Mineral Processing and Extractive Metallurgy Review

An International Journal

ISSN: 0882-7508 (Print) 1547-7401 (Online) Journal homepage: http://www.tandfonline.com/loi/gmpr20

Extraction of Ce and Th from Monazite Using REE

Tolerant Aspergillus niger

Kishor Kumar Keekan, Jayesh Chhaganbhai Jalondhara & Abhilash

To cite this article: Kishor Kumar Keekan, Jayesh Chhaganbhai Jalondhara & Abhilash (2017):
Extraction of Ce and Th from Monazite Using REE Tolerant Aspergillus niger, Mineral Processing
and Extractive Metallurgy Review, DOI: 10.1080/08827508.2017.1350956

To link to this article: http://dx.doi.org/10.1080/08827508.2017.1350956

Accepted author version posted online: 12

Jul 2017.
Published online: 12 Jul 2017.

Submit your article to this journal

Article views: 20

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=gmpr20

Download by: [117.227.2.154] Date: 16 August 2017, At: 21:29

 MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW
https://doi.org/10.1080/08827508.2017.1350956

Extraction of Ce and Th from Monazite Using REE Tolerant Aspergillus niger

Kishor Kumar Keekana, Jayesh Chhaganbhai Jalondharaa, and Abhilash b

a
Yeneopoya Research Centre, Yenepoya University, Mangalore, Karnataka, India; bCSIR-National Metallurgical Laboratory, Jamshedpur, Jharkhand, India

ABSTRACT KEYWORDS
In the present study, bioleaching of monazite, one of oldest and most water-resistant mineral to Aspergillus niger;
microbial attack, was tested for its amenability toward extraction of Ce, Th, and U using heterotrophic bioleaching; cerium;
monazite; organic acids;
fungal species, Aspergillus niger. Shake flask experiments were carried out using Bromfield, sucrose, siderophore
synthetic, and standard media containing 2%(w/v) of monazite for 60 days. Several parameters such as
media pH, redox potential, siderophore production, biomass, and metal oxidation were analyzed at
different intervals during the process of bioleaching. The maximum concentration of Ce obtained was
0.701, 0.229, 0.132, and 0.052 mg/L in Bromfield, synthetic, sucrose, and standard media(s), respectively.
Thorium was identified only in synthetic (0.026 mg/L) and sucrose (0.0175 mg/L) media, whereas
uranium was not observed in any of the media attempted. The results were corroborated with analysis
by SEM characterization of the head and residues.
Downloaded by [117.227.2.154] at 21:29 16 August 2017

1. Introduction forms of wastes generated during the chemical processing of

monazite are reported by Pillai (2007) in detail. Leach solutions
Monazite ((Ce,La,Nd,Th)PO4) belongs to a class of important rare
generated during this physicochemical process contains high con-
earth elements (REEs) bearing mineral as it contains sufficient
centrations of soluble metals and acidity. Its release into the
concentration of rare earths (Koltun and Tharumarajah, 2014).
environment has serious consequences. Treatment of such wastes
Cerium is the most abundant of the metals of the so-called rare
further adds up to expenditure in addition to other environmental
earth. REEs have huge industrial demand and are used in metal-
problems such as toxicity, soil, groundwater contamination, etc.
lurgy, glass, ceramics, illuminations, electronics, chemical, mag-
(Koltun and Tharumarajah, 2014).
nets, nuclear, and miscellaneous applications (Vijayan et al.,
At the same time, microbial hydrometallurgy is a clean,
1989). In addition to REEs, monazite also contains significant
environmentally friendly approach for extraction of metals and
amounts of thorium (Th, 4.5–9.5% as ThO2) and uranium (U,
can be exploited commercially. Bioleaching of metals including
0.2–0.4% as U3O8) (Pillai, 2007). Hence, thorium with potential
radiogenic minerals (Abhilash and Pandey, 2013; Qu and Lian,
application in the nuclear fuel cycle is also commercially recovered
2013) by different types of microorganisms (Amin et al., 2014) is
from the mineral monazite. Monazite is a unique mineral with
well reported in the literature. For many years Acidithiobacillus
interesting physical and chemical properties, such as high fusion
ferrooxidans was considered as the most important microorgan-
temperature, optical emissivity, high resistance to aqueous and
ism in commercial bioleaching of copper, uranium, and gold
molten glass corrosion, radiation damage, etc. (Clavier et al.,
(Abhilash and Pandey, 2013). However, if the ores are oxides,
2011). Several countries such as Australia, Brazil, China, Egypt,
carbonates, and silicates then microorganisms of the genus
India, Malaysia, South Africa, Sri Lanka, China, and USA are
Thiobacillus cannot be used. Such ores are bioleached using
processing monazite for the commercial production of REE and
heterotrophic bacteria and fungi, where the metals are
Th. India has a rich deposit of monazite in the beach sands of its
bioleached from the ores by means of organic acids or complex-
southeastern coast. The composition of Indian monazites is
ing/chelating agents secreted by bacteria and fungi (Abhilash
59.68% of total rare earth oxides (TREO), 0.37% of U3O8, 9.58%
and Pandey, 2013). Heterotrophic bioleaching is mediated by the
of ThO2, and 26.23% of P2O5 (Pillai, 2007).
bio-oxidation of organometallic bonds including various organic
Industrially REE is extracted from monazite either by a basic
bonds. Aspergillus niger is one of the most important fungi
process with sodium hydroxide or through the acidic process
exploited in heterotrophic bioleaching experiments (Abhilash
using concentrated sulfuric acid (Abreu and Morais, 2010). The
and Pandey, 2013). The concept of sustainable technology is
presence of radioactive elements (Th and U) and phosphorus in
very important nowadays and the importance of biohydrome-
monazite make the monazite processing more energy and chemi-
tallurgy in metals extraction finds a special place. However, the
cal intensive than other REE minerals like bastnasite due to the
advent of biohydrometallurgy in monazite processing has not
requirement of additional separation of U, Th, and phosphorous
been explored quite effectively, except few scattered studies in
unlike the former. Monazite processing results in the generation of
the literature using fungal organic acids and electrochemical
large quantities of byproducts/wastes (chemical sludges) leading to
mode (Maes et al., 2017) and few bacterial variants (Emmanuel
the radioecological problems (Nouailhetas et al., 1993). Various
et al., 2011).

