FEMS Microbiology Letters 154 (1997) 29^35
Solubilisation of some naturally occurring metal-bearing minerals,
limescale and lead phosphate by Aspergillus niger
Jacqueline A. Sayer, Martin Kierans, Geo¡rey M. Gadd *
Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK
Received 5 April 1997; accepted 24 June 1997
Abstract
The ability of the soil fungus Aspergillus niger to tolerate and solubilise seven naturally occurring metal-bearing minerals,
limescale and lead phosphate was investigated. A. niger was able to solubilise four of the test insoluble compounds when
incorporated into solid medium: cuprite (CuO2 ), galena (PbS), rhodochrosite (Mn(CO3 )x ) and limescale (CaCO3 ). A. niger was
able to grow on all concentrations of all the test compounds, whether solubilisation occurred or not, with no reduction in
growth rate from the control. In some cases, stimulation of growth occurred, most marked with the phosphate-containing
mineral, apatite. Precipitation of insoluble copper and manganese oxalate crystals under colonies growing on agar amended
with cuprite and rhodochrosite was observed after 1^2 days growth at 25³C. This process of oxalate formation represents a
reduction in bioavailability of toxic cations, and could represent an important means of toxic metal immobilisation of
physiological and environmental significance.
Keywords:
Solubilization;
Aspergillus niger ; Metal-bearing mineral; Lead phosphate; Oxalate
1. Introduction
The solubilisation of insoluble metal compounds
by fungi has biotechnological applications, e.g. the
reclamation of metals from low-grade ores and the
recovery of metals from industrial by-products [1].
Solubilisation usually occurs as a result of protonation of the anion of the metal compound by mechanisms that include proton e¥ux and organic acid
production [2]. The proton translocating ATPase of
the plasma membrane generates the electrochemical
gradients that are required for the acquisition of nutrients by active e¥ux of protons into the external
* Corresponding author. Tel.: +44 (1382) 344266;
Fax: +44 (1382) 344275; E-mail: [email protected]
medium [3^5]. The production of organic acids provides both a source of protons and an organic acid
anion, the latter often capable of forming a complex
with the metal cation which a¡ects mobility and toxicity [6,7]: citric acid increases the environmental
mobility of many potentially toxic metals [8]. Fungi
have been isolated from weathered sandstone where
28% of the isolates produced organic acids: Penicillium corylophillum was able to produce oxalic acid
when the only source of carbon and nitrogen was
derived from the alga Monoraphidium braunii [9].
Oxalate is the most studied ligand as it can form
strong complexes with aluminium and enhance silicate dissolution [10]. The weathering of rocks and
minerals is accelerated in the presence of plants
and microorganisms, especially in the microenviron-
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 9 6 - 6
30
J.A. Sayer et al. / FEMS Microbiology Letters 154 (1997) 29^35
ments of roots and hyphae [11], and solubilisation
activity by means of organic acid production is a
major factor in this process. Solubilisation can also
occur by the production of siderophores, which are
Fe(III)-speci¢c bidentate ligands used by microorganisms to accumulate iron, also having a major incidental role in Fe(III) weathering. Iron has been
solubilised from goethite, biotite and pyrite by the
production of siderophores [12] and puri¢ed bacterial siderophores have been reported to be able to
leach 1038 mol Fe m32 h31 hematite [13].
In this work, the solubilisation of seven naturally
occurring metal-bearing minerals, limescale (CaCO3 )
and lead phosphate by Aspergillus niger was
examined. A. niger was tested for the ability to tolerate and/or solubilise commercially available lead
phosphate, and galena (PbS, included in the seven
mineral compounds screened) because it is known
that although the mineralogical form of lead in soil
can a¡ect toxicity [14], lead inhibits soil microbial
respiration, and therefore the decomposition of soil
organic matter [15]. A method of screening fungal
strains for their ability to solubilise insoluble metal
compounds was used, based on observing clear zones
around fungal colonies growing on agar medium
amended with selected insoluble metal compounds
[16]. This method also provides information on relative toxicities and the metal tolerance of fungal
strains by comparison of growth rates. We have
demonstrated that A. niger is capable of the solubilisation of selected metal-bearing minerals and compounds and, in some cases, can transform the substance into an insoluble metal oxalate via an
intermediate solubilisation stage.
