CLAY-03302; No of Pages 8

Applied Clay Science xxx (2015) xxx–xxx

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Applied Clay Science

journal homepage: www.elsevier.com/locate/clay

Review article

Biological beneficiation of kaolin: A review on iron removal

Mohammad Raouf Hosseini ⁎, Ali Ahmadi
Department of Mining Engineering, Isfahan University of Technology, Isfahan, Iran
Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan, Iran

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

Article history: Application of mineral bio-beneficiation to remove iron oxyhydroxides from kaolinite clays has been the center of
Received 13 October 2014 interest in recent decades, and many research works over the world have been dedicated to study the potential of
Received in revised form 7 January 2015 this approach for upgrading the clay whiteness and refractoriness while preserving the inherent specifications
Accepted 9 January 2015
and therefore improving the commercial value of kaolin. Microbial purification methods are preferred since
Available online xxxx
they are environmentally benign (neither use nor release hazardous chemicals), not energy intensive, low in
Keywords:
initial and operational costs, and they preserve the crystal structure of the clay. The present review offers the
Kaolin general concepts and fundamentals of the process along with an outline and critical assessment of the latest
Iron studies carried out on the biological dissolution of iron from kaolinite clays.
Oxalic acid © 2015 Elsevier B.V. All rights reserved.
Bioleaching
Bioflocculation
Bioflotation

1. Introduction
Kaolin is a significant industrial clay that mainly contains a hy-
drated aluminum silicate mineral named as kaolinite (Al2Si2O5(OH)4)
(de Mesquita et al., 1996; Murray, 2006b; Zegeye et al., 2013). Other ka-
olin minerals include dickite, nacrite, and halloysite. Pure forms of these
minerals are not as ubiquitous as kaolinite, and are usually found along
with kaolinite in hydrothermal deposits (Murray, 2006b). Kaolin may
form in residual or sedimentary modes. In the former type, kaolinite is
created by in-situ weathering or hydrothermal alteration of aluminosil-
icate parent rocks like granite; though, in the latter, the mineral is pro-
duced by the deposition of kaolinite formed elsewhere (Bloodworth
et al., 1993; Zegeye et al., 2013).
Unique mineralogy, morphology, chemical and physical specifications
of kaolin make it a versatile raw material appropriate for many different
applications (Ryu et al., 1995; Kostka et al., 1999a; Štyriaková and Štyriak,
2000; Murray, 2006b), such as ceramic, paper coating and fillers, pigment
extender in water-based interior latex paints and oil-based exterior
industrial primer. In addition, kaolin is applied in non-black rubber, med-
icines and pharmaceuticals, cosmetics, crayons, fertilizers, detergents,
pesticides, white cement, ink, catalysts, and many other products
(Bloodworth et al., 1993; Ryu et al., 1995; Murray, 2006a,b). These prop-
erties are greatly affected by the mode of clay formation which controls
the kaolin quality through varying the kaolinite and impurity contents.
For instance, kaolinite content of the residual and sedimentary kaolins
⁎ Corresponding at: Department of Mining engineering, Isfahan University of Technology,
Isfahan, Iran. Tel.: +98 3133915171; fax: +98 3133912776.
E-mail address: r.hosseini@cc.iut.ac.ir (M.R. Hosseini).
differs from 20% to 60%, respectively (Bloodworth et al., 1993). High qual-
ity kaolins are also low in iron-bearing minerals.
The existence of iron oxides in kaolin adversely affects the clay color,
and reduces its brightness and refractoriness (Ryu et al., 1995;
de Mesquita et al., 1996; Lee et al., 2002; Mockovčiaková et al., 2008).
These cause a dramatic decrease in its commercial price (Guo et al.,
2010). Even an amount of 0.4% of oxides, hydroxides and hydrated
oxides of ferric iron may be enough to impart a red to yellow pig-
mentation to clay deposits. These iron oxide/hydroxides may be he-
matite (red), maghemite (reddish brown), goethite (brownish yellow),
lepidocrocite (orange), ferrihydrate (brownish red), etc. (Ambikadevi
and Lalithambika, 2000). Similarly, iron ores such as hematite may
contain clays like kaolin as contamination which cause problems in
the operation of blast furnaces. Therefore, the first beneficiation step
to make these raw materials commercially valuable is to effectively
eliminate iron oxides from kaolinite clays and vice versa (Poorni and
Natarajan, 2013).
The iron removal processes can be categorized as physical, chemical,
or a combination of both (Prasad et al., 1991; de Mesquita et al., 1996).
Physical approaches include intense magnetic separation which is
able to remove iron and titanium impurities, gravity separation, and
hydrocyclones (Prasad et al., 1991; de Mesquita et al., 1996; Guo et al.,
2010; He et al., 2011). Chemical methods involve leaching the iron con-
tent of clay by organic and inorganic acids such as oxalic, citric or hydro-
chloric acid (Prasad et al., 1991; Ambikadevi and Lalithambika, 2000;
Musiał et al., 2011), and reductive leaching by potent reducers, like so-
dium dithionite plus aluminum sulfate, sulfur dioxide plus aluminum
or zinc powder, sodium formaldehyde sulfoxylate, hydroxyl ammonium
acid sulfate, and hydrazine (de Mesquita et al., 1996; Ambikadevi and