CONTACT Abhilash abhibios@gmail.com CSIR-National Metallurgical Laboratory, Jamshedpur 831007, Jharkhand, India.
© 2017 Taylor & Francis Group, LLC
 2 K. K. KEEKAN ET AL.

Hence, developing a cost-effective, environmentally friendly Table 1. Composition of culture media used for bioleaching experiments.
process to extract valuable metals from monazite, an economically Quantity (g)
important mineral is the crux of this work. Therefore, the present Ingredients Bromfield Sucrose Synthetic Standard
study deals with the bioleaching activity of A. niger on monazite Glucose 20 — — —
minerals to extract metal constituents of Ce, Th, and U and Sucrose — 100 100 75
Yeast extract 1 1.6 — —
optimizing the microbial growth as well as leaching parameters. KH2PO4 0.25 0.5 0.1 2.5
KCl — 0.025 — —
(NH4)2SO4 0.25 — — —
NH4NO3 — — 0.45 —
2. Materials and methods (NH4)2CO3 — — —- 2.5
NaNO3 — 1.5 —- —
2.1. Chemicals MgSO4⋅7H2O 0.75 0.025 0.3 0.25
FeSO4⋅7H2O — — 0.0001 —
All the chemicals used in this experiment were procured from FeCl3⋅H2O — — — 0.0013
HiMedia™ unless otherwise stated and all aqueous solutions were Zn SO4⋅5H2O — — 0.00025 —
ZnCl2⋅2H2O — — — 0.00006
prepared using distilled water (Distil - On 2SQ-Single distillation Distilled water 1000 mL
(Infusil™). Monazite minerals (‘MK’ Grade, 96.5%) were obtained pH 6.5 5.6 5.5 7.5
from Rare Earths Division (RED)/Indian Rare Earths Limited
(IREL), Aluva District, Kerala mined and separated by
Manavalakurichi Mineral Division, IREL, India. Before using, synthetic media (SynM), and standard media (StdM, Castro
the mineral was passed through laboratory test sieve (<250 µm) et al., 2000) were used for the bioleaching experiments. The
(TIC™) to remove soil/dust and other debris and washed several
Downloaded by [117.227.2.154] at 21:29 16 August 2017