2. Materials and methods
2.1. Preparation of metal-containing minerals and
X-ray di¡raction analysis
Seven metal-containing mineral ores [apatite
(Ca5 (PO4 )3 ), chalcopyrite (CuFeS2 ), cuprite (CuO2 ),
galena (PbS), rhodochrosite (Mn(CO3 )x ), selenite
[CaSO4 2H2 O] and sphalerite (ZnS)] were obtained
from R.G. Widdowson (Scarborough, UK). These
minerals were powdered in a ball mill, and composition and purity veri¢ed by powder X-ray di¡racW
tion using a Hilton-Brooks X-ray di¡ractometer ¢tted with a curved graphite monochromator, using
Cu KK 1³ radiation. A sample of limescale (CaCO3 ,
from Garway Hill, Herefordshire, UK) was dried to
constant weight at 60³C and ground in a pestle and
mortar. Pb3 (PO4 )2 was obtained from Alfa (Johnson
Matthey, Royston, UK).
2.2. Organism, media and culture conditions
The organism used was Aspergillus niger (from our
own culture collection), maintained on malt extract
agar (MEA, lab M) at 25³C. All experiments were
carried out using MEA with the addition of the appropriate metal compound to the desired ¢nal concentration. It should be noted that the concentrations of the insoluble metal compounds are
expressed here in molar terms, which does not provide any information on the partitioning of the metal
between soluble and insoluble forms: it should be
noted that a natural equilibrium will be reached between these phases, and that soluble cations may
bind to medium components.
A. niger was inoculated, in triplicate, onto 10 cm3
MEA containing the powdered metal-containing
minerals, limescale or lead phosphate, in 90 mm diameter Petri dishes. The metal-bearing minerals were
used over a concentration range of 0^100 mM. Powdered limescale was incorporated into the agar at
concentrations of 0^0.5% (w/v). Pb3 (PO4 )2 was incorporated into the agar at concentrations of 2.5,
5 and 10 mM. Inoculations were carried out with
7 mm diameter discs of mycelium cut from colonies
which had been grown on MEA at 25³C for at least
24 h. Inoculated plates were incubated at 25³C, and
daily measurements were made of the diameter of
the colonies any zones of solubilisation present.
Rates of colony extension and extension of the solubilised zone were calculated by least squares regression over the linear portion of the data.
2.3. Crystal formation in agar under colonies
When A. niger was grown on agar amended with
cuprite and rhodochrosite, crystals formed in the
agar under and around the colonies after 1^2 days
growth at 25³C. These crystals were extracted by
gently homogenising the agar in approximately
J.A. Sayer et al. / FEMS Microbiology Letters 154 (1997) 29^35
50 ml warm distilled deionised water (ddH2 O) in a
crystallising dish. The crystals were allowed to settle
to the bottom of the dish, and the aqueous phase
was removed [6]. Small samples of the crystals were
mounted on double-sided carbon adhesive tape on
7 mm diameter aluminium stubs and dried overnight
in a vacuum desiccator. The crystals were examined
using a JEOL JSM-35 scanning electron microscope
(SEM) coupled with energy dispersive X-ray microanalysis (EDXA, link interface p1449). For SEM,
the samples were sputter-coated for 5 min using a
Polaron E5100 series II `cool' sputter coater ¢tted
with a Au/Pd target. For EDXA, uncoated samples
were analysed for at least 100 s at a voltage of 15 kV.
