Applied Clay Science 138 (2017) 8188

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Applied Clay Science

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

Research Paper

Effects of microbial cells and their associated extracellular polymeric

substances on the bio-occulation of kaolin and quartz
Ramin Akbari Ghashoghchi, Mohammad Raouf Hosseini , Ali Ahmadi
Department of Mining Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran

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

Article history: Microbes can function as bio-surfactants to selectively occulate or disperse particles, and separate minerals. In
Received 28 February 2016 the present study, Bacillus licheniformis cells and metabolite were used to change the settling behavior of kaolin
Received in revised form 12 December 2016 and quartz. Moreover, to perform a more detailed study on the metabolite, settling effect of its protein and poly-
Accepted 3 January 2017
saccharide constituents were also investigated separately. In the best condition, about 40% improvement in kao-
Available online xxxx
lin settling was observed using bacterial cells and metabolite at pH = 7 and 3, respectively. Also, enhancement in
Keywords:
quartz sedimentation using the same bio-occulants was N50% at pH = 13. In addition, polysaccharide was
Bacillus licheniformis more effective in kaolin occulation, while protein was more inuential in quartz agglomeration. Results of ad-
Flocculation sorption tests suggested that all of the bio-surfactants were more inclined to be adsorbed on quartz, and gener-
Kaolin ally, increasing the pH value decreased the reagent adsorption and mineral occulation. Moreover, although
Polysaccharide reducing the mineral concentration from 50 to 20 g/l improved the settling of both kaolin and quartz, the highest
Protein difference (50%) in their sedimentation occurred at the higher mineral concentration. Finally, according to the
Quartz statistics, clay concentration was the most inuential parameter on kaolin, and the dispersion pH was the most
effective variable in quartz bio-occulation.
2017 Elsevier B.V. All rights reserved.

1. Introduction
The environmental concerns, and requirements to reduce operating
costs, have provoked substantial research works on introducing innova-
tive methods or developing current technologies like nding more in-
uential otation and occulation reagents (Lopez et al., 2015; Poorni
and Natarajan, 2014). Regarding these vigorous attempts, biological
mineral processing approaches are ourishing every day because of
being environmentally sustainable, cost effective, and exible
(Karthiga and Natarajan, 2015; Shih et al., 2001).
The existence and behavior of microorganisms are linked to mineral,
solution and nutrient properties like particle occulation, dispersion,
grain shape and size, ion exchange capability, ionic strength and compo-
sition of solutions (Dai et al., 2014). So, microbes can be applied as col-
lectors, activators, depressants, occulants, or dispersants to selectively
separate valuable minerals from their contaminants (Khoshdast et al.,
2012; Smith and Misra, 1993). This is achieved through selective ad-
sorption of bacterial cells or their secretions onto mineral surfaces as
biolm, then altering their surface chemistry or making adhesive brid-
ges between mineral particles (Dubel et al., 1992; Raichur et al.,
1996). Therefore, two different mechanisms govern the surface bio-
treatment. One type is the direct mechanism which involves adhesion
Corresponding author.
E-mail address: r.hosseini@cc.iut.ac.ir (M.R. Hosseini).
of cells themselves to mineral particles while the other is the indirect
mechanism in which microbial metabolites act as bio-reagents
(Govender and Gericke, 2011; Smith and Miettinen, 2006). Various pa-
rameters such as the chemical or electrostatic interactions, Van der
Waals forces, surface free energy components, and the presence of dif-
ferent cations affect the attachment of bio-reagents on mineral surfaces
(Lopez et al., 2015; Sharma, 1999). Surfactant characteristics of these bi-
ological reagents are due to the presence of functional nonpolar hydro-
carbon chains, and polar carboxyl, hydroxyl, or phosphates species in
the cell wall structure or in the extracellular polymeric substances
(EPS) such as polysaccharides, proteins, glycoproteins or nucleic acids
(Diao et al., 2014; Iqbal and Edyvean, 2004; Poorni and Natarajan,
2014).
Kaolinite and silica are two important industrial minerals which
have vast applications in manufacturing ceramics, porcelain, glass, ce-
ment, paints and cosmetics (Choy et al., 2007). So, kaolinite and silica
may be present in industrial efuents that should be removed before
discharging to the environment (Karthiga and Natarajan, 2015;
Yannopoulos et al., 2015). Furthermore, they are usually contaminated
with different impurities, mostly iron (hydr)oxides (Bergaya and
Lagaly, 2013). Even silica itself is considered as an impurity in kaolin
clay. Therefore, bio-beneciation which utilizes various types of micro-
organisms like fungi and bacteria (Hosseini and Ahmadi, 2015) is the re-
cent method of choice. This approach mostly includes bio-otation (Deo
and Natarajan, 1998; Lopez et al., 2015; Sarvamangala and Natarajan,

http://dx.doi.org/10.1016/j.clay.2017.01.002
0169-1317/ 2017 Elsevier B.V. All rights reserved.
82 R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188

2011), and bio-occulation (Deo and Natarajan, 1998; Manivasagan et a

al., 2015; Poorni and Natarajan, 2014). In the most recent research K
work, Lin et al. (2016) meticulously studied the adsorption of EPS K Q
from Pseudomonas putida on montmorillonite, kaolin and goethite as a

Intensity
K
function of pH using confocal laser scanning microscopy, and isothermal K
KK K
titration calorimetry. Manivasagan et al. (2015) examined the bio-pro-
Q Q K K
duction and optimization of polysaccharide based surfactant for bio- Q Q
occulation of kaolin and green synthesis of silver nanoparticles using
Streptomyces sp. Karthiga and Natarajan (2015) conducted a compara- 10 20 30 40 50 60 70 80 90
tive study on the bio-occulant production and kaolin occulation abil- 2

ities of Bacillus licheniformis and Bacillus rmus. Zhao et al. (2012)

studied the adsorption of Escherichia coli and Streptococcus suis on b
Q
montmorillonite and kaolinite as a function of pH and ionic strength.
Also, occulation behavior of hematitekaolin dispersions in the pres-