composition of each medium is given in Table 1. Media were

times with tap water. Air-dried samples after characterization by sterilized by autoclaving at 121°C for 20 min. Shake flask experi-
electron probe microanalyses (EPMA) were used for bioleaching ments were carried out in 250 mL Erlenmeyer flask containing 100
experiments. mL of the each medium with 2%(w/v) monazite mineral in dupli-
cate. Spore suspensions of the fungi were prepared by flooding the
5–7 day old A. niger cultures slants with distilled water containing
2.2. Fungi, media, and growth conditions
0.05% Tween 80. The inoculum was enumerated (3 × 106 spores/
Several soil samples were screened initially by serial dilution/ mL) using Neubauer chamber. Flasks were incubated in an orbital
pour plate method using Potato Dextrose Agar (HiMedia™) to shaker at 30±1°C with 150 rpm for 60 days. Uninoculated flasks
obtain a candidate fungal isolate with diverse biochemical activ- served as control mixed with mercuric chloride as a biocide.
ity such as phosphate solubilization, siderophore production,
acid production, and silicate solubilization, as these biochemical
properties are normally associated with the microbial isolates 2.3. Analysis of leach liquor
with potential metal solubilization/weathering of soil/rocks.
Fungal isolates were scrutinized and only the organisms with Culture supernatants (leach liquors) were withdrawn at 0, 15, 30,
these properties were selected for further studies and others were and 60 days under sterile conditions and subjected to analysis.
ignored. Morphological features, both macroscopic and micro- Hydrogen ion (pH) concentration and redox potential (Eh) of
scopic characteristics of the best performing fungi, were matched the leach liquor was measured using Benchtop meter (HI 3512,
among the possible variants using Motic BA310 trinocular com- HANNA Instruments, USA). Concentrations of Ce, Th, and U
pound microscope with Motic Image Plus 2.01 software and were estimated using Inductively Coupled Plasma - Atomic
Zeiss Stemi DV4 Stereo Microscope, respectively (Nithiyaa Emission Spectrometer (ICP-AES, Model “Arcos,” Spectro
et al., 2012). The fungal isolate was adapted first on salts of Gmbh, Germany) fitted with charge coupled device (CCD)
cerium and thorium in the concentration range of 0.5–10 mM, detector with data acquisition using Smart Analyzer Vision
and further on 1% (w/v) monazite sample in Potato Dextrose (Version 5.01.0921) facility available at Sophisticated Analytical
media. Qualitative analysis of organic acids in different culture Instrument Facility (SAIF), Indian Institute of Technology
media was carried out using LaChrom Elite High Performance
Liquid Chromatography (HPLC) system (Hitachi®, Tokyo, Table 2. Elemental composition of monazite mineral (‘MK’ grade)
Japan) constituting LaChrom Elite L-2130 (HTA) Quaternary used.
pump with built in Degasser, Shodex C-18 4D (4.6 × 150 (mm) Chemical entities % Composition
ID × Length) column with a L-2300 Column Oven, UV Detector Ce2O3 27.84
La2O3 15.7
(L-2400) and D-2000 Elite HPLC System Manager to acquire Pr2O3 2.9
and analyze data. Separation of organic acids was achieved using Nd2O3 10.5
5 mM Phosphoric acid (H3PO4) as the mobile phase. The flow Y2O3 0.4
P2O5 28.2
rate of mobile phase was 1 mL/min with column oven main- TiO2 0.4
tained at 40°C during the analysis and the process was mon- Fe2O3 0.9
ZrO2 1.3
itored at 210 nm using a UV Detector. The leach liquor was SiO2 2.4
filtered through a nylon membrane filter (13 mm, 0.22 μm) and Al2O3 0.1
5 µL of the leach liquor was injected into the HPLC system. Cr2O3 0.001
ThO2 9.0
Four culture media, namely, Bromfield media (BM, Bromfield U3O8 0.35
and Skerman, 1950), sucrose media (SM, Amiri et al., 2010),