http://dx.doi.org/10.1016/j.clay.2015.01.012
0169-1317/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Hosseini, M.R., Ahmadi, A., Biological beneficiation of kaolin: A review on iron removal, Appl. Clay Sci. (2015),
http://dx.doi.org/10.1016/j.clay.2015.01.012
2 M.R. Hosseini, A. Ahmadi / Applied Clay Science xxx (2015) xxx–xxx
Lalithambika, 2000; Štyriaková et al., 2003; Stucki, 2013; Zegeye et al.,
2013). Also, combined remediation processes are froth flotation and
flocculation (Prasad et al., 1991; Lee et al., 2002; Guo et al., 2010; He
et al., 2011).
Among the above mentioned approaches, the reductive leaching of
iron from kaolins is more useful and results in a significant removal of
iron (Prasad et al., 1991; de Mesquita et al., 1996; Zegeye et al., 2013);
however, it destructs the crystal structure of the clay and also, lessens
the organic component of kaolin, which affects the clay plasticity and re-
fractoriness (Kostka et al., 1999a; Lee et al., 2002; Andrade et al., 2011).
On the other hand, these bleaching methods are high in capital and
operating costs (Lee et al., 2002), require complex operating conditions
(de Mesquita et al., 1996), and are environmentally hazardous (Guo
et al., 2010).
Hence, in order to dispose of these technical, environmental, and
economic problems, physical and chemical techniques should be
substituted by biological methods (Ryu et al., 1995; Chen and Lin,
2010) which are supposed to play a trivial role in iron mobilization
from clay minerals in nature. Iron oxide existence in kaolin is biological-
ly induced, so it is better to apply a biological process for removing it
from clays by exploiting microorganisms (Poorni and Natarajan,
2013). Interaction of microbes with clay minerals conserves the clay
structure, fertility, and chemistry of the soil through the cation
exchange and pH buffering (Kostka et al., 1999a). This also greatly
affects the mineral weathering, degradation of organic material, devel-
opment of aggregates, and the mobility of a wide variety of elements
(Jiang et al., 2007) that cause microorganisms to be applied for several
purposes such as valuable metal extraction, heavy metal removal from
wastes to prevent environmental pollution, and beneficiation of indus-
trial minerals (Kostka et al., 1999b; Cameselle et al., 2003).
Bio-beneficiation processes are more efficient in comparison to
chemical routes, because they are environmentally benign as they
neither use nor release hazardous chemicals, not energy intensive for
they operate at room temperature, low in initial and operational costs,
and to a large extent, maintain the crystal structure of clay.
In the present study, initially, the general concepts and fundamen-
tals of the biological removal of iron from kaolinite clays are described,
and then the literatures and findings published on this subject in the
past two decades are concisely reviewed and assessed.
2. Iron impurities in clay minerals
Iron (Fe) comprises about 6% of the earth's mass, and therefore is the
fourth most abundant element in the world after oxygen, silicon, and
aluminum (Dong, 2012; Stucki, 2013). Biotite, pyroxene, amphibole,
olivine, ilmenite, magnetite, and pyrite are the most important iron
bearing minerals in soils which in all of them, iron is predominantly in
bivalent state (Cornell and Schwertmann, 2004). Weathering causes
the release of Fe II from rocks and formation of Fe III oxide/hydroxides
which move through the crust mechanically or via complexation/
reduction by organic compounds and microbes, and then mix with clay
silicates by precipitating and coating soil grains or scatter throughout
the bulk of the clay as a separate phase (Ambikadevi and Lalithambika,
2000; Cornell and Schwertmann, 2004; Štyriaková et al., 2012; Stucki,
2013). Also, iron may present in clays as structural style either in oxi-
dized form bound the aluminosilicate lattice in both the octahedral
and tetrahedral sheets or as a compensating iron ion which substitutes
for aluminum in silicate structure (Štyriaková and Štyriak, 2000;
Mockovčiaková et al., 2008; Štyriaková et al., 2012; Stucki, 2013). Iron
oxide formation varies with the changing water and air content of
soil. For example, hematite may be found as coatings at the dry surface
of rocks, whereas goethite can occur near moist cracks or plant roots
(Cornell and Schwertmann, 2004; Murray, 2006a). The higher the
pH of the environment, the greater the iron oxidation rate, and de-
pending on the type, maturity, and iron content of the parent rock,
iron content varies between less than 0.1% to several 10% (Cornell and
Please cite this article as: Hosseini, M.R., Ahmadi, A., Biological beneficiation of kaolin: A review on iron removal, Appl. Clay Sci. (2015),
http://dx.doi.org/10.1016/j.clay.2015.01.012
Schwertmann, 2004). Reduction of structural iron affects the swelling,
cation exchange capacity, specific surface area, color, and magnetic ex-
change interactions. It also, causes the clay layers to collapse and trap
cations in the mineral matrix, which makes the valuable nutrients
unavailable for agricultural purposes (Xie and Walther, 1992; Kostka
et al., 1999b; Stucki, 2013).
3. Biological leaching
Bioleaching processes are based on the ability of microorganism or
their metabolites to transform solid compounds into soluble and ex-
tractable elements which can be recovered, subsequently. The following
is a brief history of kaolin bioleaching using different kinds of bacteria
and fungi.
3.1. Fungi
Fungi which are used in leaching processes must be able to excrete
considerable amounts of organic acids and be resistant to heavy metals.
Generally, fungi acidify the environment during growth because of
the excretion of protons via the proton trans-locating plasma mem-
brane, absorption of nutrients in exchange for protons, excretion of
organic acids, and the carbon dioxide production by their respiration
(Burgstaller and Schinner, 1993). This acidic medium and the metabo-
lites contribute to biological solubilization of metal compounds through
protonation (acidifying), complexation, or reduction (Burgstaller and
Schinner, 1993; de Mesquita et al., 1996; Cornell and Schwertmann,
2004). A fungus may also accumulate the metal ion from the solution
and cause metal detoxification. In the first mechanism, the oxygen
atoms covering the surface of a metal compound are protonated and
the metal then detaches from the surface. In complexation, a metal ion
is solubilized by forming a soluble complex with a chelating molecule
like oxalic or citric acid (Burgstaller and Schinner, 1993; Ambikadevi
and Lalithambika, 2000; Saikia et al., 2003; Hernández et al., 2013).
This is the most important characteristic of fungal leaching processes
as the ultimate purpose of leaching is to extract metal ions in dissolved
form. Additionally, reduction may also happen as reducing ferric
iron or manganese to more soluble Fe II or Mn II by oxalic acid which is
biologically excreted to the culture medium (Burgstaller and Schinner,
1993; de Mesquita et al., 1996; Cornell and Schwertmann, 2004;
Lovley et al., 2004).
Among the microorganisms of interest, the fungus Aspergillus niger
is often favored as the most efficient species producing oxalic acid
(Mulligan et al., 2004; Hosseini et al., 2007; Musiał et al., 2011; Zegeye
et al., 2013). The maximum oxalic acid production from saccharides
by this fungus is obtained by setting the pH in the range of 6 to 7, but
when the pH is lower, citric acid is dominant (Cameselle et al., 1998;
Musiał et al., 2011). Application of oxalic acid produced by fungi
for the dissolution of iron impurities from kaolinite clays has been the
subject of many experimental research works which are summarized
in Table 1. These experiments have been generally conducted in two
forms of “in-situ” or “two-stage”. When the process is done in-situ,
microbial growth and bioleaching proceed simultaneously that means
microorganisms metabolize in the presence of clay; however, in the
two-stage style, fungal fermentation is performed firstly, then the clay
bioleaching stage is carried out using the metabolites separated from
the fungal cells.
The idea of the presence of kaolin in the growing cultures (in-situ
bioleaching) affects negatively both fungus development and active
metabolite secretion. In contrast, two-stage bioleaching makes it
possible to perform the process at higher temperatures which are not
tolerable for the microorganisms in an in-situ approach. Therefore,
applying a full factorial designs, Cameselle et al. (2003) tried to make
a comparison between the kaolin bleaching efficiency through in-situ
and two-stage processes by two different strains of A. niger, CBS
246-65 which had been used previously (Cameselle et al., 1995), and
 M.R. Hosseini, A. Ahmadi / Applied Clay Science xxx (2015) xxx–xxx 3