ence of extracellular proteins and polysaccharides of Bacillus subtilis
was investigated by Poorni and Natarajan (2013, 2014). Furthermore,
selective separation of hematite from silica, and kaolin through otation
and occulation using Bacillus subtilis and Paenibacillus polymyxa was
examined (Deo and Natarajan, 1998; Sarvamangala and Natarajan,
2011; Shih et al., 2001).
Despite the valuable contributions of the above-mentioned litera-
ture to the mineral bio-occulation and bio-separation science, varia-
tions in the settling behavior and bio-occulation of kaolin and silica
in different pH ranges have not been fully investigated and compared
with each other. Such a study is necessary as both minerals may coexist
together. So, a difference in their bio-occulation behavior can substan-
tially assist their separation. On the other hand, there is no information
about the occulating ability of B. licheniformis cells. Also, the extracellu-
lar polymeric substances of this bacterium have not been examined in
silica occulation. Therefore, the present study evaluates and compares
the occulation of quartz and kaolin using bacterial cells, extracellular
metabolite, polysaccharide, and proteins of Bacillus licheniformis as
bio-occulant, at various pH values and two levels of mineral
concentrations.
2. Materials and methods
2.1. Minerals and chemicals
A high-grade raw kaolin sample was provided by Iran China Clay In-
dustries Co. from Zonouz mine located in Marand, Iran. Also, a high pu-
rity raw quartz sample was supplied by Tooma Minerals Co., Isfahan,
Iran. Both samples were ground and screened, separately using 400
mesh (38 m) sieves, and the nes accumulated under the sieve were
used in all experiments. Also, all the chemical reagents were purchased
from Titrachem, Iran in analytical grade.
The X-ray diffraction (XRD) analysis was performed on the screened
samples using Asenware AW/XDM 300, China in the 2 range of 10
90 (step size: 0.05 2, step time: 1 s, slit width: 0.5) with Cu K radi-
ation k = 1.5405 at 40 kV, 30 mA, and illustrated in Fig. 1. According to
the XRD patterns, kaolinite, Al2Si2O5(OH)4, and quartz, SiO2, are the
main constituents of kaolin (Fig. 1a), and the quartz sample is almost
pure (Fig. 1b). The reections associated with each phase are marked
and described in Fig. 1. In order to obtain the chemical compositions
of the samples, S4 Pioneer, Bruker (Germany) X-ray uorescence
(XRF) analyzer was employed. The clay sample was milled, dried at
100 C and roasted for 2 h at 1000 C to determine the Loss on Ignition
(LOI). Results are presented in Table 1. Also, specic surface area of the
samples was measured by the Brunauer, Emmett and Teller (BET)
method of adsorption of N2 gas (Belsorp mini II, Bel, Japan). The temper-
ature of the liquid nitrogen was 77 K, and all the samples were
outgassed at 150 C overnight under owing nitrogen, before measure-
ments. According to this analysis, the specic surface areas of the initial
quartz and kaolin samples were 0.46 and 4.81 m2/g. Additionally,
their total pore volumes were 6.30 10 3 and 30.66 103 cm3/g,
respectively.
Intensity
Q
Q Q Q Q
Q Q Q Q
Q Q Q QQQ Q
10 20 30 40 50 60 70 80 90
2
Fig. 1. XRD patterns of (a) kaolin and (b) quartz samples. (K: kaolin, Q: Quartz).
2.2. Bacterial growth conditions
Lyophilized strain of Bacillus licheniformis (PTCC1320) was pur-
chased from the Iranian Research Organization for Science and Technol-
ogy (IROST), Tehran, Iran. Initially, bacterial cells were cultivated on
PDA (potato, 300 g/l; dextrose, 20 g/l; and agar 20 g/l), and also in
100 ml liquid pre-culture medium containing peptone, 5 g/l; meat ex-
tract, 3 g/l; CaCl242H2O, 100 mg/l; MnCl22H2O, 10 mg/l, in a 500 ml
Erlenmeyer ask and incubated for 24 h. In order to biosynthesis the
required occulants, 10 ml of the pre-culture medium was transferred
to 90 ml of a synthesis medium consisting of glucose, 50 g/l;
Na2HPO47H2O, 12.8 g/l; KH2PO4, 3 g/l; NaCl, 0.5 g/l; NH4Cl, 1 g/l;
MgSO4, 2 mM; CaCl2, 0.1 mM, in a 500 ml ask, and incubated for
96 h. Incubation was performed at 35 C using Jaltajhiz (JTSL20) orbital
shaker at 150 rpm.
2.3. Preparation of bio-occulant solutions
In the present study, bacterial cells, metabolites, puried extracellu-
lar protein and polysaccharide were applied as bio-reagents. To separate
bacterial cells and metabolites, the cultures were centrifuged at
5000 rpm, and the precipitated cells were washed with distilled
water, centrifuged three times, and were used in experiments. The re-
maining liquor itself was used as reagent, and for protein and polysac-
charide extraction. Polysaccharides were extracted from the liquor
according to Manivasagan et al. (2015) by adding cold ethanol to the
cell free extract at a volumetric ratio of 2:1. For protein extraction, the
procedure previously described by Poorni and Natarajan (2014) was
employed by dissolving ammonium sulfate in the cell free liquor at
4 C to 65% saturation level.
2.4. Adsorption evaluation
Adsorption tests were conducted to compare the tendency of pro-
tein and polysaccharide ingredients to be adsorbed onto the mineral
particles at different pH values. So, in 50 ml of each solution, containing
bacteria (~7.2 108 cell/ml), protein or polysaccharide (~72 g/ml), 1 g
of the minerals was suspended. Also, NaCl as an electrolyte was added
(10 mM), and pH was adjusted to the levels of 2 to 9. Then, the disper-
sion was agitated for 15 min on a heater-stirrer at 35 C. Finally, the dif-
ference between initial and nal concentrations of bacterial cells,
 R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188
Table 1
Chemical compositions of the kaolin and quartz samples.
Oxide (mass %) SiO2 Al2O3 Fe2O3 TiO2
Kaolin 63 1 24 1 0.6 0.1 0.04 0.01
Quartz 98 1 0.2 0.1 0.1 0.1 0.02 0.01
a
ND: Not determined.
protein, and polysaccharide was calculated and reported as the mass of
reagent (mg) adsorbed onto the unit surface (m2) of mineral powder.
Protein concentration was measured by the Bradford (1976) method,
and phenol-sulfuric acid (Dubois et al., 1956) technique was imple-