Bombay IIT-Bombay, Powai, Mumbai. Biomass of the cultured
fungi in different bioleaching media was harvested at the end of
the experiment. Minerals if any adhered/trapped in the mycelial
network was removed by sonicating the harvested mycelium.
The mycelium was dried in hot air oven at 80°C for 24 h to
obtain a constant dry weight. Detection and estimation of side-
rophore if any produced in the leaching media by A. niger was
estimated by chrome azurol sulfonate (CAS) assay (Schwyn and
Neilands, 1987). Leach liquor of each media was centrifuged at
12,000 rpm for 20 min at room temperature. Mycelia-free super-
natant was mixed with 0.5 mL CAS solution. The color obtained
was measured using the spectrophotometer at 630 nm with
reference blank consisting of 0.5 mL uninoculated control of
respective media and 0.5 mL CAS solution.
The percentage of siderophore units was estimated using
the formula (Sayyed et al., 2005):
% Siderophore units ¼ ½ðAr  AsÞ=Ar x 100;
where ‘‘Ar” is the absorbance at 630 nm of reference (CAS assay
solution + uninoculated media) and ‘‘As” is the absorbance at
Downloaded by [117.227.2.154] at 21:29 16 August 2017
630 nm of the sample (CAS assay solution + supernatant;
Payne, 1994).
Surface features of the bioleached monazite minerals and
control samples were studied using SEM (JEOL JSM-
6380LA™). Partially bioleached monazite minerals were recov-
ered from the leach media, washed three to four times using
distilled water. The minerals were sputter coated using palla-
dium and dried before imaging.
3. Results and discussion
3.1. Chemical and microscopic characterization of
monazite mineral
Monazite mineral derived from ore body was used in the present
study and analyzed by ICP-OES as depicted in Table 2. The
fractioned monazite mineral was subjected to characterization
Figure 1. Electron probe micro-analysis of <250 μm monazite sample (*-Ag coating; inset - magnified micrograph of mineral grain in focus).
MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW 3
by EPMA. Both major and trace element analysis for monazite
were performed using a JEOL JXA-8230™ EPMA equipped with
four wavelength dispersive spectrometers at CSIR-NML,
Jamshedpur, India. Monazite spot analyses were performed at
an acceleration voltage of 15 kV, beam current of 150 nA, using a
LaB6 electron source. Both natural and synthetic standards were
used for calibration. These are: UO2 for U; ThO2 for Th; syn-
thetic glass containing rare earth elements (REE) (4 wt.%) La, Ce,
Pr, Nd, Sm, Gd, Dy, Ho; apatite for Ca and P; yttrium aluminum
garnet (YAG) for Y; hematite for Fe; corundum for Al; and Th-
glass for Si. The analysis in Figure 1 revealed the less pitted
surface (inset) of phosphate mineral, with wide spread distribu-
tion of REE (La, Ce, Nd, Sc, Y) as depicted in the EDAX analysis.
3.2. Isolation of fungal species
(1) Bioleaching of monazite and thorium-uranium concentrate was
earlier attempted by Hassanien et al. (2014) using Aspergillus
ficuum (A. ficuum) and Pseudomonas aeruginosa (P. aeruginosa).
The studies utilized the siderophore produced by P. aeruginosa
and the metabolites of A. ficuum to bioleached Th, U, and REE.
In addition, the authors highlighted the role of exogenous poly-
saccharides (EPSs) in bioleaching of monazite minerals.
However, Hassanien et al. (2014) used general purpose culture
media (Modified Czapek’s Dox broth (MCDM) and Nutrient
broth (NB) for A. ficuum and P. aeruginosa, respectively) for
bioleaching experiments, which may support the luxuriant
growth of the organisms under study but may not efficiently
leach the mineral. Heterotrophic organisms like A. niger is more
useful to bioleach the nonferrous minerals/ores like oxide, car-
bonate, and silicate. However, non-sulfide ores contain no
energy source for the microbial growth (Abhilash and Pandey,
2013). This is not always true and it depends on the minerals
types. Monazite contains sufficient quantity of phosphate
(26.23% in the form of P2O5) (Pillai, 2007), which can render
the phosphorus to the growing cells, provided it is solubilized
 4 K. K. KEEKAN ET AL.
from the matrix. Hence, forcing the organisms to feed on the
minerals, designing a good medium is a key criterion for efficient
bioleaching of mineral under study. However, only the cultures
showing positive results for biochemical tests such as phosphate
and silicate solubilization, siderophore and acid production were
retained, others were ignored. For the present work, fungal
isolates possessing all the above biochemical activities were pre-
ferred as it can solubilize minerals and metal compounds by
acidolysis, complexolysis, redoxolysis, and by metal accumula-
tion through heterotrophic leaching (Abhilash and Pandey,
2013). Of the several shortlisted fungal isolates, only one strain
was selected for the study with larger halo zone for the above
biochemical tests, which was identified as A. niger and had all the
morphological features (macroscopic and microscopic) of A.
niger described earlier (Nithiyaa et al., 2012). The growth of
the microbe was confirmed on daily basis for 10 days before
next sub-culturing, by staining, biomass weight, and HPLC-
based estimation of organic acids.
Downloaded by [117.227.2.154] at 21:29 16 August 2017
3.3. Growth and metabolite production
Growth of A. niger in Bromfield, sucrose, and synthetic media was
in the form of mycelial mat/pad, whereas in standard media the
growth was observed in the form of pellets/beads (Figure 2), in
spite of treating all the flasks in same cultural condition such as
size and type of inoculums, shaking velocity, etc., as these factors
determine the mycelial morphology in submerged liquid condi-
tions (Papagianni and Mattey, 2006). Mycelial mats show exten-
sive indignations and also observed numerous rhizoidal hyphae in
sucrose media when comparing to Bromfield and synthetic
media. Fungal growth is in the form of mat/pad over the surface
of the medium. Filamentous fungi forms freely dispersed mycelia,
loose hyphal aggregates (clumps), dense aggregates (pellets) of
different sizes, and mat of mycelium in liquid media/submerged
conditions. A. niger shows different type of mycelial morphology
under various conditions. They grow as disperse mycelia, as
Figure 2. (A–D) is the cultural and morphological characteristics of the fungal isolate A. niger used in the bioleaching experiment. (A: Colony of the A. niger on
Czapek Dox Agar (CZA), B: Globose, radiate, biseriate conidial heads, C: metulae with phialides, D: Rounded, dark brown/black, rough-walled conidia. (E–G) Light
microscopic and SEM image of monazite minerals showing the surface features (E: Light microscopic image of minerals, F: A single minerals enlarged showing pits/
cracks, G: SEM image of single mineral showing pits/cracks).
pellets in submerged cultivations (Papagianni and Mattey, 2006).
The morphology of filamentous fungi in bioleaching process is
very important; since it influences the physiology of the organism,
which in turn affects the bioleaching capacity of the organism.
Mycelial morphology depends on several factors such as the type
of medium, physicochemical factors like shear forces, surface
active agents, pH, temperature, Ca2+ ions, ionic strength, etc.
Similarly, agitation is very important in bioleaching process; as it
enhances the contact of fungal mycelium and or its metabolites
with the minerals. As shown in Figure 3A to D, monazite minerals
were found embedded in the mycelia network of A. niger during
the bioleaching process. Intimate contact of mycelium with the
mineral surface may efficiently dissolve the monazite mineral, as
observed earlier (van Breemen et al., 2000) because of the bio-
mechanical forces. Fungal hyphae have special affinity to grow on
these kinds of cracks and pits (Gadd, 1999). The growth of fungi
was not inhibited by the presence of up to 10 mM cerium depict-
ing the established tolerance. It is also important to note that the
concentration of thorium is less in this sample of monazite as
compared to cerium.
The media in all the inoculated flasks except control turned to
acidic as leaching progresses due to the production of organic
acids. Lowest pH recorded was 1.82 ± 0.02 in sucrose media. The
pH was almost stable in all media except SM, where after a
maximum decrease there is an increase in pH. Where the pH
changed from the lowest 1.84 on the 15th day to 6.41 on the 16th
day. This may be due to the exhaustion of carbon source and
shift in the utilization of a different energy source or utilization
of its own metabolite for growth. Production of acid and
decrease in the media pH is an important criterion for the
efficient bioleaching of minerals under test. It is known that
acidolysis is the main mechanism of heterotrophic bioleaching
mediated by fungi, which generates both protons and metal-
complexing organic acid anion (Gadd, 1999). Production of
gluconic acid, oxalic acid, citric acid by A. niger is well reported
in the bioleaching experiments carried out earlier (Papagianni
 MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW 5
Downloaded by [117.227.2.154] at 21:29 16 August 2017