Table 1
History of iron bioleaching from kaolin by fungi (the best result of each work is listed).

Fungus Clay Method Time Temp. (°C) Iron oxide (%) Brightness (%) Reference

Before After Before After

Candida sp. Kaolin In situ 7 days 30 0.8 0.72 – – Toro et al. (1992)
A. niger & Penicillium sp. Kaolin In situ 7 days 30 0.8 0.76 – – Toro et al. (1992)
A. niger Kaolin In situ 7 days 30 0.8 0.77 – – Toro et al. (1992)
A. niger CBS246-65 Kaolin Two-stage 5h 30 2.30 1.30 56.5 67.0 Cameselle et al. (1995)
A. niger (from talc ore) Kaolin Two-stage 3h 60 0.35 0.30 62.9 64.4 de Mesquita et al. (1996)
A. niger CBS246-65 Kaolin In situ 15 days 30 2.30 1.52 – – Cameselle et al. (2003)
A. niger CBS1120 Kaolin In situ 10 days 30 2.30 0 56.5 80.0 Cameselle et al. (2003)
A. niger CBS1120 Kaolin Two-stage 40 h 30 2.30 0 56.5 80.0 Cameselle et al. (2003)
A. niger NCIM548 Kaolin In situ 30 days 30 11.00 10.76 – – Hosseini et al. (2007)
A. niger (from pistachio shell) Kaolin In situ 30 days 30 11.00 6.27 – – Hosseini et al. (2007)
A. niger PTCC5012 Kaolin In situ 30 days 30 2.20 1.88 – – Aghaie et al. (2007)
A. niger Kaolin In situ 21 days 25 1.70 0.40 – – Arslan and Bayat (2009)
A. niger (from pistachio shell) Kaolin In situ 30 days 30 2.20 0.72 – – Aghaie et al. (2009)
A. niger XP Kaolin Two-stage 2h 50 0.51 0.44 74.0 79.4 Musial et al. (2011)
A. niger (from pistachio shell) Kaolin In situ 35 days 30 2.20 0.58 – – Aghaie et al. (2012)

CBS 1120. The factors were pH, strain type, amount of kaolin, and the
time of clay addition to the culture. For in-situ experiment, all of the
iron content was removed by A. niger CBS 1120 in less than 10 days
when the pH value was adjusted to 2, while CBS 246-65 was able to re-
move just 34% of iron in the same conditions. Considering the two-stage
operation by A. niger CBS 1120, the obtained results were the same as in-
situ experiments, but in a shorter time (40 h) at pH: 2. It was also con-
cluded that the lower the pH and clay loading in operations, the higher
the removal efficiency. The authors had also practiced the two-stage
bioleaching by the spent fermentation liquor of A. niger CBS 246-65 at
a higher temperature, formerly (Cameselle et al., 1995) to show that
iron dissolution could be improved in this condition. Moreover, they in-
vestigated the effects of temperature, agitation rate, pH, and citric acid
concentration. As a result, 43% of iron oxide impurities were removed
at 60 °C after 5 h and the whiteness index improved to 67%. Also, as
enzymes are not active in these temperatures, it was found that iron
dissolution was due to the presence of organic acids, and extracellular
enzymes were not responsible.
Furthermore, de Mesquita et al. (1996) conducted a series of iron
leaching experiments on different samples of Brazilian kaolin using
citric acid, oxalic acid and fermented medium of A. niger (two-stage)
at 60 °C. The iron contents of the samples varied between 0.34% to
3.67%, and the maximum improvement of kaolin brightness was 1.5,
4.9, and 4.7% for citric acid, oxalic acid, and fermented medium, respec-
tively. The better results that were derived by oxalic acid implementa-
tion were attributed to the Fe III reduction capability of this organic
acid. The greater iron leaching ability of oxalic acid was also described
by Mulligan et al. (2004) as being five times more than citric acid. More-
over, Arslan and Bayat (2009) compared kaolin obtained by chemical
leaching with the product of bioleaching treatment using A. niger.
Exploiting chemical method, the maximum removal of iron (~ 95%)
happened at 15% w/v pulp density, temperature of 80 °C, oxalic acid
concentration of 0.2 M, and a particle size of b63 mm after 120 min.
In contrast, bioleaching experiments resulted in removal of 77.13% of
the total iron at a much lower pulp density (1% w/v), temperature of
25 °C, 3 × 107 spore/l, and particle size of b 63 mm after 21 days. Like
the preceding reports (Cameselle et al., 2003; Hosseini et al., 2007),
the authors also indicated that increase in pulp density decreased iron
extraction efficiency.
Some researchers have tried to substitute other carbon sources for
saccharides to improve oxalic acid secretion or make the process more
cost effective. For instance, using a response surface methodology,
Musial et al. (2006, 2011), and Musiał and Rymowicz (2009) proved
that A. niger XP cultured in a biodiesel-derived waste composed of
glycerol and fatty acids metabolized both compounds and excreted
oxalic acid (48.9 g/l) to the medium without any by-product. Also, the
whiteness index of kaolin reached to 79.4% using a kaolin content of
20 g/l, a solution containing 27.5 g/l oxalic acid, and the elevated tem-
perature of 50 °C over a period of 1.7 h (Musiał et al., 2011).
Considering in-situ approach, one of the first attempts was made by
Toro and colleagues in 1992 (Toro et al., 1992) by using different fungi
and bacteria and combinations of them. They showed that cultures of
Candida sp., A. niger, and a mix culture of A. niger and Penicillium sp. in
the presence of kaolin could respectively solubilize 10%, 3.7%, and 5%
of the iron in the clay sample. Employing A. niger isolated from pistachio
shell and NCIM548, and by applying a full factorial design, a highly con-
taminated kaolin was subjected to in-situ iron leaching by Hosseini et al.
(2007) to study the influences of pulp density and time of kaolin addi-
tion to the culture. The best outcome (about 43% removal of iron) was
attained after one month using the strain isolated from pistachio shell
at the lower pulp density (20 vs. 60 g/l) when the clay was added at
the beginning of the fungal fermentation, although in runs which kaolin
was added on the third day of growth, the initial rate of iron dissolution
was higher. The reason was that when the clay and fungal spores were
added to medium simultaneously, microorganism needed a time period
to adapt to the clay, but after that, the adapted fungus performed better
compared to the one exposed to kaolin on the third day. These out-
comes confirmed the results reported by Cameselle et al. (2003). As a
complementary to the previous research, Aghaie et al. (2007) studied
the iron bioleaching ability of another strain of A. niger, PTCC5012, but
it was still lower in contrast to the strain isolated from pistachio shell.
Furthermore, they applied central composite design (Response Surface
Methodology) to illustrate the effects of initial pH, sucrose and spore
quantity on the oxalic acid and citric acid production, and dissolved
iron concentration in the fermented medium of A. niger isolated from
pistachio shell during the bioleaching of a kaolin sample. Results indi-
cated that the maximum organic acid production happened at the
highest spore concentration (35 × 107 spore/l), and a pH value around
4, whereas optimum iron removal (67.4%) happened when initial
pH was at its lowest value (pH: 2). These were also reported by de
Mesquita et al. (1996) and Cameselle et al. (2003), previously. In the
next stage, Aghaie et al. (2012) carried out a comprehensive research
on the kinetic modeling of fungal cell growth, substrate utilization,
iron bioleaching, oxalic and citric acid production during the in-situ
bioleaching of kaolin. For growth estimation a logistic equation
(Eq. (1)) was used, in which, X is cell concentration; X0 and Xm, are ini-
tial and maximum cell concentrations, respectively, μm is maximum
specific growth rate, and t is ferment time; while, for substrate con-
sumption (Eq. (2)) and product formation (Eq. (3)), Luedeking–Piret
equations were applied, in them, P and S are product and substrate
concentrations, α and β are growth and non-growth associated product
formation coefficients, respectively, S0 is initial substrate concentration,