mented for the polysaccharide assessment. In order to identify the bac-
terial adsorption to the minerals, samples were taken from the bacterial
dispersion before and after treatment with mineral powder. Then the
bacterial cells were centrifuged (4000 rpm) and transferred to a lysis
buffer containing phosphate buffer saline (PBS) and imidazole, 10 mM.
Afterward, the opaque dispersion was sonicated for 2 min (5-s pulses,
amplitude of 20 nm) until becoming clear. After centrifugation, the pro-
tein contents of the remained liquors were measured by Bradford
method.
2.5. Design of experiments
In order to conduct sedimentation experiments, a general factorial
design was selected for each mineral. Variables were the type of bio-re-
agent, pH of the sedimentation medium, and mass of the mineral pow-
der. The investigated variables and their levels are shown in Table 2.
Each run was done in duplicate, and results were assessed by Design ex-
pert 7 software.
2.6. Sedimentation tests
Sedimentation experiments were carried out using different bio-re-
agents. Therefore, to compare the inuence of the reagent type on the
mineral sedimentation, initially, a control test was conducted without
any additive at the ve specied pH levels. The mineral powder (kaolin
or quartz) was added to 80 ml of deionized water, and its pH was set to
the identied value by dilute sulfuric acid or sodium hydroxide while
steering for 5 min. Afterward, the slurry was transferred to a 100 ml
measuring cylinder and lled with deionized water to exactly 100 ml.
The cylinder was inverted ten times during 5 min and kept still for
3 min. Then, 90 ml of the dispersion was removed and the remained
sediments dried and weighted. Finally, the ratio of the precipitates to
the total powder mass was reported as sedimentation percent. The
same procedure was also implemented when bio-reagents were ap-
plied. For metabolite, one-fth (20 ml) of the separated metabolite
was added to 80 ml of the mineral dispersion during agitation, and
then pH was adjusted to the set point. For protein and polysaccharide,
each puried component obtained from one culture ask was dissolved
in 100 ml deionized water and a similar process was repeated to apply a
reagent concentration similar to the bacterial metabolite (protein
~ 7.2 g/ml, and polysaccharide ~ 0.24 mg/ml). For the bacterial cells,
all the washed precipitated cells from one culture ask, were gradually
Table 2
Variables and their levels in experimental design.
Variables Levels
1 2 3 4 5 charges (Lopez et al., 2015). Although, the presence of peptidoglycan,
A Dispersion pH 2 3 5 7 9 teichoic acids, and teichuronic acids in the cell wall of gram-positive
B Bioreagent No Bacteria Metabolite Protein Polysaccharide
reagent
C Mineral 20 g/l 50 g/l
concentration
83
CaO MgO Na2O K2O L.O.I
1.2 0.2 0.55 0.05 0.4 0.1 0.3 0.1 91
0.14 0.01 0.01 0.01 0.03 0.01 0.06 0.01 NDa
added to 100 ml agitating mineral dispersion (3.6 108 cells/ml) and
the settling test conducted after pH adjustment.
2.7. Scanning Electron Microscopy (SEM)
SEM images were taken of gold coated quartz and kaolin before and
after treatment with bacterial cells, and bacterial protein at pH = 2
using scanning electron microscope (SEM, Philips, XI30, The Nether-
lands) at 20 kV acceleration voltage.
3. Results and discussion
The nal cells and secretions obtained from the bacterial cultures
were applied as bio-occulants in order to investigate the inuences
of the dispersion pH, solid concentration, and bio-reagent type on the
settling behavior of kaolin and quartz by conducting different bio-oc-
culation experiments. Furthermore, the outcomes of the settling tests
were used in a general factorial design to analyze the signicance and
inuence of each parameter on the response variable. Finally, a mathe-
matical model was obtained to predict the bio-occulation behavior of
both minerals.
3.1. Bio-occulation experiments
Results of the settling tests on kaolin and quartz using 50 g/l solid
concentration are depicted in Fig. 2a and b, respectively. Considering
the reagent-free control experiments, kaolin sedimentation with an av-
erage of around 30% was generally lower than the mean quartz settling
percentage (37%) in the examined pH range. According to both dia-
grams, increasing the dispersion pH from 1 to 9 reduced the mineral set-
tling percentage, because at pH values less than the PZC of the minerals,
which are about 4 and 2 for kaolin and quartz, respectively (Kosmulski,
2009), the surface charge is positive and after the point of zero charge it
becomes progressively negative. Therefore, the repulsive forces be-
tween the particles caused a reduction in particle settling, although
this was not obvious for the kaolin sample.
After addition of the bacterial cells to the mineral dispersions as bio-
surfactants, an obvious augmentation in the extent of occulation was
registered. Regarding quartz, this increase was more signicant in acidic
solutions of all the applied bio-occulants. Considering kaolin disper-
sions, only 3% improvement in settling was achieved using an acidic en-
vironment in the presence of microorganisms, and changing the pH
values from 1 to 9 did not show signicant variation in the amount of
kaolin sedimentation. However, in the case of quartz dispersion, settling
was enhanced by 36% using microbial cells at pH = 1. Increasing the
medium pH to 7 and 9, lowered the extent of sedimentation to that of
the control test.
Adhesion of bacterial cells on clay minerals such as kaolinite was at-
tributed to the large specic surface area available on colloidal clay sur-
faces and their ion-exchange properties (Deo and Natarajan, 1998).
Mineral surface adheres to the functional groups of the cell wall mostly
due to the attractive electrostatic forces between opposite electric
bacteria imparts negative electric charge to their surface (Ahmad
et al., 2011), positively charged amine groups of proteinaceous com-
pounds, which are also present on the cell wall, are responsible for the
binding of quartz and aluminosilicate minerals to gram-positive
84 R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188