Figure 3. Submerged growth and mycelial morphology of A. niger in different culture media in various time intervals. A–D shows the interaction of monazite minerals with the
mycelia of A. niger in different culture media. A: Underneath view of an A. niger mycelia mat showing the attachment of monazite minerals. B: Clump of mycelia with monazite in
the periphery/embedded. C: Entrapment of monazite minerals in the mycelia network. D: Clumping of monazite minerals within the hyphal network.

and Mattey, 2006; Amiri et al., 2010). Several studies in the past 70
IA, 12.23
Intensity (mV)/Concentration of organic acid (ppm)

OA: Oxalic acid

have shown the production of organic acids during bioleaching A AA: Acetic acid
(Qu and Lian, 2013; Amin et al., 2014). Similarly, in this present 60 CA: Citric acid
SA: Succinic acid
work, the reduction in the pH is attributed to the production of KA: Kojic acid
50
organic acids. The organic acids detected by the growth of A. IA: Itaconic acid
niger in potato dextrose medium were oxalic, acetic, citric, suc-
40
cinic, and new molecules of kojic acid and itanoic acid
(Figure 4A). All the media showed amicable production of 30
organic acids when compared to the intensity in Table 3,
KA, 6.24
SA, 5.05

which attributed to the lowering of pH and rise in redox poten-

CA, 4.64

20
OA, 2.8
AA, 3.44

tial. The growth of the fungi was maximum in synthetic media

(1.55 gm/100 mL), whereas the same was minimum in 10
Bromfield media (0.91 gm/100 mL) (Figure 4B). This demon-
strates the preference of sucrose as the carbon source for the 0
growth of A. niger then glucose. The preference for sucrose as a 0 2 4 6 8 10 12 14 16 18 20 22 24
Retention Time (min)
carbon source is being utilized in the industrial production of
citric acid because A. niger has an extracellular hyphae bound 2.5
enzyme (invertase), which is active at a low pH (Xu et al., 1989).
Growth in SM (1.41 gm/100 mL) and StdM (1.36 gm/100 mL)
B
Dry weight of mycelium (g)

2
was almost comparable.
Figure 5 shows the redox potential values of all the leaching
media. Eh (ORP) values were found increasing as the leaching 1.5
progresses. The maximum values of redox potential recorded in
different sets were 284.45, 281.70, 274.25, 266.70 mV in SM, 1
SynM, StdM, BM media, respectively. Redox potential was main-
tained almost in all the culture media except sucrose media where
0.5
the curve was found declining after 30 days of incubation.

0
3.4. Siderophore production and leaching of REE BM SM SynM StdM
Figure 6 illustrates the production of siderophore in different Figure 4. Metabolite production vis-a-vis growth of biomass (A: in potato
leach media. Of the four media, SynM supported the max- dextrose media; B: mycelia dry weight of A. niger cultured in different bioleach-
imum production of siderophore, i.e., 25.81% SU, followed by ing media).
 6 K. K. KEEKAN ET AL.