Please cite this article as: Hosseini, M.R., Ahmadi, A., Biological beneficiation of kaolin: A review on iron removal, Appl. Clay Sci. (2015),
http://dx.doi.org/10.1016/j.clay.2015.01.012
4 M.R. Hosseini, A. Ahmadi / Applied Clay Science xxx (2015) xxx–xxx
YX/S is yield factor for cells on substrate, and ms is maintenance coeffi-
cient. Regarding the cell growth, A. niger followed a classical growth
trend which consisted of a lag (about 2–3 days), exponential and sta-
tionary phase, and reasonably fitted the Eq. (1), where, Xm = 4.1 g/l,
X0 = 0.423 g/l and μm = 0.120 l/day. Moreover, considering Eq. (2)
for product formation, α was − 0.668, 4.089, and 163, and β was
0.222, − 0.008, and − 2.3 for oxalic acid, citric acid and dissolved
iron concentration, respectively. Also, for the sucrose utilization
(Eq. (3)), YX/S = 0.082 g/g, mS = −0.2 g/g·day, and S0 = 25 g/l.
X0 eμm t
X¼
:
1−ðX 0 =X m Þ 1−eμ m t
0 1
 
B e μm t C X X  μ t
P ¼ αX 0 B
@   −1C
A þ β m ln 1− 0 1−e m : ð2Þ
X
 μm Xm
1− 0 1−eμ m t
Xm
X eμm t
S ¼ S0 −  0 
X