45 0.8

Adsorbed bioflocculant (mg/m2)

Adsorbed cells (1010 cells/m2)
0.7
40
Mass settling percent

0.6

35 0.5

0.4
30
0.3

25 0.2

0.1
20
0
1 3 5 7 9 2 3 4 5 6 7 8 9
(a) Dispersion pH (a) pH

7
85

Adsorbed bioflocculant (mg/m2)

Adsorbed cells (1010 cells/m2)
6
Mass settling percent

75
5
65
4
55
3
45
2
35
1
25
0
(b) 1 3 5 7 9
(b) 2 3 4 5 6 7 8 9
Dispersion pH pH

Fig. 2. Settling behavior of (a) kaolin, and (b) quartz as a function of pH and type of bio-
reagent using slurry concentration of 50 g/l. (square, solid line: cells; circle, dashed line:
protein; triangle, dotted line: polysaccharide; diamond, dash-dotted line: metabolite;
gray solid line: control).
bacteria (Deo and Natarajan, 1998). Aluminum as the natural constitu-
ent of clay minerals can be hydrolyzed to electrically positive species
like AlOH+ or Al13(OH32)7+ complexes which act as a bridge between
the two negatively charged groups (Cao et al., 2011; Poorni and
Natarajan, 2013).
The dependence of cell adsorption on the dispersion pH are illustrat-
ed in Fig. 3a and b. According to these diagrams, the lower was the pH of
the mineral dispersion, the higher was the reagent adsorption. The only
exception was cell adsorption on kaolin particles which culminated at
pH = 7. At this pH, kaolin occulation was also at a maximum. A similar
trend was also reported for kaolin and quartz by Deo and Natarajan
(1998), and for quartz by Sarvamangala and Natarajan (2011), while
Jiang et al. (2007) indicated a maximum cell adsorption on kaolin at
pH = 3.
A pH value between 2 and 4 was declared as the PZC of bacterial cells
by some researchers (Claessens et al., 2006; Hong and Brown, 2008;
Jiang et al., 2007; Lopez et al., 2015); therefore, in an alkaline solution,
where repulsive forces were dominant between extremely negative
surface charges, less adsorption would happen. However, at acidic pH
values around PZC of the minerals and microbial cells, cell-mineral ad-
hesion was enhanced. Nevertheless, the mentioned increase in the cell
adsorption to kaolin particles at neutral to alkaline pH, and subsequent
improvement in its precipitation (Fig. 2a) implied that other forces like
chemical, Van der Waals or hydrogen bonding might play a role in cell-
mineral aggregation (Deo and Natarajan, 1998; Jiang et al., 2007; Lopez
et al., 2015). Regarding Fig. 3a and b, the tendency of kaolin particles for
the adsorption of bacterial cells was much lower than quartz, and this
phenomenon was the reason why the quartz sedimentation was better
than kaolin after occulation by the cells. Attachment of B. licheniformis
cells on quartz particles and their occulation effect was discernible in
Fig. 4b. Also, the bacterial cells adhered to the mineral particles were
clearly distinguishable in Fig. 4c.
Fig. 3. Adsorption of bio-reagents on (a) kaolin and (b) quartz as a function of pH. (Square,
solid line: cells; Circle, dashed line: protein; Triangle, dotted line: polysaccharide).
When bacterial culture broth used as bio-reagent, a signicant in-
crease in occulation of the both minerals was observed. Although
after the bio-treatment, kaolin settling only increased to 41% at pH =
5, the greatest improvement in quartz sedimentation percent was
achieved using bacterial metabolite which caused 90% of quartz to be
settled at pH = 1. Neglecting the nal rise in kaolin sedimentation at
pH = 9, increasing the dispersion pH generally led to decline in clay set-
tling percent. At neutral pH, settling of quartz and kaolin reached the
value obtained in the control test. Similar results about kaolin-metabo-
lite interaction in acidic (Deo and Natarajan, 1998) and neutral (Poorni
and Natarajan, 2013; Shih et al., 2001) pH ranges were reported in liter-
ature. Considering quartz mineral, a signicant reduction in sedimenta-
tion around neutral pH was reported by Deo and Natarajan (1998), and
Sarvamangala and Natarajan (2011).
Soil microorganisms excrete extracellular polysaccharide molecules
to their growth environment in order to increase the physical strength
of the soil by making the soil minerals and microstructures bind togeth-
er for supporting themselves in water uctuation, and dryness periods
(Gomoiu and Catley, 1996). These extracellular polymeric substances
(EPS) may be expelled to the culture medium as bacterial metabolites,
formed onto the cell wall, or adhered on mineral surfaces as biolms.
They are made of proteins and polysaccharides synthesized from carbon
and nitrogen sources of the growth medium and contain different func-
tional groups such as carboxyl, phosphoryl, amide, amino and hydroxyl
groups (Manivasagan et al., 2015; Poorni and Natarajan, 2014). In the
bio-occulation process, EPS molecules are attached to mineral surfaces,
inter-bridge the divides between particles and creates steady three-di-
mensional network of ocks which improves the settling rate of mineral
particles (Karthiga and Natarajan, 2015).
According to the adsorption graph depicted in Fig. 3a, and b, the
overall decrease in precipitation of minerals at neutral to alkaline pH
values was associated with the adopted negative charge of metabolite
constituents as a result of proton dissociation of carboxyl, phospho-
lipids, phosphodiester, hydroxyl and amino groups. This phenomenon
 R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188
(a)
Fig. 4. SEM micrographs of quartz (a) before and (b) after treatment with B. licheniformis at pH = 2. (c) Attachment of bacterial cells to the quartz surface after bacterial treatment. Scale
bars: (a, b) 100 m, (c) 2 m.
