus and oxygen
peaks).
3.3.2. HRTEM and EDX analyses
HRTEM was applied to determine the cellular localization of
lead precipitates and to elucidate the key mechanisms through
which fungi interact with metals.
Images obtained through HRTEM showed the presence of
electron-dense precipitates on the fungal cell walls after 8 weeks
incubation. EDX spectra confirmed that the minerals produced by
this fungal strain were mainly composed of lead, phosphorus and
oxygen (Fig. 8). The copper peak appeared due to the copper grid
used to support the specimen. The presence of the silicon peak
would be attributed to specimen-burned by electron beam. The
carbon peak resulted from the metallization during the sample
preparation procedure.
4. Discussion
Lead is considered one of the most toxic heavy metals that has
historically caused serious health and pollution problems world-
wide. This element is present in different industrial products such
as lead shot used in hunting or shooting reactional activities
Please cite this article in press as: Povedano-Priego, C., et al. Fungal biomineralization of lead phosphates on the surface of lead metal. Miner. Eng. (2016),
http://dx.doi.org/10.1016/j.mineng.2016.11.007
(Rooney et al., 2007). Environments contaminated with these
lead-containing products, and other heavy metals, are character-
ized by a high fungal diversity and activity, due to their high toler-
ance to these constraining conditions. Thus, fungi are increasingly
considered as bioremediation agents for their ability to remove
contaminants with harmful concentrations through several inter-
action mechanisms (Gadd et al., 2012). Remediation techniques
have been proposed to reduce lead exposure in the environment
through immobilization by the formation of lead phosphate min-
eral phases (e.g. pyromorphite, the most stable lead mineral)
(Rhee et al., 2014).
In the present work, Pb(II) tolerance by three fungal species P.
chrysogenum, A. niger and T. viride was characterized determining
the MIC of this cation. These three fungal strains exhibited differ-
ent levels of tolerance to Pb. A. niger and P. chrysogenum could tol-
erate high Pb concentrations, being able to resist lead up to 8 mM
of this toxic heavy metal, while T. viride showed no mycelium
development at this concentration. Besides, Pb-treated Penicillium
mycelia showed phenotypic changes evidencing the occurrence
of metal-fungal interaction mechanisms. Hence we have chosen
P. chrysogenum as a model microorganism to evaluate and deter-
mine the role of fungi in the transformations of metallic lead (lead
shots) to lead-containing secondary minerals.
In natural environments, abiotic factors such as air, water, and
varying pH conditions result in the corrosion of metallic lead and
the liberation of mobile lead compounds (Cao et al., 2003;
Dermatas et al., 2004; Vilomet et al., 2003). In this work, lead oxide
crystals were observed by ESEM on the lead shot surfaces after two
months incubation under abiotic conditions. Similar results were
reported by Rhee et al. (2012), who observed the deposition of typ-
ical lead corrosion products on the surface of abiotically incubated
 C. Povedano-Priego et al. / Minerals Engineering xxx (2016) xxx–xxx
Fig. 7. Environmental scanning electron micrographs of the mycelium grown around the Pb pellet after 2 weeks incubation. Details of the surface at 57X (A), 920X (B) and
3996X (C). EDX spectrum of the secondary minerals deposited between fungal hyphae (C).
lead shots. The nature of these products depends on the incubation
time: while minium (Pb3O4) and hydrocerussite (Pb3[CO3]2[OH]2)
were deposited after two months incubation, litharge (PbO) and
cerussite (PbCO3) were formed after three months incubation.
Under biotic conditions, the presence of P. chrysogenum led to
the precipitation of Pb-phosphate mineral phases as botryoidal
aggregates on the surface of the lead shots after 2 and 8 weeks
incubation. The morphology of these biotically deposited sec-
ondary minerals showed significant differences in shape in com-
parison to those produced under abiotic conditions, appeared
meanly globular, prismatic and acicular crystals.
On another hand, HRTEM observations confirmed the deposi-
tion of lead mineral phases at the fungal cell walls. One possible
explanation of this fact would be that the microorganism has
enhanced biosorption mechanisms, leading after to the precipita-
tion of the metal as Pb-phosphate minerals. This process seems
to be the first defense of P. chrysogenum against lead toxicity. In
fact, polymeric substances found in the fungal cell walls such as
chitin and pigments (like melanin) and their functional groups
(e.g. phosphate and carboxyl groups) play an important role in
the biosorption process (Gadd, 2007). Sarret et al. (1999), for
instance, reported that phosphate groups were responsible for
the 95% lead bound to P. chrysogenum. Biomineralization of lead
phosphates (e.g. pyromorphite) by fungi is a well-known process
(Leitão, 2009), which has been described to be mainly mediated
by acidic phosphatase activity. This enzyme releases inorganic
Please cite this article in press as: Povedano-Priego, C., et al. Fungal biomineralization of lead phosphates on the surface of lead metal. Miner. Eng. (2016),
http://dx.doi.org/10.1016/j.mineng.2016.11.007
7
phosphate from organic phosphate substrates (Liang et al., 2015).
All in all these results confirm that fungi are effective biosorbents
of a variety of heavy metals, including Pb (Gadd et al., 2012).
The interaction of metallic lead with P. chrysogenum resulted in
morphological and growth alterations. At high lead concentration,
darkening of mycelium was observed. After 9 days incubation, the
mycelium covering the lead shot presents a greenish color indicat-
ing that there is a high sporulation degree in the metal-fungal
interaction area. A possible explanation for this fact might be an
increase in the synthesis of fungal pigments such as melanin. In
fact, according to Gadd et al. (2012), several toxic metals can
induce or accelerate melanin production in fungi, conducting to
the blackening of colonies and chlamydospore development. This
lead fungal response could be associated to a need of a biological
adaptation for the cells to synthesize some enzymes essential for
the lead uptake (Ezzouhri et al., 2009).
Our findings extend the spectrum of fungal strains with ability
to tolerate Pb-contaminated soils and transform metallic lead into
lead phosphate mineral phases, what is relevant for bioremedia-
tion of soils contaminated with lead shots. These stable minerals
could reduce the bioavailability of lead, avoiding ionic lead forms
complex with dissolved organic matter and transfer to other envi-
ronmental compartments (mineral soil, underground water) and
organisms (Akhtar et al., 2013). Therefore, pyromorphite formation
using fungal strains could be an efficient bioremediation strategy,
converting the contaminant metals into their less toxic forms.
8 C. Povedano-Priego et al. / Minerals Engineering xxx (2016) xxx–xxx
Fig. 8. High resolution transmission electron micrograph of biominerals formed by Penicillium chrysogenum A15 around the Pb shot after 8 weeks incubation and energy-
dispersive X-ray analysis of the electron-dense precipitates. Bar: 5.000 Å.
Acknowledgements
This work was funded by grant CGL2012-36505 (80% by FEDER)
(Ministerio de Ciencia e Innovación, Spain). We acknowledge the
assistance of María del Mar Abad Ortega, and Concepción Hernán-
dez Castillo (Centro de Instrumentación Científica, University of
Granada, Spain) for help with HRTEM measurement; and of Isabel
Sánchez Almazo (Centro de Instrumentación Científica, University
of Granada, Spain) for his help with ESEM measurements.
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