Y X=S 1− 0 1−e μ m t
Xm
   
X X m X μ t
þ 0 − m s ln 1− 0 1−e m : ð3Þ
Y X=S μm Xm
Collecting the iron dissolution, oxalic and citric acid production data
obtained from the three previous studies, Pazouki et al. (2012) per-
formed an optimization using artificial neural network (ANN). It was
suggested that the highest iron removal (about 65%) would be obtained
at sucrose and spore concentration of 60 g/l and 3.5 × 107 spore/l, re-
spectively; also, the best oxalic and citric acid concentration would be
gained at pH: 4–6, and sucrose and spore concentration of 50–60 g/l
and 3–4 × 108 spore/l, respectively.
3.2. Bacteria
Iron oxide/hydroxides may be eliminated from kaolinite clays by
dissimilatory Fe III reduction. Bacterial Fe III reduction has been recog-
nized as a significant process which catalyzed many natural and con-
taminant biogeochemical cycles (Kostka et al., 2002). In this process,
microorganisms reduce Fe III, even in the structural form, for purposes
other than assimilation of iron (Kostka et al., 1996; Lovley et al., 2004;
Zegeye et al., 2013). This happens under anaerobic conditions and the
soluble Fe II is produced by bacteria and removed from the kaolin (Lee
et al., 1999, 2002; Guo et al., 2010; Štyriaková et al., 2012). These micro-
organisms can utilize hydrogen or organic compounds like sugars,
amino acids and even mono-aromatic compounds and long-chain
fatty acids as electron donors and oxidize them to carbon dioxide with
Fe III serving as the sole electron acceptor (Lee et al., 1999; Maurice
et al., 2001; Lovley et al., 2004; Zegeye et al., 2013). Formerly, it
had been stated that the reactivity of Fe III as an electron acceptor was
related to its degree of crystallinity. For instance, it was supposed that
ferric hydroxide (Fe(OH)3) was microbially reduced more easily than
goethite (FeOOH), and hematite (Fe2O3) (Lovley and Phillips, 1986;
Phillips et al., 1993; Lee et al., 2002); however, later studies proved
that the extent of Fe III reduction mostly depended on the surface area
of iron oxide and not their crystallinity (Kostka and Nealson, 1995;
Roden and Zachara, 1996; Kostka et al., 2002). Nevertheless, in the
structural form, Fe III impurities are less bio-available in kaolin and
since are hardly reduced in contrast to other clay minerals such as smec-
tite, illite, chlorite, and nontronite (Zegeye et al., 2013). A brief history of
iron dissolution from clays by bacterial leaching is presented in Table 2.
Toro et al. (1992) described a microbial method to remove iron im-
purities from kaolin ore using different bacteria including Agrobacter sp.,
Bacillus sp., Enterobacter sp., Escherichia coli, and their mix cultures to-
gether or with some fungi. In the best conditions, 37% and 81% of iron
Please cite this article as: Hosseini, M.R., Ahmadi, A., Biological beneficiation of kaolin: A review on iron removal, Appl. Clay Sci. (2015),
http://dx.doi.org/10.1016/j.clay.2015.01.012
was removed by a single culture of Agrobacter sp. and a mix culture of
Agrobacter sp. & Candida sp., respectively. Moreover, conducting the ex-
periments using the microbial metabolites of Agrobacter sp. and Bacillus
sp. in elevated temperature (80 °C) negatively affected the iron removal
percent, which disagrees with the result of fungal leaching published
later (de Mesquita et al., 1996; Musiał et al., 2011), and suggests that
in contrast to fungi, the bacterial iron reduction and dissolution mostly
depends on enzymatic reactions. In order to contrast the ability of
Shewanella oneidensis for reducing structural iron III in clays with other
forms of iron compounds, Kostka et al. (2002) performed a comparative
study. Nearly one-third of the structural Fe III was reduced after three
ð1Þ
days, compared to approximately all of the Fe III citrate, and half of
amorphous oxyhydroxide (FeOOH). Once more, different Shewanella
species (Shewanella alga BrY, S. oneidensis MR-1, Shewanella putrefaciens
CN32, and S. putrefaciens CIP 8040) were employed by Zegeye et al.
(2013) to evaluate the Fe III removal from English kaolin. They showed
that the reductive dissolution of iron followed a first order kinetic
model and although the strains CIP8040, and CN32, respectively had
the highest, and lowest reduction rate, the final reduced iron content
by both strains after five days was the same. It was also suggested that
increasing the temperature would substantially improve the rate and
extent of bioleaching. Using a mixed culture of dissimilatory Fe III reduc-
ing microorganisms, Lee et al. (1999) successfully removed 44–45% of
iron at the sugar concentration of 5% (w/w) under anaerobic conditions.
They also reported that removal percent and rate linearly increased
with increasing concentration of sugars, irrespective of sugar species
used; this was also reported latterly by Aghaie et al. (2009) in bleaching
by A. niger. In another process (Groudev, 1999), the iron contaminants
leached from kaolin at about 90 °C by hydrochloric acids and lixiviant
containing oxalic acid biologically produced by Bacillus circulans. As a re-
sult, the iron contents (Fe2O3) of different kaolins were lowered from
levels of 0.65–1.49% to 0.44–0.75%. Also, the whiteness was increased
from values of 55–87% to values of 86–92%. Using the same procedure,
iron content of another sample was lowered from 6.25% to 1.85%, and
this increased the refractoriness from 1670 °C to 1750 °C.
In addition to the attempts to increase iron removal efficiency and
improve the clay whiteness and quality, the impact of structural iron re-
duction on the clay structure and properties has also been a matter of
concern. Some researchers have reported substantial alterations (Xie
and Walther, 1992), though other mentioned minor variations. Kostka
et al. (1999a,b) observed that the swelling pressure declined as well
as the particle surface area, and surface charge density increased as a re-
sult of rise in cation exchange capacity, when S. putrefaciens, Geobacter
metallireducens, Pseudomonas putida, and an iron-reducing strain from
rice paddy soil were used to reduce iron compounds of a clay (Kostka
et al., 1999b). Additionally, it was shown that the maximum iron reduc-
tion (55%) was performed by using the last microorganism, while it was
23–46% for the first bacteria, and 20% and 1% for the second and third
ones, respectively. Changes of kaolinite structure after bioreduction
were further assessed by Mockovčiaková et al. (2008) using lixiviant
produced by cultivating organic acid-producing bacteria of Bacillus
cereus and Bacillus pumilus. They stated that a small amount (around
1–2%) of Al and SiO2 was dissolved along with iron extraction which
led to the destruction of kaolinite structure. Also, as reported formerly
(Kostka et al., 1999b), they observed a decrease in surface area due
to the loss of iron ions from the mineral surface, and some small in-
crease in pore volume and redistribution in meso- and macropores.
Štyriaková and Štyriak (2000), measured the structural and free iron
oxide existed in three mineral samples and reported that B. cereus
strains were able to remove 43% of free iron and approximately 15% of
iron bound in mica after 1 month of bioleaching. They also, concluded
that the extraction of amorphous (free) form of iron was easier than
iron bound in mica as was previously suggested by some authors
(Phillips et al., 1993; Kostka et al., 1999a; Lee et al., 2002). However,
after a prolonged bioleaching time, removal of 53% of structural iron
was observed along with a partial destruction of mica structure and
 M.R. Hosseini, A. Ahmadi / Applied Clay Science xxx (2015) xxx–xxx 5

Table 2
History of iron bioleaching from kaolin and clay minerals by bacteria (the best result of each work is listed).