prevented negative clay and quartz particles from adhering to the bio-
surfactants (Cao et al., 2011; Deo and Natarajan, 1998). However, in
acidic environment, EPS functional species became protonated and ac-
quired positive charge which adsorbed negative quartz and clay parti-
cles. The lower sedimentation of kaolin compared to quartz in the pH
range of 13 was related to the higher PZC value of kaolin, which caused
the surface charge of kaolin to be positive and repulsive against cell free
extracts. Despite the fact that these ndings indicated electrostatic in-
teractions as important driving forces in EPS adsorption on both min-
erals (Cao et al., 2011; Lin et al., 2016; Poorni and Natarajan, 2013,
2014), a slight increase in polysaccharide and protein adsorption to ka-
olin at pH = 9 along with great improvement in its settling behavior
compared to pH = 7 suggested that other bonds like Van der Waals
or chemical interactions might attach cell metabolite to kaolin surface.
Regarding Fig. 2a, and b, the application of bacterial exo-
polysaccharides has led to the lowest settling efciency of two minerals
at pH = 7. At this point, the particle sedimentation was even lower than
what observed in the control test. For quartz, this settling reduction con-
tinued at higher pH values, but for kaolin particles, another 14% increase
was observed at pH = 9. This peculiar uctuation was not specic to
exopolysaccharides of B. licheniformis, because it was also previously re-
ported using B. subtilis (Poorni and Natarajan, 2014), and B. nivea
(Gomoiu and Catley, 1996). Additionally, 82% of quartz was settled at
pH = 5, which was the highest quartz sedimentation using polysaccha-
ride among all 5 pH values.
Adsorption of polysaccharide to kaolin at different pH values, which
is illustrated in Fig. 3a, revealed that increasing pH decreased adsorption
until pH = 7, but after that there was another increase at pH = 9. This
trend was also shown by Cao et al. (2011), and Lin et al. (2016). So, in-
crease in kaolin occulation efciency at pH = 9 could be associated
with the higher adsorption of polysaccharide at this pH compared to
pH = 7. Containing COO, OH and PO3 4 groups, exopolysaccharides
are negatively charged in a wide range of pH values. So, they can interact
with aluminosilicate minerals, and form stable complexes (Deo and
Natarajan, 1998). In addition, an acidic environment improves the ex-
tracellular polysaccharide adsorption onto the mineral particles, be-
cause of the positive surface charge of minerals which electrostatically
attracts negative saccharide polymers. Nevertheless, as repulsive forces
rule at high pH values, adsorption declines and mineral occulation de-
creases. Augmentation in adsorption to kaolin and settling performance
85
(b)
(c)
at pH = 9 in spite of the repulsive forces could again be attributed to
bonding forces other than electrostatics (Jiang et al., 2007; Poorni and
Natarajan, 2014). Lin et al. (2016) conrmed that there was a large
drop in enthalpy from pH = 5 to 9 for the montmorillonite and kaolinite
systems that indicated the adsorption of EPS to clay minerals was less
exothermic at higher pH (Lin et al., 2016). Moreover, the difference in
polysaccharide adsorption between Fig. 3a and b, specied that quartz
was much more inclined to adsorb polysaccharide in comparison to ka-
olin (Deo and Natarajan, 1998). So, the higher occulation efciency of
quartz in the presence of bacterial polysaccharides was justied.
Considering protein as bio-surfactant, about 78% quartz sedimentation
was achieved at pH = 1. Further increasing the settling pH resulted in a
sudden drop in quartz occulation at pH = 5 to below the amount regis-
tered in the control test. Generally, as polysaccharide adhesion to quartz
was more than protein (Fig. 3b) polysaccharide was more inuential in
quartz settling, especially at pH = 5. Variations in kaolin settling were sim-
ilar to what was observed for polysaccharide with a minimum point at
pH = 7. The highest kaolin settling percent (about 45%) was obtained at
pH = 9 using both extracellular proteins and polysaccharides. These out-
comes seem rational if the results of adsorption tests are regarded
(Fig. 3a). Considering the curves presented in Fig. 3a and b, protein attach-
ment was prevented at pH values higher than mineral PZC except for ka-
olin which showed a high protein adsorption at pH = 9. Although the
same observation was reported by some researchers (Cao et al., 2011;
Lin et al., 2016), Poorni and Natarajan (2014) presented a completely dif-
ferent pattern of protein and polysaccharide adsorption on kaolin. In their
adsorption curve, EPS adhesion to the clay was at maximum at pH = 7
where the lowest extent of settling happened. Also, in contrast to Deo
and Natarajan (1998), protein adsorption to quartz was signicantly bet-
ter than kaolin at all the pH ranges (Fig. 3).
Proteins are comprised of amine and carboxyl groups. At low pH,
amine groups are protonated and gain positive charge; on the other
hand, carboxyl groups of proteins lose hydrogen at high pH, and acquire
negative charge (Ahmad et al., 2011). Therefore, attachment of proteins
to the kaolin and quartz surfaces was easier at pH values under 7, in
which an opposition might exist between surface charges of positive
proteins and negative minerals. SEM images of kaolin particles before
and after interaction with proteins are depicted in Fig. 5. Obviously,
the micro-sized kaolin akes agglomerated after treatment with bacte-
rial protein as bio-occulant.
86 R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188