Table 3. Quantified concentrations of various organic acids detected in experi-

mental medias against calculated limit of detection in potato dextrose media
(OA: oxalic acid; AA: acetic acid; CA: citric acid; SA: succinic acid; KA: kojic acid;
IA: itaconic acid).
Organic acid concentration in ppm (Intensity in mV)
Media OA AA CA SA KA IA
Potato dextrose media 2.80 3.44 4.64 5.05 6.24 12.23
(11) (9) (8) (14) (15) (65)
Bromfield media (14) (7.2) (5.8) (4.2) (3.5) (2)
Sucrose media nd (55) nd nd nd nd
Synthetic media nd (13) nd nd nd nd
Standard media nd (6) nd nd nd nd

6.08% SU. Siderophore was not detected in StdM. The pro-

duction of siderophore was estimated to be 2.39% SU in BM,
however, the peaks were not detected in the spectrophoto-
metric assay. The role of siderophores is primarily to scavenge
Fe, but they also form complexes with other metals ions such
Figure 6. Quantitative analysis for siderophore production in different culture
as Cd2+, Cu2+, Ni2+, Pb2+ and Zn2+, Mn3+, Co3+ and Al3+, media (BM: Bromfield media, SM: Sucrose media, SynM: Synthetic media; StdM:
including actinides such as Th4+, U4+, and Pu4+ (Payne, 1994; Standard media).
Albrecht-Gary and Crumbliss, 1998). Siderophore has a
Downloaded by [117.227.2.154] at 21:29 16 August 2017

greater role in soil mineral weathering, even more, effective

when comparing to organic acids because siderophores form
more stable complexes with Fe (Reichard et al., 2007). Hence,
the production of siderophore is one of the important criter-
ions for efficient bioleaching of minerals, which is supplemen-
ted with the high intensity of organic acids produced.
The quantity of metals (Ce, Th) leached in different media
at various time intervals using 2% PD is given in Figure 7.
Cerium concentration was maximum (0.70 mg/L) in BM after
30 days of leaching recording 100% extraction. However, the
concentration decreased to 0.59 mg/L on 60 days of leaching.
Maximum quantity of Ce obtained were 0.22 (49%), 0.13
(28%), 0.05 (~5%) mg/L in SynM, SM, StdM, respectively.
Thorium was estimated only in SynM (0.026 mg/L and SM
(0.01 mg/L). Uranium was not found in any of the media
tested. Table 4 further substantiates to the result wherein the
presence of respective elements in different media have been
shown. The reduction in the concentration of metals in all the
leach liquor may be due to the biosorption of metals by fungal
mycelium, as fungal biomass is known to be a good biosor-
bent (Rezza et al., 2001). This was further justified by the
SEM-EDAX revealing the adsorption of leached REE (Ce)
onto the fungal hyphae (Figure 8C,D) against a control set
without metal ions (Figure 8A,B). Monazite mineral after
leaching was subjected to SEM evaluation for changes in
morphology. There was a visible change in the color from
brown to pale yellow in the bioleaching sets (not shown in
figure) when comparing to the unreacted mineral. To corro-
borate the same and to study the surface features, both

Figure 5. pH and redox potential (ORP) of the leach liquor (A: Bromfield media, B: Sucrose media, C: Synthetic media, D: Standard media) during the
bioleaching process.
 MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW 7

Figure 7. Change in concentration of Ce and Th in the leach liquor of bioleaching process mediated by A. niger in different culture media (BM: Bromfield media, SM:
Sucrose media, SynM: Synthetic media, StdM: Standard media).

indicating the hardness of the material. However, Figure 9C

Table 4. Detection of different metal constituents of monazite in different leach
media on completion of leaching at 2%PD, at 35°C in 60 days.
shows some vents, which essentially act as active sites for
fungal mycelia to enable metal dissolution. SEM image of
Different leach media
bioleached minerals (Figure 10A) shows clear, larger poros-
Metals Bromfield Sucrose Synthetic Standard
ity/pits with white fungal spores attaching on the surface of
Downloaded by [117.227.2.154] at 21:29 16 August 2017

Cerium ✓ ✓ ✓ ✓
Lanthanum ✓ ✓ ✓ ✓ mineral, which further results in attack of metabolites.
Praseodymium – – – – Figure 10B–C shows high degree of corrosion and vent for-
Phosphorus ✓ ✓ ✓ ✓
Titanium – – ✓ ✓ mation as compared to Figure 9C, owing to the action of
Iron ✓ ✓ ✓ ✓ microbial metabolites and prolonged attachment of fungal
Zirconium – – – – biomass.
Silicon ✓ ✓ ✓ ✓
Aluminium – – ✓ –

unreacted and leached samples were analyzed using SEM as
shown in Figures 9A–C and 10A–C. Scanning electron
micrograph of the unreacted monazite under increasing mag-
nification reveals the scarce pits on the mineral rock
4. Conclusions
The present study demonstrates the amenability of the fungus
to leach metals from monazite minerals by bioleaching.
Preliminary study has shown the A. niger is able to grow
normally in the culture media (without phosphate) used in