3. Results
3.1. Solubilisation of naturally occurring
metal-bearing minerals, limescale and lead
phosphate by A. niger
The results of the powder X-ray di¡raction
showed that apatite [Ca5 (PO4 )3 (OH)], chalcopyrite
(CuFeS2 ), cuprite (CuO2 ), galena (PbS), rhodochrosite (Mn(CO3 )x ), selenite [Ca(SeO3 )] and sphalerite
31
(ZnS) were all pure samples of the mineral (results
not shown). Solubilisation of galena and cuprite by
A. niger is shown in Fig. 1. The results in Tables 1
and 2 are presented as ratios of the colony extension
rate in the presence of a given metal compound (Rm )
to that of the control (Rc ), and the rate of extension
of the clear zone of solubilisation (Rs ) in relation to
the extension rate of that colony (Rm ). A ratio of 1.0
indicates that the colony extension rate in the presence of a metal compound (Rm ) is the same as the
control extension rate (Rc ) and that the rate of extension of the clear zone of solubilisation (Rs ) on a
given metal compound is the same as the colony
extension rate (Rm ). A. niger was able to grow on
all the minerals tested, with no apparent reduction in
growth rate. There was a slight stimulation of
growth by galena, cuprite and chalcopyrite, and an
approximate 50% stimulation of growth with apatite
on all concentrations tested. As the concentration of
apatite increased, growth stimulation increased,
probably due to the presence of released
phosphate. A. niger partially solubilised cuprite and
galena (the zone of solubilisation was not completely
clear) (see Fig. 1). There was no zone of solubilisation around colonies growing on rhodochrosite, but
a zone of crystal formation was clearly visible under
Fig. 1. Growth of A. niger on (a) 60 mM galena, (b) 60 mM cuprite and (c) 60 mM rhodochrosite when incorporated into MEA. In (a)
and (b), the clear zone of solubilisation can be seen at the colony edge; in (c), there is no zone of solubilisation, but extensive crystal formation under the colony. The photographs were taken after 6 days growth at 25³C, scale bar = 1 cm.
J.A. Sayer et al. / FEMS Microbiology Letters 154 (1997) 29^35
32
Table 1
Growth on and solubilisation of metal-bearing minerals (apatite, chalcopyrite, cuprite, galena, rhodochrosite, selenite and sphalerite) in
MEA by
A. niger
20 mM
40 mM
60 mM
80 mM
100 mM
1.54
1.61
1.62
1.62
1.65
^
^
^
^
^
1.21
1.15
1.15
1.16
1.20
^
^
^
^
^
Apatite
R
m :Rc
s m
R :R
Chalcopyrite
R
m :Rc
s m
R :R
Cuprite
R
m :Rc
s m
1.31
R :R
1.33
1.27
b
0.63
a
1.15
1.10
b
1.03
a
0.91
c
1.09
1.02
b
1.03
a
1.08
c
1.07
0.91
b
1.04
a
1.04
c
1.05
b
Galena
R
m :Rc
s m
1.20
R :R
1.21
1.24
1.21
1.20
1.23
a
Rhodochrosite
R
m :Rc
s m
R :R
1.06
1.12
^
1.06
1.10
1.10
1.15
1.07
1.08
1.10
1.10
1.11
^
0.93
^
^
^
1.05
1.05
1.04
1.05
1.08
^
^
^
^
^
c
Selenite
R
m :Rc
s m
R :R
Sphalerite
R
m :Rc
s m
R :R
m :Rc )
Results are presented in the form of ratios of the rate of growth on mineral-amended media in relation to growth on control media (R
s
m
and the rate of extension of the solubilised zone in relation to growth on that given mineral (R :R ). Control growth rate for
31 (average of 3 replicates). ^, no solubilisation.
a Partial solubilisation only.
b Crystallisation under colony, and in zone of solubilisation.
c Crystallisation under colony, but no zone of solubilisation.