Strain Clay/clay Iron Method Time Temp. (°C) Iron (%)a Iron Whiteness (%) Reference
minerals phase comp.
BLb ALb BL AL

Agrobacter sp. Kaolin Free In situ 7 days 30 0.80 0.50 Oxc – – Toro et al. (1992)
Bacillus sp. Kaolin Free In situ 7 days 30 0.80 0.60 Ox – – Toro et al. (1992)
B. subtilis Kaolin Free In situ 7 days 30 0.80 0.67 Ox – – Toro et al. (1992)
Enterobacter sp. Kaolin Free In situ 7 days 30 0.80 0.71 Ox – – Toro et al. (1992)
Escherichia coli Kaolin Free In situ 7 days 30 0.80 0.75 Ox – – Toro et al. (1992)
Agrobacter sp. & Candida sp. Kaolin Free In situ 7 days 30 0.80 0.15 Ox – – Toro et al. (1992)
Bacillus sp. & Yeast sp. Kaolin Free In situ 7 days 30 0.80 0.3 Ox – – Toro et al. (1992)
Enterobacter sp. & E. coli Kaolin Free In situ 7 days 30 0.80 0.75 Ox – – Toro et al. (1992)
Bacillus sp. Kaolin Free 2-stage 1.5 h 80 0.80 0.74 Ox – – Toro et al. (1992)
Agrobacter sp. Kaolin Free 2-stage 1.5 h 80 0.80 0.61 Ox – – Toro et al. (1992)
Thiobacillus ferrooxidans Marine clay Free In situ 8 days 28 2.39 0.07 Pyc – – Ryu et al. (1995)
S. putrefaciens MR-1 Smectite Bound In situ 14 days 30 19.85 10.72 Fec – – Kostka et al. (1999a)
S. putrefaciens MR-1 Montmorillonite Bound In situ 14 days 30 2.92 0.29 Fe – – Kostka et al. (1999a)
S. putrefaciens MR-4 Smectite Bound In situ 14 days 30 19.85 11.32 Fe – – Kostka et al. (1999a)
G. metallireducens Smectite Bound In situ 14 days 30 19.85 15.88 Fe – – Kostka et al. (1999a)
Pseudomonas putida Smectite Bound In situ 14 days 30 19.85 19.65 Fe – – Kostka et al. (1999a)
IRB from rice paddy soil Smectite Bound In situ 14 days 30 19.85 8.93 Fe – – Kostka et al. (1999a)
S. putrefaciens MR-1 Smectite Bound 2-stage 14 days 30 19.85 19.57 Fe – – Kostka et al. (1999b)
Mixed culture of IRB Kaolin – In situ 11 days 30 3.22 1.76 Ox 58.84 77.46 Lee et al. (1999)
Bacillus circulans Kaolin – 2-stage 6h 90 1.49 0.75 Ox 55 86 Groudev (1999)
Bacillus cereus Kaolin Free In situ 28 days 30 0.92 0.53 Ox – – Štyriaková and Štyriak (2000)
Bacillus cereus Kaolin Bound In situ 28 days 30 1.43 1.29 Ox – – Štyriaková and Štyriak (2000)
S. oneidensis Smectite Bound In situ 3 days 30 19.85 13.23 Ox – – Kostka et al. (2002)
Mixed culture of IRB Kaolin – In situ 70 h 30 3.22 2.57 Ox 64.49 71.50 Lee et al. (2002)
B. cereus & B. pumilus Kaolin – 2-stage 100 days 28 1.17 1.03 Ox – – Mockovčiaková et al. (2008)
B. cereus, B. sphaericus, & B. mycoides Kaolin – In situ 10 days 30 0.94 0.46 Ox 61 82 Guo et al. (2010)
B. cereus, B. sphaericus, & B. mycoides Kaolin – In situ 10 days 30 0.94 0.40 Ox – – He et al. (2011)
S. alga BrY Kaolin – In situ 5 days 20 1.05 0.46 Ox – – Zegeye et al. (2013)
S. oneidensis MR-1 Kaolin – In situ 5 days 20 1.05 0.67 Ox – – Zegeye et al. (2013)
S. putrefaciens CN32 Kaolin – In situ 5 days 20 1.05 0.46 Ox – – Zegeye et al. (2013)
S. putrefaciens CIP 8040 Kaolin – In situ 5 days 20 1.05 0.46 Ox – – Zegeye et al. (2013)
a
It is supposed that the reduced iron is dissolved out of the clay.
b
AL: after leaching, BL: before leaching.
c
Ox: iron oxide/hydroxide, Py: pyrite, Fe: iron in a structural form.

surface, and illite formation that was confirmed by X-ray analysis and
SEM micrographs. In contrast, Zegeye et al. (2013) reported that the
XRD patterns showed very little structural modification during the
bioleaching of the kaolin sample by Shewanella species. And, according
to XRD and SEM studies done by Guo et al. (2010), not only no structural
damages were reported, but also improvement in crystallinity was
observed.
Furthermore, it has been suggested that the presence of ligands and
chelators in the growth medium facilitated the dissolution of reduced
iron from clay by serving as an electron shuttle or by making the iron
more available for reduction via chelating and solubilizing Fe III. Also
organic ligands like oxalate may stimulate the reaction by being used
as a carbon source. Kostka et al. (1999a) added organic ligands to a cul-
ture of S. putrefaciens. Consequently, the Fe III reduction was doubled
when nitrilotriacetic acid (NTA) was used, and about 37% of the total
iron in clay was reduced. Oxalate, citrate, and malate also stimulated
the process in the mentioned order. Other ligands like humic acid
and anthraquinone disulfonate (AQDS), also showed similar results
(Kostka et al., 2002). Considering the effect of additives on the
bioleaching process, He et al. (2011) exploited several organic acids,
complexing agents and heavy metals. Results indicated that except
for oxalate and oxalic acid which enhanced the iron dissolution, addi-
tion of other organic acids inhibited the culture growth. Similarly,
heavy metals like Fe II, Fe III, and Cu II had an inhibitory effect; though,
Mn II slightly enhanced the process probably due to its roles as an inter-
mediate electron transporter and beneficial trace element for enzyme
activation (Lovley et al., 2004). Moreover, in contrast to other studies
(Lovley and Woodward, 1996; Kostka et al., 1999a, 2002), addition of
complexing agents like NTA and EDTA did not show any improvements
(He et al., 2011).
Some researchers have investigated the effect of carbon source
and its quantity on the bio-reduction of Fe2O3 in kaolin. Lee et al.
(2002) used the same culture as used before (Lee et al., 1999) in addi-
tion to maltose, sucrose, glucose, and galactose as carbon source. Re-
sults showed that increasing the carbon source from 1 to 5%,
enhanced the bioreduction, but after that, due to the increase in the os-
motic potential of the microorganism, an inhibitory effect was observed
as was later similarly seen by Aghaie et al. (2009). In addition, all the
carbon sources contributed to the dissolution of the same amount of
Fe III except galactose which dissolved the least amount. In another
attempt, exploiting bacteria (B. cereus, Bacillus sphaericus, and Bacillus
mycoides) isolated from a kaolin deposit, Guo et al. (2010) evaluated
the effects of different carbon and nitrogen sources on iron removal
rate as: monosaccharide N disaccharide N polysaccharide. The reason is
that monosaccharide is directly oxidized to pyruvate, an electron
donor that is used by bacteria; however, other saccharides have more
complicated pathways. Additionally, (NH4)2SO4 was introduced as the
best nitrogen source because it provides nitrogen as well as sulfur
which is also essential for growth.
If the iron mineral in the clay is in the sulfide form (pyrite), bio-
oxidation is a proper solution. Ryu et al. (1995) exploited Thiobacillus
ferrooxidans to oxidize both sulfur and iron for removing 93–97% of py-
rite impurities from a pulp with 20% solid after 8 days. They found that
this biological process could be performed in distilled water instead of
nutrient medium without a significant change in efficiency as the clay
itself contained necessary salts for microbial growth. However, overuse
of the mineral salts reduced the rate and extent of pyrite removal with
the insoluble sulfate compound (such as jarosite) formed on the pyrite
surface. Furthermore, in contrast to pyrite removal from coals (Andrews
and Maczuga, 1982), and other studies reviewed here (Cameselle et al.,