Figure 8. Scanning electron microscopy with energy dispersive X-ray spectroscopy data demonstrating the binding of metals during bioleaching [chromatogram (A)
and figure of mycelia (B) of control samples and chromatograms (C) and figure of mycelia (D) of leached samples].
 8 K. K. KEEKAN ET AL.
Downloaded by [117.227.2.154] at 21:29 16 August 2017

Figure 9. SEM of the unreacted monazite under different magnifications (A: 50

Figure 10. SEM of the bioleached monazite by A. niger (PSSG 8) under different
μm, B: 10 μm, C: 2 μm).
magnifications (A: 50 μm, B: 10 μm, C: 2 μm).

the present study but in the presence of powdered monazite,
the uptake of phosphate from monazite is exhibited. Synthetic
media supported the maximum production of siderophores.
Cerium leaching was maximum (0.70 mg/L) in Bromfield
media after 30 days of leaching with thorium estimated as
0.01–0.03 mg/L. The production of organic acids and concur-
rent advancement in redox potential attributed to the dissolu-
tion of metals. Changing the physicochemical parameters and
cultural conditions may enhance the yield of metals. Advance
statistical tools like response surface methodology (RSM) can
be used to optimize the culture media and leaching experi-
ments to improve metal dissolution.
Disclosure statement
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the article.