(a) (b)

Fig. 5. SEM micrographs of kaolin (a) before and (b) after treatment with extracellular proteins of B. licheniformis at pH = 2. Scale bars: 20 m.

Another aim of this study was to investigate the role of mineral mass
in settling efciency. Hence, the applied mineral concentration in all of
the experiments was decreased to 20 g/l while the amounts of bio-re-
agents remained unchanged. Although, increasing the bio-occulant
to mineral ratio improves the sedimentation percentage of the minerals,
using high bio-occulant dosages causes oversaturation of bio-surfac-
tants on mineral surface binding sites which reduces particle sedimen-
tation (Karthiga and Natarajan, 2015). The nal consequences are
presented in Fig. 6a and b. Comparing the data presented in this gure
and Fig. 2, substantial enhancement in the extent of kaolin occulation
was achieved compared to previously examined 50 g/l concentration.
Despite this large change in kaolin settling, little improvement in the
settling of quartz particles was discerned, especially at high pH values.
Using 20 g/l mineral concentration, cell-free extract showed the
greatest occulating ability among other bio-occulants, which was
83% for kaolin at pH = 3, and 92% for quartz at pH = 1.
Considering Figs. 2 and 6, the maximum difference between quartz
and kaolin settling varied for the two mineral concentrations. When
90
80
Mass settling percent
the mineral concentration was 20 g/l, the highest settling difference
was 36%, which reached at neutral pH using bacterial cells as bio-surfac-
tant, while at 50 g/l mineral concentration, about 4550% difference in
mineral settling was observed in the pH range of 31, when bacterial
metabolite was applied. So, despite the better occulation efciency at
lower mineral concentration, the separation of quartz from kaolin
might be more effective at higher mineral concentration.
As reported by Cao et al. (2011), Lin et al. (2016), and Poorni and
Natarajan (2014), protein adsorption to the both mineral surfaces was
higher in comparison to polysaccharide. However, based on the Figs. 2
and 6, adsorption of protein and polysaccharide on the both minerals
were pH dependent and affected their occulating performance. Ac-
cording to Fig. 3a, in acidic region, protein adsorption to kaolin was
lower than polysaccharide, but after pH = 6.5, protein was adsorbed
more. This phenomenon was also reected in the occulation efcien-
cies illustrated in Fig. 2a. The same trend was also distinguished for
quartz. At pH = 2 that protein adsorption was higher than polysaccha-
ride (Fig. 3b), quartz sedimentation by protein was also greater (Figs. 2b
and 6b). After this point, polysaccharide adhesion exceeded protein till
around pH = 6.5 where it became the reverse. Exactly similar occur-
rence was observed in settling graphs depicted in Figs. 2b and 6b. Nev-
ertheless, since the variation in occulating performance of metabolite