Please cite this article as: Hosseini, M.R., Ahmadi, A., Biological beneficiation of kaolin: A review on iron removal, Appl. Clay Sci. (2015),
http://dx.doi.org/10.1016/j.clay.2015.01.012
6 M.R. Hosseini, A. Ahmadi / Applied Clay Science xxx (2015) xxx–xxx
2003; Hosseini et al., 2007; Arslan and Bayat, 2009), changing the pulp
density from 5 to 70% did not show any negative impact on iron removal
rate and extent, because of the low organic contents of the clay which
eliminated their inhibitory effect.
4. Bio-flocculation and bio-flotation
As an alternative approach to bioleaching, microbially induced
separation of kaolin and iron oxides can be achieved via selective
bioflocculation or bioflotation which mainly depend on the surface
chemistry of microorganisms and minerals (Poorni and Natarajan,
2013). Therefore, any changes in the surface chemistry brought about
by adsorption and adhesion of bacterial cells and their metabolic
products would be important (Deo and Natarajan, 1998). The negative
charge of bacterial cells would provoke electrostatic interaction with
positively charged minerals at pH values lower than the mineral IEP
values (Deo and Natarajan, 1998); also, wide variety of gram-negative
and positive bacteria produce extracellular polymeric substances (EPS)
during their growth period. EPS are biomolecules which are mainly
composed of polysaccharides and proteins, with nucleic acids and lipids
as minor components (Wilkinson, 1958; Cao et al., 2011). EPS may at-
tach to the cell surface, secrete into solution or connect to the hydrated
matrix of biofilms (Wingender et al., 1999; Cao et al., 2011). EPS play an
important role in biofilm formation, migration of bacterial cells, mineral
dissolution, biomineralization and heavy metal accumulation (Fang
et al., 2010; Cao et al., 2011). EPS adsorb on mineral surfaces through hy-
drophobic, electrostatic, covalent, and polymer–polymer interactions
(Whitfield, 1988; Cao et al., 2011). Adsorption of bacterially generated
proteins on mineral surface renders them more hydrophobic; whereas,
polysaccharides make them hydrophilic (Poorni and Natarajan, 2013).
Table 3 puts together the use of these mechanisms for separation of
iron minerals from clays which are published in some recent literatures.
Regarding the bioflocculation of kaolin alone, there are a lot of liter-
atures published worldwide (Salehizadeh and Shojaosadati, 2001; Shih
et al., 2001; Rong et al., 2008; Li et al., 2009) which are not included in
the present review as it focuses on the separation of kaolin from iron
minerals. For example, one of the oldest was carried out by Gomoiu
and Catley (1996) using biopolymers elaborated by Byssochlamys
nivea. They explained that the flocculant isolated from the microorgan-
ism was predominantly (97%) polysaccharide bound to clay through a
carboxyl moiety; however, for iron separation, polysaccharides adsorb
on iron minerals, and flocculate them from clay particles which are
suspended throughout the pulp as a consequence of protein adsorption.
Later, Deo and Natarajan (1998) in a comprehensive research ob-
served that Paenibacillus polymyxa and its cationic metabolites positive-
ly changed the IEP of quartz, while anionic metabolites caused a
substantial negative shift in measured IEP of corundum and hematite.
Flotation tests, and contact angle measurements demonstrated that
interaction of cells and their metabolites with minerals resulted in en-
hancing the hydrophobicity of quartz and kaolinite unlike the hematite
and corundum which were rendered more hydrophilic. Regarding the
Table 3
History of iron and clay minerals bioflocculation/bioflotation (the best result of each work is listed).
Microorganism Minerals/clay minerals
Paenibacillus polymyxa Kaolinite/hematite
Paenibacillus polymyxa Kaolinite/hematite
Paenibacillus polymyxa Hematite/corundum
Paenibacillus polymyxa Quartz/hematite
Paenibacillus polymyxa Quartz/hematite
Paenibacillus polymyxa Quartz/corundum
Bacillus subtilis Goethite
Pseudomonas putida Montmorillonite, kaolinite, and goethite
Bacillus subtilis Montmorillonite, kaolinite, and goethite
Pseudomonas putida Montmorillonite, kaolinite, and goethite
Bacillus subtilis Kaolinite/hematite
Please cite this article as: Hosseini, M.R., Ahmadi, A., Biological beneficiation of kaolin: A review on iron removal, Appl. Clay Sci. (2015),
http://dx.doi.org/10.1016/j.clay.2015.01.012
density of adsorbed organisms, kaolinite showed the highest attraction
followed by hematite, corundum, and quartz. This was justified by the
presence of positively charged groups of proteinaceous compounds on
the cell wall, and the ion-exchange function of the clay surface. Finally,
it was suggested that it was possible to separate efficiently silica and
silicates from alumina and iron oxide through bioflocculation. Also,
using corundum-adapted cells at pH 7.0 more than 99% separation
was obtained between hematite and corundum. Additionally, efficient
separation of hematite from kaolinite (85%) could be brought about
through selective flotation at the size ranges of 75–110 μm after
biotreatment with cells of P. polymyxa. Using the same bacteria, in
another works, Poorni and Natarajan (2013, 2014) performed a re-
search on the flocculation of hematite from kaolinite. Adapted to 10%
kaolinite or hematite containing pulp, bacteria was studied for the se-
cretion of extracellular proteins and polysaccharides and their adsorp-
tion affinity to the mineral surfaces under different conditions. Growth
of cells in the presence of hematite enhanced the secretion of polysac-
charides and their adsorption on hematite surface; while, presence of
kaolinite during bacterial growth promoted the exertion of stress pro-
teins and adsorption of them on kaolinite. Also, considering the surface
properties, mineral grown cells or their metabolites shifted the IEP of
hematite and kaolinite to a lower value of 1.5, and higher value of 3.0,