A. niger = 7.59
( þ 0.11) mm day
the colony (Fig. 1). Selenite was only solubilised at a
extremely di¤cult to see in the agar at the lower
concentration of 40 mM, although the selenite was
concentrations : any solubilisation at a concentration
of 20 mM may not have been discerned. The solubilisation of limescale and lead phosphate is shown in
Table 2
3
42
Growth on and solubilisation of limescale and Pb (PO )
A. niger when incorporated in MEA
R
m :Rc
s
R :R
by
m
0.2% (w/v)
1.19
1.67
0.3% (w/v)
2.36
1.02
0.4% (w/v)
2.38
1.03
0.5% (w/v)
2.39
1.06
A. niger to more than double
that of the control. The limescale was solubilised at
2.5 mM
1.01
^
5.0 mM
1.00
1.01
10.0 mM
1.00
1.01
sation being at the lower concentrations. Lead phosphate did not a¡ect the growth rate of
A. niger, and
was either only partially solubilised, or immediately
re-precipitated [17].
42
Pb (PO )
a
a
Results are presented in the form of ratios of the rate of growth on
amended media in relation to control growth (R
m :Rc ) and the rate
of extension of the solubilised zone in relation to rate of growth
s m
31 (average
on amended media (R :R ). Control growth rate of
7.00 ( þ 0.49) mm day
a Partial
hanced the growth of
all concentrations tested, the fastest rate of solubili-
Limescale
3
Table 2. Limescale (at 0.3^0.5% w/v) greatly en-
solubilisation only.
of three replicates).
A. niger :
3.2. Crystal formation in agar under colonies
When
A. niger was grown on agar amended with
cuprite and rhodochrosite, crystals formed in the
agar under and around the colonies after 1^2 days
growth at 25³C (Fig. 2). The crystals formed under
colonies growing on agar amended with rhodochro-
J.A. Sayer et al. / FEMS Microbiology Letters 154 (1997) 29^35
33
A. niger
growing on MEA amended with (a)
Fig. 2. Scanning electron micrographs of crystals puri¢ed from the agar under colonies of
60 mM rhodochrosite, (b) 60 mM cuprite. Scale bars = 100
Wm
and 10
Wm
respectively.
site (Figs. 1 and 2a) were fan-like crystal structures
growing on agar amended with cuprite (Fig. 2b)
(600^800
form around the mineral to give a coating of crystal
of
aggregates approximately 7
Wm in length) composed of elongated plates
approximately 100^150 Wm in length and 30^50
Wm
in width. The crystals formed under colonies
Wm
in length and 2
Wm
in width. X-ray microanalysis of the crystals showed
that only the appropriate cation was present in each
sample, i.e. manganese for the crystals formed in
agar amended with rhodochrosite and copper for
the crystals formed in agar amended with cuprite
(Fig. 3).
4. Discussion
In contrast to autotrophic leaching, there has been
relatively little attention paid to the heterotrophic
solubilisation of insoluble metal compounds (other
than metal phosphates) in the natural environment
and the incidence of such abilities in microorganisms. There has been some interest, however, in the
solubilisation of some naturally occurring metalbearing minerals. 50% of Ni reserves, for example,
are found in lateritic ores and Tzeferis et al. [18]
recovered 60% Ni from non-sul¢de nickel ores using
strains of
Fig. 3. Typical spectra obtained by energy dispersive X-ray microanalysis of crystals produced by
A. niger
growing on MEA
amended with (a) 60 mM rhodochrosite and (b) 60 mM cuprite.
Aspergillus
and
Penicillium,
probably by
the production of citric and oxalic acids and subsequent complex formation. Attempts have also been
made to leach Ni from lateritic nickel ore with in-
34
J.A. Sayer et al. / FEMS Microbiology Letters 154 (1997) 29^35
digenous micro£ora (strains of Aspergillus), as microbial leaching was purported to be less energy demanding and less expensive, but it was found that
abiotic organic acid applications were more e¡ective
than the microbes [19,20].