on kaolin in the applied pH range conformed to that of polysaccharide,

70
especially at 20 g/l kaolin concentration (Fig. 6a), the polysaccharide
60 was probably the inuential component of the metabolite in kaolin oc-
culation. According to Gomoiu and Catley (1996), the mechanism of ka-
50 olin occulation lies with the polysaccharide component of metabolite
rather than protein. On the contrary, considering Figs. 2b and 6b, occu-
40 lation of quartz by the bacterial metabolite in the applied pH range,
mostly followed the protein pattern. So, in the quartz occulation pro-
30 cess, protein was possibly the effective constituent.
1 3 5 7 9
(a) Dispersion pH 3.2. Analysis of the experimental design for kaolin sedimentation

100 To analyze the effects of pH, bio-surfactant, and clay mass on the ka-
90 olin settling percent as the response factor, a general factorial design
Mass settling percent

80 with 50 tests (all of them conducted in duplicate) was applied using

the conditions previously introduced in Table 2. Results of the analysis
70 of variance (ANOVA) of the experimental design are presented in
60 Table 3.
The F-value and p-value of the model were 143.27, and b 0.01%, re-
50
spectively. So, the obtained model was statistically signicant with
40 99.99% condence level. Also, considering model parameters, the p-
30 value of b0.05 implied that with 95% condence level, the terms were
signicant (Valipour, 2012a, 2012b). Therefore, according to the data
20
presented in Table 3, all the main variables and their two/three-factor
1 3 5 7 9
(b) Dispersion pH
interactions were signicant with a high condence level. Based on
the F-values, the clay concentration (3631.65) was the most effective
factor on kaolin sedimentation. After that, there were bio-reagent type
Fig. 6. Settling behavior of (a) kaolin, and (b) quartz as a function of pH and type of bio-
reagent using slurry concentration of 20 g/l. (square, solid line: cells; circle, dashed line:
(270.75), and pH (184.52), respectively.
protein; triangle, dotted line: polysaccharide; diamond, dash-dotted line: metabolite; Specications of the kaolin settling model are summarized in
gray solid line: control). Table 4. The determination coefcient (R-squared) was 0.99. R-squared
 R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188 87

Table 3 90
ANOVA results for kaolin sedimentation experiments. 80

Mass settling percent

Source Sum of Degree of Mean F value p-Value
70
squares freedom square 60
Model 25,342.25 49 517.19 143.27 b0.0001 50
A - Dispersion pH 2664.50 4 666.13 184.52 b0.0001 40
B - Bio-reagent 3909.60 4 977.40 270.75 b0.0001
30
C - Kaolin conc. 13,110.25 1 13,110.25 3631.65 b0.0001
AB 2106.40 16 131.65 36.47 b0.0001 20
AC 447.50 4 111.88 30.99 b0.0001 10
BC 2355.80 4 588.95 163.14 b0.0001 0
ABC 748.20 16 46.76 12.95 b0.0001 (a) Bacterial cell Metabolite Protein Polysaccharide Control
Pure error 180.50 50 3.61
Cor total 25,522.75 99
65
60

Mass settling percent

value was an indication of the ability of the experimental variables and
their interactions to explain the variability in the observed response
values (Mahdizadeh Khasraghi et al., 2015). There was a very good
agreement between the adjusted (0.99) and predicted (0.97) R-
squared. The closer the R2 quantity is to 1, the better the model predicts
the response (Valipour et al., 2015). Also, adequate precision (43.17)
measures the signal to noise ratio, and a ratio N4 is generally required
(Krbahti and Rauf, 2008). The high value of the predicted sum of
squares which is abbreviated to PRESS showed that the extracted
model t each point in the design very well (Isar et al., 2006; Valipour,
2016).
Regarding the factor interactions presented in Table 3, the type of
bio-occulant and kaolin concentration (BC) had the highest inuence
on each other. After that, the interaction between pH and bio-occulant
(AB), and then, pH and kaolin concentration (AC) were important, re-
spectively. For example, BC interaction was clearer at pH = 5 (Fig. 7a),
where the extent of settling was generally higher at kaolin concentra-
tion of 20 g/l except for protein which lowered the settling percent to
the value observed for 50 g/l clay concentration. This was also, percep-
tible from AC interaction in Fig. 7b.
3.3. Analysis of the experimental design for quartz sedimentation
Another general factorial design including 50 runs (all of them were
conducted in duplicate) was implemented using the factors and levels
listed in Table 2. Mass settling percent was regarded as the response
variable. Results of the analysis of variance (ANOVA) of the applied ex-
perimental design are shown in Table 5.
The obtained model was statistically signicant with the F-value of
286.68 which associated with a condence level of 99.99%. Considering
the data presented in Table 5, all of the main variables and their interac-
tions were signicant too, with 99.99% condence level. Comparing the
F-values of the main factors, the dispersion pH (1871.87) had the
highest inuence on quartz settling. Type of bio-reagent was the second,
and as expected, quartz concentration was the least effective parameter
with the F-values of 591.46 and 146.71, respectively.
55
50
45
40
35
30
25
20
1 3 5 7 9
(b) pH
Fig. 7. Interaction graphs of (a) BC at pH = 5, and (b) AC at protein (circle: 20 g/l, square:
50 g/l).
The obtained model was evaluated by checking its statistics listed in
Table 4. The determination coefcient (R-squared) of the quartz settling
model was almost 1.00. The adjusted and predicted R-squared were ap-
proximately equal to each other.
The mutual effect between the settling pH and the type of bio-re-
agent (AB) was the most effective interaction (F-value = 237.74), and
after that, there was reagent-mineral concentration interaction (BC)
with F-value of 33.02 in the second place with a large distance from
the former (Table 5).
Regarding the mutual action between reagent and pH (Figs. 2b and
6b), changing the bio-occulant at lower pH was more effective than
doing the same at higher pH values. In other words, varying the bio-oc-
culant type did not change the response after pH = 7. Taking the BC in-
teraction into consideration (Fig. 8a), manipulation of quartz
concentration did not improve mineral settling percent either in bacte-
rial metabolite or control tests; while when other bio-occulants were
used, the difference between two mineral concentration levels became
signicant. Interaction between pH and quartz concentration (AC)
which was clearer in the bacterial cell tests represented in Fig. 8b.
Usage of bacteria as reagent, and quartz concentration of 20 g/l in low
pH ranges led to better settling results, but at high pH values, changing
the mineral concentration did not inuence the response, signicantly.
Table 5
ANOVA results for quartz sedimentation experiments.