respectively. The microbe–mineral interaction also had an influence
on bacterial cells themselves, as the kaolinite grown cells were reported
to be more hydrophobic than the hematite grown ones, and the adsorp-
tion density of cells onto hematite was almost three times higher than
that observed on kaolinite under all pH conditions. Finally, the maxi-
mum settling of hematite was attained using hematite adapted cells
or their extracellular polysaccharides. Likewise, the highest dispersion
of kaolinite was achieved using kaolinite adapted cells or their extra-
cellular proteins or cell free extract.
Furthermore, adsorption behavior of EPS and microbes on iron
oxides and clay minerals had also been the subject of research in some
literatures. Considering iron oxide, Omoike and Chorover (2006) con-
ducted a research on the adsorption of EPS produced by Bacillus subtilis
to the goethite surface. They found out that the adsorption of carbon
(EPS-C), nitrogen (EPS-N), and phosphorus (EPS-P) containing mole-
cules to goethite was decreased with increasing pH, because the adsorp-
tion was electrostatically favorable at 2 b pH b 8 where goethite and EPS
are oppositely charged. In addition, according to the isotherm plots,
raising the ionic strength using NaCl electrolyte solution declined the
EPS adsorption because of the increased competition from counter-
ions in the electrolyte (Cl− to goethite, and Na+ to EPS). Moreover,
FTIR studies indicated that the mechanism of EPS adsorption included
the formation of P–O–Fe bonds between phosphoryl groups and
goethite-surface iron metal centers which caused the preferential ad-
sorption of EPS-P relative to –C and –N. Also, further analysis proved
the preferential uptake of proteins and nucleic acids compared to poly-
saccharides; so, nucleic acids and proteins play a significant role in
bacterial adhesion to iron oxide surfaces. Adsorption of EPS and their
constituents extracted from B. subtilis on kaolinite, montmorillonite,
Method Time Separation efficiency (%) Reference
Flotation 1 min 85 Deo and Natarajan (1998)
Flocculation 1 min 54.5 Deo and Natarajan (1998)
Flocculation 1 min 99.2 Deo and Natarajan (1998)
Flocculation 1 min 95.5 Deo and Natarajan (1998)
Flotation 5 min 88.3 Deo and Natarajan (1998)
Flocculation 1 min 99.6 Deo and Natarajan (1998)
– – – Omoike and Chorover (2006)
– – – Jiang et al. (2007)
– – – Cao et al. (2011)
– – – Wu et al. (2011)
Flocculation 5 min 85 Poorni and Natarajan (2013)
 M.R. Hosseini, A. Ahmadi / Applied Clay Science xxx (2015) xxx–xxx
and goethite was also investigated by Cao et al. (2011). It was found that
the affinity of EPS-C and EPS-N (from proteins) to adsorb on montmoril-
lonite was greater compared to goethite and kaolinite, respectively.
Considering EPS-P (from nucleic acids), there was much attraction
to goethite than montmorillonite and kaolinite. Moreover, although in-
creasing the pH value decreased the adsorption of EPS-N and –P, it had
no effect on EPS-C adsorption. Also, in contrast to what was reported by
Omoike and Chorover (2006), presence of Na+ and Ca2+ promoted the
adsorption of EPS on all three minerals (Cao et al., 2011).
Additionally, attachment of microbes on the mineral particles and
its effects on the surface properties has been examined by some re-
searchers (Jiang et al., 2007; Wu et al., 2011). They illuminated that
P. putida preferred to adsorb on goethite rather than montmorillonite
and kaolinite (Jiang et al., 2007). This was because of the positive charge
of the goethite surface which attracted the negatively charged bacteria.
Also, the maximum adsorption of cells at pH 3.0 (PZC of P. putida), sug-
gested that except for electrostatic force, hydrophobic force and other
interactions such as van der Waals force and hydrogen bonding might
be effective. The addition of cations promoted the bacteria adsorption
as the cations increased contact between bacteria and mineral surfaces
by suppressing the formation of diffuse electrical double layers around
clay particles (Jiang et al., 2007). Also, it was proved that addition of
any concentration of organic ligands such as acetate, tartrate, oxalate
and phosphate in pH 7 had an inhibitory effect on bacterial adsorption
on goethite, montmorillonite, and kaolinite. Changed to 5.5, the pH
did not alter the trend except for acetate which promoted the adsorp-
tion on goethite and kaolinite (Wu et al., 2011). This depressing effect
was in the following sequence for all three minerals: citrate N
tartrate N oxalate N acetate, and the probable reasons were firstly, the
electrostatic repulsion between bacteria and minerals because of the
complex formation between ligands and surface hydroxyls that in-
creased the negative charges on mineral surfaces. Secondly, the compe-
tition between ligands and bacteria for adsorption blocked the binding
of bacterial surface groups to the mineral surface. Nevertheless, the
promoting effect of acetate might be described by neutralizing the
negative charges on the two minerals through surface protonation
(Wu et al., 2011).
5. Conclusion
The purpose of this review was to depict an overview of the status
of biological removal of iron from clays, evaluate and compare the
results, and to introduce the further potentials for the future research
on this environmentally friendly approach. Therefore, all of the bio-
beneficiation methods applied during the past two decades were
collected and divided into three categories named as fungal leaching,
bacterial leaching, and bioflocculation. Then, the last findings in each
area were revealed, and the influence of effective parameters on both
the process efficiency and clay specifications was described. Most of
the involved studies have offered valuable knowledge about the tech-
nique, and have presented very promising results which are worthy of
note to continue in higher scales.
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