The ability of A. niger to solubilise a range of
other metal-bearing minerals and limescale has
been demonstrated in this work. A. niger solubilised
three out of the seven minerals tested, and limescale
was solubilised at all concentrations tested. The ef£ux of protons and the production of organic acids
appear to be the most signi¢cant for the solubilisation of insoluble metal compounds by A. niger [7,16].
Growth of A. niger in liquid culture results in major
production of organic acids, and typical ¢nal concentrations of citric acid produced industrially by
A. niger can reach 600 mM [21]. This solubilisation
would presumably lead to enhanced mobility and
increased availability of Ca2‡ , Cu2‡ and Pb2‡ ,
although no reduction in the growth rates of A. niger
were recorded. In the terrestrial environment, the
dissolution of hematite by simple organic acids (citric
and oxalic) occurs by changes in surface micro-topography, such as pitting and pore formation [22].
The presence of acetate (0.07 M) and oxalate
(0.005 M) promoted secondary pore development
and increased feldspar dissolution rates in sandstones [23], due to the formation of Al-organic acid
anion complexes. Organic acids can interact with
mineral surfaces in the environment in two ways,
either coating the mineral and protecting it from
dissolution or increasing rates of dissolution, depending on the nature of the interaction. Hering
[24] proposed a mechanism for ligand-promoted dissolution of aluminium oxide: (1) ligand adsorption
and surface complex formation, (2) slow detachment
of the metal from the surface as a complex with the
ligand, (3) regeneration of the surface. Low molecular mass organic acids have been reported in forest
litter leachates at a concentration of 1.2 mM, and
oxalate concentrations up to 1 mM have been reported in soil solutions [24].
After initial solubilisation of the insoluble metal
compound, precipitation of crystals in the agar
under the colonies growing on cuprite and rhodochrosite has been demonstrated. Crystals of this nature have been observed under colonies growing on
agar amended with a range of insoluble metal phos-
phates and oxides and have previously been identi¢ed as insoluble metal oxalates [7]. The crystals reported here are therefore likely to be manganese and
copper oxalates and EDXA showed them to contain
only the relevant metal. While calcium oxalate is
known to be degraded only by anaerobic bacteria
of the gastrointestinal tract and by a few aerobic
actinomycetes, bacteria and fungi [25], there is little
information on the degradation of insoluble oxalates
containing potentially toxic metal cations. The formation of metal oxalates could therefore be an e¡ective means of immobilisation of toxic metal ions, and
has implications for tolerance [7].
In this study, A. niger appeared not to be able to
solubilise lead phosphate, and it is claimed that if
lead is present in soil as lead phosphate it has a
limited bioavailability [14]. Growth of the fungus
was not a¡ected by the presence of lead phosphate
in the medium. A. niger will produce organic acids
and acidify the medium whether a metal compound
is present or not [7], and it is possible that some
complexation could have occurred. In addition, it
should be noted that any solubilised Pb2‡ would
have readily precipitated with other medium components, e.g. chloride [2]. However, PbO has been solubilised with Clostridium sp. by the production of
acetic, butyric and lactic acids [15].
In conclusion, this work has demonstrated the
ability of A. niger to tolerate and solubilise a range
of metal-bearing minerals when incorporated into
MEA. The production of organic acids provides
both protons and an organic acid anion, the latter
capable of forming a complex with the metal cation,
and, in some cases the subsequent formation of an
insoluble oxalate. The environmental signi¢cance of
this latter process of immobilisation awaits further
study.
Acknowledgments
G.M.G. gratefully acknowledges ¢nancial support
from the Biotechnology and Biological Sciences Research Council (SPC 02812) and NATO (Linkage
Grant Envir. Lg. 950387). J.A.S. gratefully acknowledges receipt of a NERC postgraduate research studentship. Dr. F. Hubbard, Department of Civil Engineering, University of Dundee is thanked for
J.A. Sayer et al. / FEMS Microbiology Letters 154 (1997) 29^35
assistance with powder X-ray di¡raction of the minerals.
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