Table 4 Source Sum of Degree of Mean F value p-Value

Specications of the derived settling models for kaolin and quartz. squares freedom square

Model 48,041.84 49 980.45 286.68 b0.0001

Statistic Kaolin Quartz
A - Dispersion pH 25,607.14 4 6401.79 1871.87 b0.0001
Standard deviation 1.90 1.85 B - Bio-reagent 8091.14 4 2022.79 591.46 b0.0001
Mean 46.15 53.54 C - Quartz conc. 501.76 1 501.76 146.71 b0.0001
Coefcient of variation % 4.12 3.45 AB 13,009.06 16 813.07 237.74 b0.0001
PRESS 722.00 684 AC 104.54 4 26.14 7.64 b0.0001
R-Squared 0.99 1.00 BC 451.74 4 112.94 33.02 b0.0001
Adjusted R-squared 0.99 0.99 ABC 276.46 16 17.28 5.05 b0.0001
Predicted R-squared 0.97 0.99 Pure error 171.00 50 3.42
Adequate precision 43.17 47.41 Cor total 48,212.84 99
88 R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188
100
90
Mass settling percent
80
70
60
50
40
30
(a) Bacterial cell Metabolite Protein Polysaccharide
100
90
Mass settling percent
80
70
60
50
40
30
(b) 1 3 5 7
pH
Fig. 8. Interaction graphs of (a) BC at pH = 2, and (b) AC at bacteria (circle: 20 g/l, square:
50 g/l).
4. Conclusion
Bacterial cells and secretions of B. licheniformis culture were applied
as bio-occulants in order to assess the inuences of pH, mineral con-
centration, and type of bio-reagent on the settling behaviors of kaolin
and quartz. According to the control tests, increasing the dispersion
pH would reduce the mineral settling percent due to the repulsive
forces between the negative surfaces of mineral particles. However,
after bio-surfactant addition, an obvious augmentation in the extent of
occulation was observed, especially at lower pH values, because of
the higher reagent adsorption in this pH range. These outcomes indicate
that electrostatic interactions are the most important driving forces in
the surfactant adsorption to the both minerals. Nevertheless, a slight in-
crease in cell, protein and polysaccharide adsorption to kaolin particles
at neutral to alkaline pH, and the subsequent improvement in its set-
tling is probably awing to chemical, Van der Waals, hydrogen bonding
or depletion forces. The greatest improvement in quartz sedimentation
(90%) was obtained using bacterial metabolite at pH = 1. Using polysac-
charide at all 5 pH levels, 82% of quartz was settled at pH = 5. Consider-
ing protein as bio-surfactant, about 78% quartz sedimentation was
achieved at pH = 1. Variations in kaolin settling by protein and polysac-
charide were similar with the highest settling percent (about 45%) at
pH = 9 for both. According to the ndings of this study, polysaccharide
constituent of bacterial metabolite is more effective on kaolin occula-
tion, while protein is the inuential component in quartz occulation.
Decreasing solid concentration to 20 g/l resulted in substantial enhance-
ment in the occulation efciency of the both minerals, especially kao-
lin. Finally, according to ANOVA results, kaolin concentration, bio-
reagent type, and dispersion pH are the most effective parameters on
kaolin sedimentation, respectively. Also, regarding quartz, the pH has
the highest inuence on quartz settling, and the type of bio-reagent,
and quartz concentration are other effective parameters, respectively.
References
Ahmad, I., Ahmad, F., Pichtel, J., 2011. Microbes and Microbial Technology: Agricultural
and Environmental Applications. Springer, New York.
Bergaya, F., Lagaly, G., 2013. Handbook of Clay Science. Elsevier.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72 (1), 248254.
Cao, Y., Wei, X., Cai, P., Huang, Q., Rong, X., Liang, W., 2011. Preferential adsorption of ex-
tracellular polymeric substances from bacteria on clay minerals and iron oxide. Col-
loids Surf. B 83 (1), 122127.
Choy, J.-H., Choi, S.-J., Oh, J.-M., Park, T., 2007. Clay minerals and layered double hydrox-
ides for novel biological applications. Appl. Clay Sci. 36 (13), 122132.
Claessens, J., van Lith, Y., Laverman, A.M., Van Cappellen, P., 2006. Acidbase activity of
live bacteria: implications for quantifying cell wall charge. Geochim. Cosmochim.
Acta 70 (2), 267276.
Dai, Q., Zhao, Y., Dong, F., Wang, B., Huang, Y., 2014. Interaction between bentonite and
Bacillus litoralis strain SWU9. Appl. Clay Sci. 100, 8894.
Control Deo, N., Natarajan, K., 1998. Studies on interaction of Paenibacillus polymyxa with iron ore
minerals in relation to beneciation. Int. J. Miner. Process. 55 (1), 4160.
Diao, M., Nguyen, T.A., Taran, E., Mahler, S., Nguyen, A.V., 2014. Differences in adhesion of
A. thiooxidans and A. ferrooxidans on chalcopyrite as revealed by atomic force micros-
copy with bacterial probes. Miner. Eng. 61, 915.
Dubel, J., Smith, R., Misra, M., Chen, S., 1992. Microorganisms as chemical reagents: the
hematite system. Miner. Eng. 5 (3), 547556.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P., Smith, F., 1956. Colorimetric method for
determination of sugars and related substances. Anal. Chem. 28 (3), 350356.
Gomoiu, I., Catley, B.J., 1996. Properties of a kaolin-occulating polymer elaborated by
Byssochlamys nivea. Enzym. Microb. Technol. 19 (1), 4549.
Govender, Y., Gericke, M., 2011. Extracellular polymeric substances (EPS) from
bioleaching systems and its application in biootation. Miner. Eng. 24 (11),
11221127.
Hong, Y., Brown, D.G., 2008. Electrostatic behavior of the charge-regulated bacterial cell
surface. Langmuir 24 (9), 50035009.
9 Hosseini, M.R., Ahmadi, A., 2015. Biological beneciation of kaolin: a review on iron re-
moval. Appl. Clay Sci. 107, 238245.
Iqbal, M., Edyvean, R., 2004. Biosorption of lead, copper and zinc ions on loofa sponge
immobilized biomass of Phanerochaete chrysosporium. Miner. Eng. 17 (2), 217223.
Isar, J., Agarwal, L., Saran, S., Saxena, R.K., 2006. A statistical method for enhancing the pro-
duction of succinic acid from Escherichia coli under anaerobic conditions. Bioresour.
Technol. 97 (13), 14431448.
Jiang, D., Huang, Q., Cai, P., Rong, X., Chen, W., 2007. Adsorption of Pseudomonas putida on
clay minerals and iron oxide. Colloids Surf. B 54 (2), 217221.
Karthiga, K., Natarajan, K.A., 2015. Production and characterization of bioocculants for
mineral processing applications. Int. J. Miner. Process. 137, 1525.
Khoshdast, H., Sam, A., Mana, Z., 2012. The use of rhamnolipid biosurfactants as a
frothing agent and a sample copper ore response. Miner. Eng. 26, 4149.
Krbahti, B.K., Rauf, M., 2008. Response surface methodology (RSM) analysis of photoin-
duced decoloration of toludine blue. Chem. Eng. J. 136 (1), 2530.
Kosmulski, M., 2009. Surface Charging and Points of Zero Charge. CRC Press.
Lin, D., Ma, W., Jin, Z., Wang, Y., Huang, Q., Cai, P., 2016. Interactions of EPS with soil min-
erals: a combination study by ITC and CLSM. Colloids Surf. B 138, 1016.
Lopez, L.Y., Merma, A.G., Torem, M.L., Pino, G.H., 2015. Fundamental aspects of hematite o-
tation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. 75, 6369.
Mahdizadeh Khasraghi, M., Gholami Sedkouhi, M.A., Valipour, M., 2015. Simulation of
open- and closed-end border irrigation systems using SIRMOD. Arch. Agron. Soil
Sci. 61, 929941.
Manivasagan, P., Kang, K.-H., Kim, D.G., Kim, S.-K., 2015. Production of polysaccharide-
based bioocculant for the synthesis of silver nanoparticles by Streptomyces sp. Int.
J. Biol. Macromol. 77, 159167.
Poorni, S., Natarajan, K., 2013. Microbially induced selective occulation of hematite from
kaolinite. Int. J. Miner. Process. 125, 92100.
Poorni, S., Natarajan, K., 2014. Flocculation behaviour of hematitekaolinite suspensions
in presence of extracellular bacterial proteins and polysaccharides. Colloids Surf. B
114, 186192.
Raichur, A., Misra, M., Bukka, K., Smith, R., 1996. Flocculation and otation of coal by ad-
hesion of hydrophobic Mycobacterium phlei. Colloids Surf. B 8 (1), 1324.
Sarvamangala, H., Natarajan, K., 2011. Microbially induced otation of alumina, silica/cal-
cite from haematite. Int. J. Miner. Process. 99 (1), 7077.
Sharma, P.K., 1999. Physico-Chemical Characterisation of Microbial Cell Surface and
Biootation of Sulde Minerals. Lule Tekniska Universitet.
Shih, I., Van, Y., Yeh, L., Lin, H., Chang, Y., 2001. Production of a biopolymer occulant from Ba-
cillus licheniformis and its occulation properties. Bioresour. Technol. 78 (3), 267272.
Smith, R.W., Miettinen, M., 2006. Microorganisms in otation and occulation: future
technology or laboratory curiosity? Miner. Eng. 19 (6), 548553.
Smith, R.W., Misra, M., 1993. Recent developments in the bioprocessing of minerals.
Miner. Process. Extr. Metall. Rev. 12 (1), 3760.
Valipour, M., 2012a. Comparison of surface irrigation simulation models: full hydrody-
namic, zero inertia, kinematic wave. J. Agric. Sci. 4 (12), 6874.
Valipour, M., 2012b. Sprinkle and trickle irrigation system design using tapered pipes for
pressure loss adjusting. J. Agric. Sci. 4 (12), 125133.
Valipour, M., 2016. Optimization of neural networks for precipitation analysis in a humid
region to detect drought and wet year alarms. Meteorol. Appl. 23, 91100.
Valipour, M., Sedkouhi, M.A.G., Eslamian, S., 2015. Surface irrigation simulation models:
a review. Int. J. Hydrol. Sci. Technol. 5, 5170.
Yannopoulos, S., Lyberatos, G., Theodossiou, N., Li, W., Valipour, M., Tamburrino, A.,
Angelakis, A., 2015. Evolution of water lifting devices (pumps) over the centuries
worldwide. Water 7, 5031.
Zhao, W., Liu, X., Huang, Q., Walker, S.L., Cai, P., 2012. Interactions of pathogens Escherichia
coli and Streptococcus suis with clay minerals. Appl. Clay Sci. 69, 3742.