Funding
The authors acknowledge the financial support by DAE-BRNS, Govt of
India for sponsoring the project (No. 2013/34/7/BRNS/0442).
ORCID
Abhilash http://orcid.org/0000-0003-1147-4677
References
Abhilash, and Pandey, B. D., 2013, “Microbially assisted leaching of
uranium – a review.” Mineral Processing and Extractive Metallurgy
Review, 34, pp. 81–113. doi: 10.1080/08827508.2011.635731
Abreu, R. D., and Morais, C. A., 2010, “Purification of rare earth
elements from monazite sulphuric acid leach liquor and the produc-
tion of high-purity ceric oxide.” Minerals Engineering, 23(6), pp. 536–
540. doi: 10.1016/j.mineng.2010.03.010
Albrecht-Gary, A. M., and Crumbliss, A. L., 1998, Coordination chemistry of
siderophores: Thermodynamics and kinetics of iron chelation and release,
In Iron Transport and Storage in Microorganisms, Plants, and Animals (A.
Sigel, and H. Sigel, Eds.), New York, USA: Marcel Dekker, pp. 239–327.
Downloaded by [117.227.2.154] at 21:29 16 August 2017
Amin, M. M., Elaassy, I. E., El-Feky, M. G., Sallam, A. M., Talaat, M. S.,
and Kawady, N. A., 2014, “Effect of mineral constituents in the
bioleaching of uranium from uraniferous sedimentary rock samples,
Southwestern Sinai, Egypt.” Journal of Environmental Radioactivity,
134C, pp. 76–82. doi: 10.1016/j.jenvrad.2014.02.024
Amiri, F., Yaghmaei, S., and Mousavi, S. M., 2010, “Comparison of different
methods in bioleaching of tungsten-rich spent hydro-cracking catalyst
using adapted penecillum simplicissimum BBRC-20019.” Chemical
Engineering Transactions, 21, pp. 1483–1488. doi: 10.3303/CET1021248
Bromfield, S. M., and Skerman, V. B. D., 1950, “Biological oxidation of
manganese in soils.” Soil Science, 69, pp. 337–347.
Castro, I. M, Fietto, J. L. R., Vieira, R. X., Trópia, M. J. M. R., Campos, L. M. M.,
Paniago, E. B., Brandão, R. L., 2000, “Bioleaching of zinc and nickel from
silicates using Aspergillus niger cultures.” Hydrometallurgy, 57(1), pp. 39–
49. doi: 10.1016/S0304-386X(00)00088-8
Clavier, N., Podor, R., and Dacheux, N., 2011, “Crystal chemistry of the
monazite structure.” Journal of the European Ceramic Society, 31, pp.
941–976. doi: 10.1016/j.jeurceramsoc.2010.12.019
Emmanuel, E. S. C., Vignesh, V., Anandkumar, B., and Maruthamuthu,
S., 2011, “Bioaccumulation of cerium and neodymium by Bacillus
cereus isolated from rare earth environments of Chavara and
Manavalakurichi, India.” Indian Journal of Microbiology, 51(4), pp.
488–495. doi: 10.1007/s12088-011-0111-8
Gadd, G. M., 1999, “Fungal production of citric and oxalic acid:
Importance in metal speciation, physiology and biogeochemical pro-
cesses.” Advances in Microbial Physiology, 41, pp. 47–92. doi: 10.1016/
S0065-2911(08)60165-4
Hassanien, W. A. G., Desouky, O. A. N., and Hussien, S. S. E., 2014,
“Bioleaching of some rare earth elements from Egyptian monazite using
Aspergillus ficuum and Pseudomonas aeruginosa.” .Walailak Journal of
Science and Technology, 11(9), pp. 809–823. doi: 10.2004/wjst.v11i6.481
Koltun, P., and Tharumarajah, A., 2014, “Life cycle impact of rare earth
elements.” ISRN Materials, 2014, Article ID 907536, 10 pages.
10.1155/2014/907536
MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW 9
Lian, B., Wang, B., Pan, M., Liu, C., and Teng, H. H., 2008, “Microbial
release of potassium from K-bearing minerals by thermophilic fungus
Aspergillus fumigatus. ” Geochimica et Cosmochimica Acta, 72(1), pp.
87–98. doi: 10.1016/j.gca.2007.10.005
Maes, S., Zhuang, W. Q., Rabaey, K., Alvarez-Cohen, L., and Hennebel,
T., 2017, “Concomitant leaching and electrochemical extraction of
rare earth elements from monazite.” Environmental Science &
Technology, 51(3), pp. 1654–1661. doi: 10.1021/acs.est.6b03675
Nithiyaa, P., Nur Ain Izzati, M. Z., Umi Kalsom, Y., and Salleh, B.,
2012, “Diversity and morphological characteristics of Aspergillus
species and Fusarium species isolated from cornmeal in Malaysia.”
Pertanika Journal of Tropical Agricultural Science, 35(1), pp.
103–116.
Nouailhetas, Y., Lauria, D. C., Godoy, J. M., Reis, V. R. G., and Zenaro, R.,
1993, “Radioecological questions concerning the monazite sand cycle
wastes in Brazil.” Radiation Protection in Australia, 11, pp. 177–181.
Papagianni, M., and Mattey, M., 2006, “Morphological development of
Aspergillus niger in submerged citric acid fermentation as a function
of the spore inoculum level. Application of neural network and cluster
analysis for characterization of mycelial morphology.” Microbial Cell
Factories, 5, p. 1–12. doi: 10.1186/1475-2859-5-3
Payne, S. M., 1994, Detection, isolation, and characterization of side-
rophores.” Methods in Enzymology, 235, pp. 329–344. doi: 10.1016/
0076-6879(94)35151-1
Pillai, P. M. B., 2007, “Naturally occurring radioactive material (NORM)
in the extraction and processing of rare earths.” In Proc. 5th Int.
Symp. Naturally occurring radioactive material (NORM V), Seville,
Spain, IAEA, pp. 197–222.
Qu, Y., and Lian, B., 2013, “Bioleaching of rare earth and radioactive
elements from red mud using Penicillium tricolor RM-10.” Bioresource
Technology, 136, pp. 16–23. doi: 10.1016/j.biortech.2013.03.070
Reichard, P. U., Kretzschmar, R., and Kraemer, S. M., 2007, “Dissolution
mechanisms of goethite in the presence of siderophores and organic
acids.” Geochimica et Cosmochimica Acta, 71, 5635–5650. doi:
10.1016/j.gca.2006.12.022
Rezza, I., Salinas, E., Elorza, M., Sanz de Tosetti, M., and Donati, E.,
2001, “Mechanisms involved in bioleaching of an aluminosilicate by
heterotrophic microorganisms.” Process Biochemistry, 36, 495–500.
doi: 10.1016/S0032-9592(00)00164-3
Sayyed, R. Z., Badgujar, M. D., Sonawane, H. M., Mhaske, M. M., and
Chincholkar, S. B., 2005, “Production of microbial iron chelators
(siderophores) by fluorescent Pseudomonads.” Indian Journal of
Biotechnology, 4, pp. 484–490.
Schwyn, B., and Neilands, J. B., 1987, “Universal chemical assay for the
detection and determination of siderophores.” Analytical
Biochemistry, 160(1), pp. 47–56. doi: 10.1016/0003-2697(87)90612-9
van Breemen, N., Finlay, R. D., Lundström, U., Jongmans, A. G., Giesler, R.,
and Olsson, M., 2000, “Mycorrhizal weathering: A true case of mineral
plant nutrition?” Biogeochem, 49(1), pp. 53–67. doi: 10.1023/
A:1006256231670
Vijayan, S., Melnyk, A. J., Singh, R. D., and Nuttall, K., 1989, “Rare
earths: Their mining, processing and growing industrial usage.”
Minerals Engineering, 41(1), pp. 13–18.
Xu, D. B., Madrit, C. P., Röhr, M., and Kubicek, C. P., 1989, “The
influence of type and concentration of the carbon source on the
production of citric acid by Aspergillus niger. ” Applied Microbiology
and Biotechnology, 30(6), pp. 553–558. doi: 10.1007/BF00255358